doctor of philosophy effects of phytogenics on intestinal

DOCTOR OF PHILOSOPHY

Effects of phytogenics on intestinal pathogenic bacteria and microbiota of non-ruminants

McMurray, Rebekah

Award date:2021

Awarding institution:Queen's University Belfast

Link to publication

Terms of useAll those accessing thesis content in Queen’s University Belfast Research Portal are subject to the following terms and conditions of use

• Copyright is subject to the Copyright, Designs and Patent Act 1988, or as modified by any successor legislation • Copyright and moral rights for thesis content are retained by the author and/or other copyright owners • A copy of a thesis may be downloaded for personal non-commercial research/study without the need for permission or charge • Distribution or reproduction of thesis content in any format is not permitted without the permission of the copyright holder • When citing this work, full bibliographic details should be supplied, including the author, title, awarding institution and date of thesis

Take down policyA thesis can be removed from the Research Portal if there has been a breach of copyright, or a similarly robust reason.If you believe this document breaches copyright, or there is sufficient cause to take down, please contact us, citing details. Email:[email protected]

Supplementary materialsWhere possible, we endeavour to provide supplementary materials to theses. This may include video, audio and other types of files. Weendeavour to capture all content and upload as part of the Pure record for each thesis.Note, it may not be possible in all instances to convert analogue formats to usable digital formats for some supplementary materials. Weexercise best efforts on our behalf and, in such instances, encourage the individual to consult the physical thesis for further information.

Download date: 13. Apr. 2022

https://pure.qub.ac.uk/en/studentTheses/04477e20-2490-43a7-adae-dedbdc5ec533

Effects of phytogenics on intestinal pathogenic

bacteria and microbiota of non-ruminants

By

Rebekah Lana McMurray

BSc (Hons) Biological Sciences

A Thesis Submitted in Fulfilment of the Requirements for the

Degree of Doctor of Philosophy in Agri-Food and Biosciences

At

Queen’s University Belfast

School of Biological Sciences

Supervised by: Dr. Chen Situ, Dr. Elizabeth Ball and Professor

Michael Tunney

01.03.2021

Abstract

Increased levels of multi-drug resistant bacteria worldwide pose a significant

threat to human and animal health. The recommendations in the WHO’s global

action plan were to phase out and research alternatives to using antibiotics in

animal farming. This study aims to identify natural plant supplements that can

be used in animal feeds as sustainable alternatives to conventional antibiotics

to promote animal health and performance.

Plant extracts from traditional Chinese medicine were screened and their

antibacterial activity was demonstrated against Campylobacter jejuni, Listeria

monocytogenes, Escherichia coli, and Salmonella enterica subsp. enterica

serovar Enteritidis. Agrimonia pilosa Ledeb, Smilax glabra Roxb, Anemone

chinensis Bunge, and Iris domestica (L.) Goldblatt and Mabb were found to

exhibit strong broad-spectrum antibacterial activity against the above

pathogens. A. pilosa Ledeb demonstrated antibacterial activity comparable to

erythromycin and ampicillin. The synergistic antibacterial activity of

combinations of these plant extracts and with antibiotics was also investigated

against the above pathogens. Only one combination of extracts of A. chinensis

Bunge and A. pilosa Ledeb indicated potential synergistic effects against both

C. jejuni and E. coli. Combinations of A. chinensis Bunge and S. glabra Roxb,

and A. pilosa Ledeb and S. glabra Roxb also indicated potential synergistic

effects against E. coli. A. pilosa Ledeb, A. chinensis Bunge and S. glabra Roxb

extracts each in combination with ampicillin and erythromycin indicated

additive effects against C. jejuni, L. monocytogenes, E. coli, and S. enteritidis.

In vitro assays were used to examine the bactericidal activity of A. pilosa

Ledeb, S. glabra Roxb, A. chinensis Bunge, and I. domestica (L.) Goldblatt

and Mabb over 24 hours in broth and in selective broth with chicken caecum

content. The findings showed A. pilosa Ledeb, A. chinensis Bunge, S. glabra

Roxb reduced caecum colonisation of E. coli, L. monocytogenes, and S.

enteritidis in vitro within 0.5 hours. The results indicated a significant reduction

of ≥99.9% of the above pathogens and rapid bactericidal activity comparable

to ampicillin (P <0.001). The final part of this study investigated the effect of

plant supplementation to broiler chicken diets on their performance, nutrient

digestibility, and gastrointestinal tract microbiota. The results of the poultry trial

highlighted the selective antibacterial activity of S. glabra Roxb, which

decreased pathogenic bacteria, E. coli and Campylobacter spp. on day 21

(P <0.05) and 35 (P <0.01) and increased lactic acid bacteria relative

abundance compared to the antibiotic group on day 14 (P <0.001) and 35 (P

<0.01). In addition, S. glabra Roxb, and A. chinensis Bunge significantly

increased weight gain compared to the negative control and the positive

control receiving the recommended nutrient specification diet (P <0.001) and

this was comparable to the positive amoxicillin control group. A. pilosa Ledeb,

A. chinensis Bunge, and S. glabra Roxb significantly decreased feed

conversion ratios compared to the negative control group and had comparable

feed conversion ratios to the positive amoxicillin control group (P <0.001).

The study contributes to addressing gaps in the research literature on the use

of plant extracts as potential alternatives to antibiotics in poultry farming. It

provides additional information on the antibacterial activity of many plant

extracts against pathogens commonly found in the poultry gastrointestinal tract

and new information about others including A. pilosa Ledeb, S. glabra Roxb,

A. chinensis Bunge, and I. domestica (L.) Goldblatt and Mabb. The results of

the poultry trial also indicate the potential of S. glabra Roxb and A. chinensis

Bunge in reducing pathogens, enhancing the bird’s beneficial microbiota, and

improving bird performance. Overall, the findings of this study highlight the

potential of using plant extracts as a supplement to poultry diets.

Declaration

This thesis has been completed by the author and has not been presented in

any previous application for a degree. I confirm that the work presented in this

thesis has been completed by myself. The use of all materials from other

sources has been noted by the appropriate use of references or

acknowledgements.

Rebekah Lana McMurray

01/03/2021

Publications

McMurray, R. L., Ball, M.E.E., Tunney, M.M., Corcionivoschi, N., and Situ, C.

(2020) ‘Antibacterial activity of four plant extracts extracted from traditional

Chinese medicinal plants against Listeria monocytogenes , Escherichia coli,

and Salmonella enterica subsp. enterica serovar Enteritidis’, Microorganisms,

8(962), pp. 1–12. doi: 10.3390/microorganisms8060962

Conferences

McMurray, R. L., Ball, M.E.E., Tunney, M.M., Corcionivoschi, N., Situ, C.,

Linton, M., Pinkerton, L., Kelly, C. and Balta, I. (2021) ‘The effect of Agrimonia

pilosa Ledeb, Anemone chinensis Bunge, and Smilax glabra Roxb on broiler

performance and gastrointestinal tract microbiota’, World Poultry Science

Association Annual Meeting (UK Branch).


(2020) ‘Effects of phytogenics on intestinal pathogenic bacteria and microbiota

of livestock non-ruminants’, Food Safety and Nutrition postgraduate

research symposium (QUB).


(2019) ‘In vitro antibacterial activity of botanicals against Campylobacter jejuni,

Listeria monocytogenes, Escherichia coli and Salmonella enterica’, World

Poultry Science Association Annual Meeting (UK Branch), 34.


(2019) ‘In vitro bactericidal activity of botanicals against Campylobacter jejuni,

Listeria monocytogenes, Escherichia coli and Salmonella enterica’,

Campylobacter, Helicobacter, and Related Organisms Conference.



of livestock non-ruminants’, Department of Agriculture, Environment, and

Rural Affairs symposium.



of livestock non-ruminants’, Food Safety and Nutrition postgraduate

research symposium (QUB).



of livestock non-ruminants’, Agri-Food and Biosciences Institute symposium.



of livestock non-ruminants’, Department of Agriculture, Environment, and

Rural Affairs symposium.

Awards

The World’s Poultry Science Association (2021) President’s Prize for the

presentation entitled ‘The effect of Agrimonia pilosa Ledeb, Anemone

chinensis Bunge, and Smilax glabra Roxb on broiler performance and

gastrointestinal tract microbiota’ at the annual meeting of the WPSA UK

branch.

Acknowledgements

I have a few people that I would like to acknowledge for their part in helping

me build my future and guiding me along the path to the completion of my PhD.

I would like to thank The Department of Agriculture, Environment and Rural

Affairs (DAERA) for funding my PhD studentship and accepting the proposal.

Many thanks to QUB, AFBI, and the City Hospital for providing me with clinical

isolates and reference strains for use in my PhD.

I would like to thank my supervisory team, Dr. Chen Situ, Dr. Elizabeth Ball,

and Professor Michael Tunney. Thank you for your guidance, feedback, and

for sharing your multiple perspectives and expertise to allow me to develop a

comprehensive research project.

Many thanks to Professor Nicolae Corcionivoschi for your continued guidance

and support throughout this project.

Thanks to all my peers and colleagues at AFBI, QUB, DAERA, and Devenish.

Thank you for your technical support, and expertise.

My profound gratitude goes to my family. Thanks Mum and Dad for your

unconditional support, continuous encouragement, and guidance. Many

thanks to my brother, I really appreciated having someone to go through the

same academic journey with, thanks for the relaxing videogames, and the

motivation to meet deadlines! Thanks to my Auntie Lorna and Andy, Uncle

Stephen and Heather, and Uncle Mickey and Corinne, and Rachel. Thank you

for always showing interest, reading my publications, and your unfaltering

support. Many thanks to my partner David for your support, patience, for

always believing in me, and most importantly being my rock.

Lastly, thanks to my friends for their humour, and motivation throughout my

PhD.

Dedication

This thesis is dedicated to my granny Elizabeth (Lily) McMurray. You always

believed in me.

List of Abbreviations

AAC N-acetyltransferases

ADI Acceptable daily intake

AFBI Agri-Food and Biosciences Institute

AGP Antibiotic growth promoter

AME Aminoglycoside-modifying enzyme

AMR Antimicrobial resistance

ANOVA Analysis of variance

ANT O-adenyltransferases

APH O-phosphotransferases

ATCC American Type Culture Collection

CDC Centres for Disease Prevention and Control

CDDEP Centre for Disease Dynamics, Economics & Policy

CFU Colony forming unit

DDD Defined daily doses

DDGS Distillers' dried grain with solubles

DM Dry matter

DNA Deoxyribonucleic acid

EC European Commission

ECDC European Centres for Disease Control and Prevention

EFSA European Food Safety Authority

EO Essential oils

ERM Erythromycin ribosomal methylation

EU European Union

EUCAST European Committee on Antimicrobial Susceptibility Testing

FAOUN Food and Agricultural Organisation of the United Nations

FCR Feed conversion ratio

FDA Food and Drug Administration

FIC Fractional inhibitory concentration

FICI Fractional inhibitory concentration index

GE Gross energy

GI Gastrointestinal

HGT Horizontal gene transfer

HM Her Majesty

HPLC High performance liquid chromatography

ICGA The Interagency Coordination Group on Antimicrobial

Resistance

ISO International Organisation for Standardisation

LAB Lactic acid bacteria

LMC Litter moisture content

MBC Minimum bactericidal concentration

MCCDA Modified charcoal-cefoperazone-deoxycholate agar

MDR Multi drug resistant

MHA Mueller Hinton agar

MHB Mueller Hinton broth

MH-F Mueller Hinton fastidious

MIC Minimum inhibitory concentration

MGE Mobile genetic elements

MRD Maximum recovery diluent

MRL Maximum residue limits

MRS De Man, Rogosa and Sharpe

MRSA Methicillin-resistant Staphylococcus aureus

NARMS National Antimicrobial Resistance Monitoring System

NCTC National Collection of Type Cultures

NI Northern Ireland

OECD Organisation for Economic Co-operation and Development

OIE World Organisation for Animal Health

PALCAM Polymyxin acriflavine lithium chloride ceftazidime aesculin

mannitol

PBP Penicillin binding protein

PCR Polymerase chain reaction

PHE Public Health England

QUB Queen’s University Belfast

RNA Ribonucleic acid

SD Standard deviation

SEM Standard error of the mean

TBX Tryptone bile X-glucuronide

TCM traditional Chinese medicine

USA United States of America

WHO World Health Organisation

Table of Contents

Declaration 5

Publications 6

Acknowledgements 8

Dedication 9

1. Chapter 1 1

Effects of phytogenics on intestinal pathogenic bacteria and microbiota of non-ruminants 1

1.1. Literature review .......................................................................... 1

1.1.1. The antibiotic resistance crisis .................................................... 1

1.1.2. The use of antibiotics in animal farming .................................... 18

1.1.3. Antibiotic resistance and threats to public health ...................... 20

1.1.4. Alternatives to using antibiotics in poultry feed .......................... 26

1.1.5. Alternative poultry feed supplements: plant extracts and phytochemicals.......................................................................... 38

1.1.6. Effects of plant extracts and phytochemicals on intestinal bacterial pathogens and microbiota in poultry ........................... 44

1.2. Aims and objectives ................................................................... 50

2. Chapter 2 51

Evaluation of antibacterial activity of medical plants against Campylobacter jejuni, Listeria monocytogenes, Escherichia coli and Salmonella enterica subsp, enterica serovar Enteritidis ........................... 51

2.1. Introduction ......................................................................................... 51

2.2. Materials and methods ............................................................... 52

2.2.1. Selection of plant extracts ......................................................... 52

2.2.2. Bacterial isolates and reference strains used for screening ...... 57

2.2.3. Preparation of plant extracts: Ultrasound assisted extraction .... 58

2.2.4. Antibacterial testing: Broth microdilution method ...................... 60

2.2.5. Statistical analysis....................................................................... 62

2.3. Results ....................................................................................... 63

2.3.1. Identification of bacterial isolates and reference strains used for screening ................................................................................... 63

2.3.2. Antibacterial efficacy of plant extracts against pathogens ......... 77

2.4. Discussion ................................................................................. 89

2.4.1. Purpose of study and methods .................................................. 89

2.4.2. Plants that possess strong antibacterial properties ................... 90

2.4.3. Plants that possess weak to moderate antibacterial properties . 93

2.5. Conclusion ................................................................................. 95

3. Chapter 3 96

In vitro synergistic antibacterial activity of plant extracts and antibiotics against Campylobacter jejuni, Listeria monocytogenes, Escherichia coli and Salmonella enterica subsp. enterica serovar Enteritidis. .................. 96

3.1. Introduction ......................................................................................... 96

3.1.1. Aim and objectives ....................................................................... 99

3.2. Materials and methods ............................................................... 99

3.2.1. Bacterial isolates used for screening ......................................... 99

3.2.2. Preparation of plant extracts ..................................................... 99

3.2.3. Broth microdilution of individual and combined plant extracts . 100

3.2.4. Statistical analysis .................................................................... 102

3.3. Results ..................................................................................... 103

3.3.1. Combined antibacterial activity of plant extracts ........................ 103

3.3.2. Combined antibacterial activity of plant extracts with antibiotics 108

3.4. Discussion ............................................................................... 113

3.4.2. Indifferent activity of combinations of plant extracts ................... 114

3.4.3. Additive activity of plant extracts and antibiotics ........................ 116

3.5. Conclusion ............................................................................... 118

4. Chapter 4 119

Antibacterial activity of extracts from A. pilosa Ledeb, S. glabra Roxb, A. chinensis Bunge, and I. domestica (L.) Goldblatt and Mabb against Campylobacter jejuni, Listeria monocytogenes, Escherichia coli and Salmonella enterica subsp. enterica serovar Enteritidis using a poultry caecum model 119

4.1. Introduction ....................................................................................... 119

4.1.2. Aims and objectives ................................................................... 120

4.2. Materials and methods ............................................................. 121

4.2.1. Bacterial isolates used for screening ....................................... 121

4.2.2. Preparation of plant extracts ................................................... 121

4.2.3. Time kill assay in broth ............................................................ 121

4.2.4. Time kill in an in vitro caecum model ....................................... 124

4.2.5. Statistical analysis ................................................................... 127

4.3. Results ..................................................................................... 127

4.3.1. Time kill ................................................................................... 127

4.3.2. Bactericidal activity in an in vitro cecum model against L. monocytogenes ....................................................................... 134

4.3.3. Bactericidal activity in an in vitro cecum model against E. coli 138

4.3.4. Bactericidal activity in an in vitro cecum model against S. enteritidis ................................................................................. 141

4.4. Discussion ............................................................................... 144

4.4.1. Purpose of study and methods ................................................ 144

4.4.2. Time kill in broth ...................................................................... 145

4.4.3. In vitro caecum content model ................................................ 146

4.5. Conclusion ............................................................................... 148

5. Chapter 5 149

The effect of A. pilosa Ledeb, A. chinensis Bunge, and S. glabra Roxb on broiler performance, nutrient digestibility, and gastrointestinal tract microbiota 149

5.1. Introduction ....................................................................................... 149

5.2. Methods ............................................................................................ 151

5.2.1. Experimental diets ..................................................................... 151

5.2.2. Experimental design, birds, and management ........................... 155

5.2.3. Laboratory analysis .................................................................... 157

5.2.4. Lactic acid bacteria, Campylobacter spp., and E. coli ................ 157

5.2.5. Statistical analysis and calculations ........................................... 159

5.3. Results ............................................................................................. 160

5.3.1. Nutritional composition of plants ................................................ 160

5.3.2. Performance, and nutrient digestibility ....................................... 161

5.4. Discussion ........................................................................................ 173

5.4.1. Purpose of study and methods .................................................. 173

5.4.2. A. chinensis Bunge, S. glabra Roxb and amoxicillin improved performance ............................................................................ 175

5.4.3. Lactic acid bacteria, Campylobacter spp., and E. coli ................ 176

5.4.4. Morbidity and mortality of birds .................................................. 178

5.4.5. Nutrient digestibility .................................................................... 179

5.4.6. Litter quality and ammonia output .............................................. 180

5.5. Conclusion ........................................................................................ 182

6. General Discussion 183

7. References 190

Literature Review

1

1. Chapter 1

Effects of phytogenics on intestinal pathogenic bacteria and microbiota

of non-ruminants

1.1. Literature review

1.1.1. The antibiotic resistance crisis

Antibiotic resistance is a worldwide crisis that threatens animal and human

health (IAGC, 2019). This is reflected throughout the research literature and

in government and international reports (WHO, 2015; O’Neill, 2016; CDC,

2019; HM Government, 2019). In 2014 the WHO warned of a post-antibiotic

era in the 21st century in which frequent and minor infections would be life

threatening because pathogens are becoming increasingly resistant to

antibiotics. This reduces options for treating infectious diseases and reduces

their therapeutic effectiveness against bacterial pathogens (WHO, 2014).

Currently, high and concerning levels of antibiotic resistance and multidrug

resistance in several bacterial pathogens have been reported in all countries.

It was estimated that antibiotic resistance results in 33, 000 deaths yearly in

the EU (ECDC, 2019) and 35, 000 deaths yearly in the USA (CDC, 2020).

Twenty-two foodborne bacterial pathogens were estimated to have caused

around 350, 000 deaths in 2010. Campylobacteriosis, Salmonellosis,

Escherichia coli, and Listeria infections account for around 25% of total

deaths due to foodborne pathogens (Kirk et al., 2015).

Literature Review

2

Increased exposure to antibiotics is a primary contributary factor to the

emergence and spread of acquired antibiotic resistance in bacterial

pathogens. This is because bacteria can acquire and transfer resistance

through genetic mutations and HGT when exposed to bacteria resistant to an

antibiotic (Bennett, 2008). Two of the main causes of the antibiotic crisis is

the overuse and misuse of antibiotics in agriculture and medicine (Čižman,

2003; Morgan et al., 2011).

Since the widespread use of antibiotics in the 1930s the global consumption

of antibiotics has continued to increase annually. It was estimated that global

human consumption of antibiotics rose by 65% within 15 years from 2000 to

2015 from 21.1 billion to 34.8 billion Defined Daily Doses (DDD) (Klein et al.,

2018). Over 70% of the antibiotics classified as important to humans are used

for livestock in the USA and over 50% are used for livestock in most other

countries (O’Neill, 2016; WHO, 2019). Many of the critical and essential

antibiotics such as colistin and vancomycin are also used in some countries

in animal farming. The widespread use of critically essential antibiotics in

animal farming increases the risk of bacteria acquiring resistance, and cross-

resistance from animals to humans, and is associated with the emergence of

new types of multi-resistant foodborne bacteria such as Salmonella,

Campylobacter, E. coli, and L. monocytogenes (O’Neill, 2016; Olaimat et al.,

2018).

There is worldwide consensus amongst scientific, veterinary, and medical

experts that using critically important related antibiotics for human health for

non-essential use in agriculture is a dangerous threat to public health and has

https://www.independent.co.uk/topic/colistin

Literature Review

3

led the WHO to recommend the reduction of medically important antibiotics

in animal farming and regulation and restriction on the use of these antibiotics

for the promotion of growth (WHO, 2017).

1.1.1.1. Origins and development of antibiotic resistance

Antibiotic resistant genes existed in nature a long time before the antibiotic

era and their use in clinical settings. Antibiotic resistant genes result from

complex interactions of organisms with their environment (Petrova et al, 2009;

Munita and Arias, 2016; Santiago-Rodriguez et al., 2017). However, the

discovery and widespread use of antibiotics such as the sulphonamides in the

late 1930s and the β-lactams beginning with penicillin in the 1940s has

created an antibiotic resistance crisis (Podolsky, 2018). While many

pathogenic bacteria have a natural or intrinsic resistance to antibiotics they

can also acquire resistance through the acquistion of new genes or genetic

mutations (Peterson and Kaur, 2018). This is illustrated in the shared timeline

of antibiotic discovery and resistance (Figure 1.1.) The timeline shows that

the discovery and widespread use of new antibiotics is followed some time

later by the development of pathogenic bacterial strains which have acquired

resistance to the antibiotic (Lowy, 2003; Ventola, 2015).

Literature Review

4

Figure 1.1. Timeline, development of antibiotics and antibiotic resistance

Figure 1.1. is based on a previous diagram (Taneja et al., 2019) with the new addition of

Teixobactin (Piddock, 2015). This illustration outlines the dates of the discovery of new

classes of antibiotics and the concomitant discovery of resistance to these antibiotics.

1.1.1.2. Intrinsic resistance

The majority of antibiotics are natural compounds. They are produced by

bacteria which co-exist with other bacterial species which have genes that

encode intrinsic resistance to the compounds they produce. This is

independent of exposure to antibiotics and unrelated to HGT (Davies and

Davies, 2010). This is part of a natural selection process enabling bacteria to

select for self-defence mechanisms to overcome the antibiotic effect of their

neighbours in order to survive. The natural environment acts as a reservoir

and provides pathways for antibiotic resistance genes produced by diverse

pathogenic bacteria populations, to acquire and transfer resistance to new

hosts (Allen and Stanton, 2014).

Literature Review

5

1.1.1.3. Acquired antibiotic resistance: Genetic mutations and horizontal

gene transfer (HGT)

Acquired resistance occurs when bacteria develop the ability to resist and

block the activity of an antibiotic that they were once susceptible to. Bacteria

can acquire resistance when exposed to even low levels of antibiotics (Cantón

and Morosini, 2011). Bacterial pathogens have both intrinsic and acquired

resistance. The two ways in which bacteria acquire resistance to antibiotics is

through genetic mutations and HGT. Genetic mutations and HGT enable the

transfer of intrinsic antibiotic resistance elements to bacterial pathogens.

These mechanisms are associated with higher levels of antibiotic resistance

in the new host and to the rapid spread and transfer of antibiotic resistance

genes in clinically relevant strains of bacterial pathogens (Munita and Arias,

2016). Bacteria can acquire resistance through spontaneous genetic

mutations. Some bacterial cells from the susceptible population develop gene

mutations which allow the cells to survive in the presence of the antibiotics.

The antibiotic destroys susceptible bacteria, leaving resistant bacteria to

thrive. Mutations typically have short generation times which means bacteria

can rapidly acquire resistance to an antibiotic (Woodford and Ellington, 2007).

Once bacteria have acquired resistance, they can transfer resistance by

exchanging genetic material with other bacteria (Figure 1.2.). This can lead to

multi-drug resistance (MDR) when bacteria acquire resistance to one

antibiotic in three or more drug classes (CDC, 2019). Acquired resistance

occurs in one of three ways:

Literature Review

6

1. Transformation - uptake of naked dead or degraded DNA from the

environment

2. Transduction - transfer of DNA from a donor bacterium to the recipient via

bacteriophages

3. Conjugation - requires contact from cell to cell and involves DNA

transfer via sexual pilus from donor to recipient

Figure 1.2. Bacteria: HGT mechanisms

1.

2.

3.

(Vernikos et al., 2014). Figure 1.2. the mechanisms of HGT in bacteria as described above

including: 1. Transformation; 2. Transduction; and 3. Conjugation.

1.1.1.4. Antibiotic resistance mechanisms

Bacteria which produce antibiotics are encoded with antibiotic resistance

genes which protect them against the biologically active molecules which they

have synthesised (Mak et al., 2014). The genetic determinants encoding

resistance are nearly always clustered alongside biosynthesis genes.

Literature Review

7

Generally, each cluster of genes encodes for one or more resistant genes

which corresponds to its antibiotic counterpart and are co-regulated with the

expression of resistance occurring before or alongside biosynthesis.

Antibiotics or biochemical intermediates from biosynthetic pathways can also

induce cluster-encoded resistance genes in response to increasing antibiotic

concentrations resulting in upregulating its resistance. Another possibility is

that resistance genes which are not producing the antibiotic can be activated

by cell to cell signally mechanisms this ensures their survival by protecting

them from their antibiotic producing neighbours (Mak et al., 2014). Genetic

encoding biosynthesis and resistance helps to explain the timeline of the

development of different antibiotics and the inevitably and corresponding

emergence of antibiotic resistance (Figure 1.1).

Bacteria which produce antibiotics have developed a variety of complex self-

defence mechanisms against their own antibiotics and these can be acquired

by other bacteria through genetic mutations and HGT. Antibiotic resistance

mechanisms inlcude, inactivation of antibiotic via modification or degradation,

alteration of the antibiotic target, altering the cell wall and efflux pumps (Figure

1.3). Commonly, bacteria such as those in this study, C. jejuni (Elhadidy et

al., 2018), L. monocytogenes (Baquero et al., 2020), E. coli (Yasir et al.,

2020), and S. enteritidis (Higgins et al., 2020) have genetically encoded

resistance genes and may deploy several of these mechanisms to provide

protection against a range of antibiotics (Peterson and Kaur, 2018). The

biosynthetic origins of antibiotics provide insight into the development of the

resistance mechanisms that currently exist or emerge in the future.

Literature Review

8

Figure 1.3. Mechanisms of Antibiotic Resistance

Figure 1.3. (Gullberg, 2014) different mechanisms of antibiotic resistance including: A

(inactivation of antibiotic via modification or degradation due to enzymatic inactivation); B

(alteration of the antibiotic target); C (altering the cell wall leading to decreased

permeability); and D (efflux pumps reducing antibiotic concentration in the cell).

A. Inactivation of Antibiotic via Modification or Degradation

Modification of the Antibiotic Molecule

Modification or destruction of the antibiotic structure is a common resistance

mechanism involving enzymes. This resistance mechanism is commonly

deployed by bacteria against bactericidal aminoglycoside antibiotics such as,

kanamycin, gentamycin, and streptomycin by aminoglycoside-modifying

enzymes (AMEs). Aminoglycosides are broad spectrum antibiotics these

include gentamicin, kanamycin, neomycin, and streptomycin. These typically

work by binding to the A-site on the 16S ribosomal RNA of the 30S ribosome

which inhibits protein synthesis (Munita and Arias, 2016). In response bacteria

can produce a range of AMEs confered by diversity of AME genes, which are

usually found on plasmids and transpons. AMEs work by covalently modifying

the hydroxyl or amino groups of the aminoglycoside molecule (Figure 1.4).

B

C D

A

Literature Review

9

AMEs are typically classified according to their biochemical reactions they

catalyse:

(i) N-acetyltransferases (AACs)

(ii) O-adenyltransferases (ANTs)

(iii) O-phosphotransferases (APHs)

The binding affinity of the drug for its target is decreased by these

modifications and leads to a reduced antibacterial potency (Krause et al.,

2016).

Literature Review

10

Figure 1.4. adenylation of streptidine moiety in streptomycin

Figure 1.4. adenylation catalysed by ANT in streptomycin. In this case, ANT(6) catalyses the

adenylation of streptidine moiety (red) in streptomycin (1) antibiotic, which results in an

inactive AMP-streptomycin (Latorre et al., 2016).

Degradation of the Antibiotic Molecule

One of the most common examples of enzymatic destruction of antibiotic

molecules by bacteria is the action of β-lactamase enzymes against broad

spectrum, bactericidal β-lactam antibiotics, which include penicillins,

monobactams, cephalosporins, and carbapenems (Munita and Arias, 2016).

The genes encoding for β-lactamases such as, blaxy have been found in the

Literature Review

11

chromosome or localized in mobile genetic elements (MGEs) and in

integrons. β-lactams are characterised by a β-lactam ring at the core of their

structure (Figure 1.5.) and the key to their mechanisms of action.

Figure 1.5. Chemical structures of β-lactam antibiotics

Figure 1.5. chemical structures of β-lactam antibiotics including: (a) penicillins; (b) ampicillin;

(c) cephalosporins; (d) carbapenems; and (e) monobactams. The β-lactam ring is highlighted

in red in all of the antibiotics (Lee et al., 2016).

β-lactam antibiotics target penicillin-binding proteins (PBPs). They typically

work by inhibiting the bacterial cell wall peptidoglycan layer by binding to

different PBPs and irreversibly acetylating the active site serine of the

transpeptidases. This prevents them the PBPs performing their role in cell

Literature Review

12

wall synthesis leading to the death of the bacterial cell due to osmotic

instability or autolysis (Konaklieva, 2014). However, bacteria have developed

antibiotic resistance to a wide range of β-lactam antibiotics. The main

resistance mechanism is based on producing β-lactamases which destroy the

amid bond of the β-lactam ring, through hydrolysis thereby, inactivating the

antibiotic (Figure 1.6.) (Rice, 2012; Harris, 2015; Eiamphungporn et al., 2018).

Figure 1.6. β-lactamase action on penicillin

Figure 1.6. hydrolysis reaction catalysed by the enzyme β-lactamase resulting in an altered

structure of penicillin rendering it inactive (Harris, 2015).

There are over 1,000 different β-lactamases with a wide range of biochemical

activities. Many enzymes are capable of destroying almost all available β-

lactams, including carbapenems and confer multi-resistance to these

antibiotics (Munita and Arias, 2016). Mutations can also contribute to the

evolution of these into novel enzymes. An example of this is the extended

spectrum β-lactamases (ESBLs). The ESBL TEM-3 enzyme evolved from the

original TEM-1 penicillinase. Its function was altered as a result of developing

two amino acid substitutions and acquiring the ability to hydrolyse third

generation cephalosporins, and aztreonam (Paterson and Bonomo, 2005).

The emergence and subsequent increase in bacteria which produce ESBLs

Literature Review

13

since the mid 1980s represent a growing threat to human health and the

effecive treatment of a range of diseases (Tamma and Mathers, 2021).

B. Alteration of Antibiotic Target Sites

Examples of antibiotics which work by altering bacterial target sites include

tetracycline and fluoroquinolone. Tetracycline works by blocking aminoacyl-

tRNA by binding to the 30S and 50S subunit of microbial ribosomes and

preventing new amino acids being added to the peptide chain.

Fluoroquinolones interfere with DNA replication by inhibiting the ligase activity

of type II topoisomerases and producing DNA with single- and double-strand

breaks which causes cell death (Iovine, 2013).

Protecting the Target

Bacteria can alter the antibiotic target site to avoid the action of antibiotics.

Mechanisms include preventing the antibiotic reaching its binding site by

protecting the target. Tetracycline, and Fluoroquinolones are affected by this

mechanism. Widely distributed amongst bacterial species are Tet(M) and

Tet(O) encoded Tetracycline resistance genes. These are located in plasmids

and conjugative transposons (Connell et al., 2003). Tet(M) and Tet(O) interact

with the ribosome to dislodge tetracycline from its binding site and prevent it

from rebinding. Tet(O) also supports the continuation of protein synthesis by

the displacing antibiotic molecule from the ribosome (Li et al., 2013). The

quinolone plasmid-mediated resistance protein Qnr also works by competing

for the DNA binding site of the DNA gyrase and topoisomerase IV. This

compromises the ability of quinolone molecule to produce and stabilize the

Literature Review

14

gyrase-cleaved DNA-quinolone complex which causes cell death (Rodríguez-

Martínez et al., 2011). Qnr-encoding genes also promote the selection of

highly resistant isolates through point mutations in genes encoding the DNA

gyrase and/or topoisomerase IV (Aldred et al., 2014).

Modification of Target

Point Mutations

Point mutations can occur in genes protecting the target for example,

rifamycin blocks bacterial transcription by inhibiting the DNA-dependent RNA

polymerase. This is a complex enzyme with a α2ββ’σ subunit structure. The

rifamycin binding pocket is in the β subunit of the RNA polymerase and

encoded by rpoB. The antibiotic molecule, after binding, then blocks the path

of the nascent RNA, interrupting transcription. High level rifampicin resistance

occurs through single step point mutations which result in amino acid

substitutions in the rpoB gene. This decreases affinity for the antibiotic while

catalysing polymerase activity and enabling transcription to continue (Munita

and Arias, 2016).

Another example is resistance to fluoroquinolones which work by inhibiting

DNA gyrase and topoisomerase IV, thereby altering DNA replication, and

causing cell death. Resistance to fluoroquinolones occurs through

chromosomal mutations in the genes gyrA-gyrB and parC-parE encoding

subunits for DNA gyrase and topoisomerase IV which resulting in alteration

of the target proteins. Higher levels of resistance fluoroquinolones is achieved

through additional mutations in the gyrase or topoisomerase IV genes (Munita

and Arias, 2016).

Literature Review

15

Enzymatic Alteration of the Target Site

Bacteria can also produce enzymes which modify the target site of antibiotics.

For example, macrolides are broad spectrum antibiotics which work by

binding to the bacterial 50S ribosomal unit preventing protein synthesis. After

binding, the antibiotic prevents mRNA translation and the growth of the

peptide chain by stopping the addition of tRNA amino acids to the chain

(Vázquez-laslop and Mankin, 2019). In the case of macrolide resistance the

target site is modified by methylation of the ribosome catalyzed by an enzyme

encoded by the erythromycin ribosomal methylation (erm) genes. This impairs

the ability of the antimicrobal molecule to bind to its target. In the 23S rRNA

expression of erm genes also confers cross-resistance to lincosamides, and

streptogramin B due to their due to their overlapping binding sites (Munita and

Arias, 2016). Over 30 erm genes have been discovered mainly in mobile

genetic elements and widely distributed in at least 30 bacterial genera

(Leclercq, 2002).

Replacement or Bypass of the Target Site

Target bypass involves the bacteria overwhelming the antibiotic by increasing

the production of the antibiotic target. An example of this mechanism is

trimethoprim-sulfamethoxazole resistance. The mechanism of action of

trimethoprim-sulfamethoxazole is to alter the ability of the bacteria to produce

folate thereby impairing purine and important amino acids synthesis

(Huovinen, 2001) Folate synthesis involves two main enzymes. These are

dihydropteroic acid synthase, and dihydrofolate reductase. Dihydropteroic

Literature Review

16

acid synthase forms dihydrofolate from para-aminobenzoic acid which is

inhibited by trimethoprim-sulfamethoxazole while dihydrofolate reductase

catalyses the formation of tetrahydrofolate from dihydrofolate which is

inhibited by trimethoprim. This resistance mechanism is associated with

mutations in the promoter region of DNA which encodes these enzymes

(Huovinen, 2001).

Bacteria can also evolve new targets with similar biochemical functions to the

real target but will not be inhibited by the antibiotic molecule. One example of

this is Staphylococcus aureus resistance to methicillin (MRSA). β-lactams

work by disrupting cell wall synthesis by inhibiting penicillin binding proteins

(PBPs). These are enzymes responsible for the transpeptidation and

transglycosylation of peptidoglycan from the cytoplasm. Staphylococcus

aureus develops resistance to methicillin by acquiring a foreign gene, mecA,

possibly from Staphylococcus sciuri. MecA encodes for a new PBP, PBP2a

which has a low affinity for β-lactams, and carbapenems rendering them

ineffective (Lowy, 2003; Krawczyk-Balska and Markiewicz, 2016; Munita and

Arias, 2016).

Another example is bacterial resistance to vancomycin. Vancomycin also kills

bacteria by inhibiting cell wall synthesis but in a different way to β-lactams.

The antibiotic molecule binds to the terminal D-alanine of the pentapeptide

moiety of nascent peptidoglycan precursors (lipid II). This prevents PBP-

mediated cross-linking inhibiting cell wall synthesis, leading to bacterial death.

In this case, resistance occurs by acquiring a van gene cluster which encode

Literature Review

17

for biochemical remodelling peptidoglycan synthesis. This changes the last

D-alanine ending precursors for either D-serine (low-level resistance) or D-

lactate (high-level resistance) and prevents vancomycin binding to the cell

wall precursors (Munita and Arias, 2016).

C. Altering the Cell Wall: Decreased Permeability

Many antibiotics have intracellular bacterial targets. In gram-negative bacteria

the antibiotic has to penetrate the outer and/or cytoplasmic membrane in

order to reach its target. Gram-negative bacteria have an outer membrane

which provides a permeablity barrier which offers intrinsic resistance aginst

hydrophilic antibiotics such as β-lactams and vancomycin. Bacteria can alter

the cell wall to prevent the antimicrobial from reaching its target by reducing

its ability to penetrate the cell. Many antibiotics used have intracellular or

periplasmic bacterial targets. One mechanism which bacteria use to prevent

the antibiotic reaching its target is by limiting the influx of the antimicrobial. β-

lactams, tetracyclines, and some fluoroquinolones which use porin channels

to cross the barrier are particularly susceptible to outer membrane

permeability changes. Vancomycin for example, is ineffective against Gram-

negative bacteria because it cannot penetrate the outer cell membrane.

Resistance is mediated by reducing the level of porin expression or disrupting

their function. This resistance mechanism usually results in low levels of

resistance and commonly occurs alongside other mechanisms such as efflux

pumps (Munita and Arias, 2016; Reygaert, 2018).

Literature Review

18

D. Efflux Pumps

Gram-positive and Gram-negative bacteria use a range of efflux pumps which

enable bacteria to regulate their environment and to flush out antibiotics.

(Piddock, 2006.). Thereby, reducing the antibiotic concentration in the cell and

increasing resistance. Antibiotic resistance due to efflux pumps occurs in two

ways. One, the expression of the efflux pump protein is increased or two,

amino acid substitutions in the protein increases efflux efficiency. Efflux pump

systems can be substrate specific for a particular antibiotic such as Tet

determinates for tetracycline and Mef genes for macrolides or have broad

substrate specificity which is common in MDR bacteria (Poole, 2005)(Poole,

2005). Efflux pumps are a common mechanism in MDR bacteria and effect a

range of classes of antibiotics including, carbapenems, aminoglycosides, β-

lactams, fluoroquinolones, and polymyxins (Muller et al., 2011).

1.1.2. The use of antibiotics in animal farming

Antibiotics are conventionally classified according to their use into three

categories, therapeutic, prophylactic and growth promotion. Antibiotics are

used therapeutically in agriculture to treat sick animals. One of the most

controversial uses of antibiotics is for growth promotion because the antibiotics

are added at sub-therapeutic levels to healthy animals’ water and feed

continuously over their life span to promote animal growth and increase

productivity (Collignon and McEwen, 2019). Additionally, antibiotics classified

as prophylactics have also been used for growth promotion with prophylactic

use having different interpretations. Therefore, the distinction between the use

of antibiotics for the promotion of growth and disease prevention is not always

Literature Review

19

clear (Kirchhelle, 2018). Prophylactic use can mean the routine and

unnecessary use of antibiotics as prophylactics without veterinary oversight or

targeted use when prescribed by a veterinarian at therapeutic levels for

specific situations and limited time (OECD, 2019).

Although scientists and pharmaceutical companies were aware of the

problem of antibiotic resistance since the 1930s, it was not until the early

1960s that concerns began to be raised. In part, prompted by research which

showed the use of antibiotics in animal farming led to resistance selection and

transfer across species on British farms, when MDR S. typhimurium caused

outbreaks of severe food poisoning (Podolsky, 2018). In 1986 Sweden was

the first country to ban all antibiotic growth promoters (AGPs), followed by

Denmark (2000) and EU countries (2006). Other countries which have

banned AGPs include, Mexico (2007, partial ban), South Korea (2011) and

Thailand (2012). New Zealand prohibit the use of critically important ones,

Australia has a partial ban, and in 2017 the USA introduced new measures

which mean AGPs can only be attained by prescription from a veterinarian

(Maron et al., 2013).

Recently there has been a rise in support for reducing the global use of AGPs

in animal farming by imposing greater restrictions and phasing out their use

(WHO, 2015; O’Neill, 2016). Although some countries have imposed tighter

restrictions or banned the use of AGPs in animal farming the World

Organisation for Animal Health report found 45 out of 150 countries continue

to use AGPs (OIE, 2018).

Literature Review

20

1.1.3. Antibiotic resistance and threats to public health

1.1.3.1. The use of antibiotics in animal farming and antibiotic resistance

All humans and animals carry normal or commensal gut flora. The widespread

use of antibiotics in animal farming is associated with some bacteria acquiring

antibiotic resistance and colonising the GI tract of the host (Marshall and Levy,

2011). The extended use of antibiotics at sub-therapeutic levels in animal

farming selects for increasing resistance to the antibiotic (Koch et al., 2017).

Cross-resistance occurs when antibiotics used to treat humans are also

administered to animals. This contributes to bacteria acquiring MDR and

resistance to other antibiotics which have not previously been used on the farm

(Singer, 2003) through genetic mutations and HGT. The majority of antibiotics

used in animal farming are similar to those used in human medicine and

bacteria can develop cross-resistance to these medically important antibiotics.

Surveillance and monitoring reports from many countries (CDDEP, 2015;

ECDC, 2019; NARMS, 2018) on the use of antibiotics in animal farming and

antibiotic resistance show a trend of increasing levels of resistance in strains

of C. jejuni, E. coli, L. monocytogenes, and Salmonella in humans, pigs,

calves, and poultry to a range of antibiotics and MDR. This includes resistance

to ampicillin, aminoglycosides, sulphonamides, tetracyclines, macrolides, and

quinolones (Buckland et al., 2016; Yang et al., 2019).

Resistant strains of C. jejuni, E. coli, L. monocytogenes, and Salmonella can

then be transmitted to humans through the environment, or by direct contact

with infected animals or by ingesting contaminated animal foodstuffs (Figure

1.7., A, B, and F). These pathogens cause infections which are more difficult

Literature Review

21

to treat and may become life threatening because they have acquired

increased resistance to a range of antibiotics. Consequently, leading to

increased failure of treatment, increased frequency and severity of infections

and increased morbidity (Scallan et al., 2011; WHO et al., 2018; EFSA, 2019).

Figure 1.7. Zoonoses and transmission of antibiotic resistance to humans

Figure 1.7. different transmission routes of antibiotic resistant bacteria from animals to humans

and across the environment. These include: A (consumption of contaminated meat or animal

products); B (transfer of resistant bacteria from slaughter worker to the meat or animal

products); C (transfer of resistant bacteria from excreta of wildlife, farm animals, and domestic

animals to the surrounding environment); D (transfer of resistant bacteria from infected

persons to the hospital and within the hospital to patients); E (transfer of resistant bacteria

between wastewater treatment facilities and the environment); F (transfer of resistant bacteria

from farm animals to agricultural workers); G (transfer of resistant bacteria between farm

animals to wildlife); and H (transfer of resistant bacteria between farm animals to companion

animals).

Literature Review

22

1.1.3.2. Transmission of antibiotic resistance: Direct contact with

infected animals

Studies have shown that farmworkers and farm animals on farms using

antibiotics at subtherapeutic levels as a feed additive have more resistant

bacteria in their intestinal flora compared to people and animals that do not

use antibiotics on farms (Marshall and Levy, 2011). Other studies indicate

those most at risk include farmworkers, their families, veterinarians, and

slaughter house workers (Smith and Crabb, 1957). Further studies have

confirmed the transmission of antibiotic resistant bacteria from animals to

farmers and others in direct contact with animals occurs shortly after the

introduction of antibiotics in animal feed (Levy et al., 1976; Bogaard et al.,

2002; Padungtod et al., 2006).

1.1.3.3. Transmission of antibiotic resistance: Consumption of food

contaminated with bacteria

The main transmisson route of resistant bacteria such as C. jejuni, E. coli, L.

monocytogenes, and S. enteritidis from animals to humans is through

consuming contaminated food in the food chain (Lazarus et al., 2015). This

presents a significant threat to food safety and public health. Contamination of

animal foodstuffs can occur during slaughter, processing, preparation or

transport. Non-animal products such as fruit and vegetables can be cross

contaminated through contact with infected animal products or through other

environmental sources (EFSA, 2010; CDC, 2013; CDC, 2019). Numerous

worldwide studies have reported resistant strains of C. jejuni, E. coli, L.

monocytogenes, and Salmonella in a variety of animal and non-animal

Literature Review

23

foodstuffs (Singh et al., 2007; Alexander et al., 2008; Alonso-Hernando et al.,

2012; Wasiński et al., 2013; Kaakoush et al., 2015; CDDEP, 2015; Ye et al.,

2018; Noll et al., 2018; Rahman et al., 2018; Wieczorek et al., 2018; Yang et

al., 2019).

1.1.3.4. Transmission of antibiotic resistance: The environment

Around 75% to 90% of antibiotics are not metabolised and are excreted in

animal waste along with resistant bacteria which contaminates soil, ground

water and streams (Figure 1.7., C and E) (Kim et al., 2011). This exposes more

species of susceptible bacteria to antibiotics and antibiotic resistance genes.

This exerts a selective pressure and the spread of resistance though genetic

mutations and HGT, enabling pathogens to become multidrug resistant

(Munita and Arias, 2016; Manyi-Loh et al., 2018; Kraemer, et al., 2019).

1.1.3.5. Food safety: Antibiotic residues

The addition of antibiotics at sub-therapeutic levels to farm animals over a long

period of time can accumulate in different concentrations in parts of the animal

that will be eaten as food, eggs, and milk (Mund et al., 2017). Antibiotic

residues in food pose another serious threat to public health (EC, 2013). To

ensure food safety and protect consumer health the joint Food and Agriculture

Organisation of the United Nations and the World Health Organisation

(FAOUN/WHO) established international standards in the Codex Alimentarius

(WHO and FAOUN, 2018). This consists of guidelines, standards, and codes

of practices. The Codex sets Maximum Residue Limits (MRLs) and Acceptable

Literature Review

24

Daily Intake (ADI) levels. Various studies mainly from lower and middle income

countries, where there is usually a scarcity of regulation on the use of

antibiotics in animal farming, have found antibiotic residues in a range of

animal foodstuffs (Ramatla et al., 2017; Jammoul and Darra, 2019; Oyedeji et

al., 2019). Antibiotic residues in food increases the risk of people developing a

number of health problems. These include hypersensitivity, anaphylaxis (Sachi

et al., 2019), reduced fertility (Prajwal et al., 2017), renal dysfunction (Shahrbaf

and Assadi, 2015), bone marrow toxicity, carcinogenic effects, organ lesions

(Liu et al., 2017), and obesity (Li et al., 2017).

1.1.3.6. Effects of antibiotics on poultry microbiota and intestinal

pathogens

The main reasons for using antibiotics in poultry feed are to enhance the

growth, performance, immune response, and health of poultry. Although the

mechanism of action of antibiotics is not clearly understood, studies show this

is in part due to the modifying effect on the GI tract microbiota (Maki et al.,

2019). Antibiotics selectively modify and improve microbiota in the poultry gut

which can inhibit the colonisation by bacterial pathogens such as

Camplobacter, E. coli, Salmonella and C. perfringens. This prevents infectious

diseases and improves the bird’s health, digestion, nutrient utilisation and feed

efficiency which may lead to enhanced performance and growth (Dibner and

Richards, 2005; Lee et al., 2012). It is also suggested that antibiotics used for

growth promote an optimal balanced microbiota which can reduce the

inflammatory response in poultry (Lin, 2011). This is a natural immune

response which can be caused by poor gut integrity, heat stress, and other

Literature Review

25

factors. If prolonged this can reduce feed intake and performance and increase

the bird’s vulnerability to pathogens (Pont et al., 2020).

Several studies refer to the effect of antibiotics on poultry microbiota in relation

to growth performance, immunity, and health. Virginiamycin was found to

increase the amount of Lactobacillus in the ileum (Dumonceaux et al., 2006).

It has also been associated with decreased diversity in caecal microbiota, and

enrichment of Lactobacillus and Faecalibacterium in the caecum of chickens

receiving virginiamycin in feed compared to a controlled feed without

antibiotics (Neumann and Suen, 2015). The increase in Faecalibacterium may

improve energy utilisation in birds by producing butyrate which is beneficial as

an energy source for intestinal epithelial cells which have an important role in

digestion.

The effects of these bacteria may improve feed efficiency and growth

promotion in birds (Pourabedin et al., 2015). Salinomycin was reported to

increase the abundance of Bifidiobacterium and Lactobacillus in comparison

to the untreated control group of poultry (Fung et al., 2013). Penicillin was

found to increase the proportion of Firmicutes and decrease Bacteroidetes in

chickens in comparison to the control group (Singh et al., 2013). The ratio of

Firmicutes to Bacteroidetes is often used as a poultry performance indicator.

A higher ratio of Firmicutes to Bacteroidetes in the caecum is associated with

increased growth. This results in an increased capacity for fermentation and

increased fermentation of volatile fatty acids which promotes fat deposition

(Hong et al., 2019).

Literature Review

26

However, antibiotics are not specific to one target, and they kill or inhibit any

microorganisms that are susceptible to them. This results in a reduction of

beneficial microorganisms that are involved in important functions such as

digestion and immunological response. Significant changes to the composition

of the microbiota can occur in the GI tract due to antibiotic use. These

significant modifications can catalyse dysbiosis, this can have a negative effect

on the metabolic performance and physiology of the host that could result in

GI disease (Allen and Stanton, 2014; Le Roy et al., 2019).

The use of antibiotics in poultry selects for persistence and transfer of antibiotic

resistant genes between bacteria. Therefore, the poultry microbiota acts as an

environmental reservoir for antibiotic resistant genes. Resistant genes can

spread from farm animals to humans through the ingestion of contaminated

food, water or contact with sewage waste. Therefore, the low dosage of

antibiotics in poultry feed has a detrimental effect on both the microbiota of

humans and poultry.

1.1.4. Alternatives to using antibiotics in poultry feed

Although many countries continue to use antibiotics as growth promoters in

animal farming, at a global level there is a commitment to reducing and

ultimately phasing out their use (WHO, 2015; O’Neill, 2016; ICGA, 2019). This,

in addition to growing concerns about public health regarding the use of

antibiotics in animal farming in certain countries has stimulated increased

interest in exploring alternatives to antibiotics (Lillehoj et al., 2018).

Literature Review

27

Researched alternatives to antibiotics in poultry feed include prebiotics,

probiotics, postbiotics and synbiotics, enzymes, acidifiers (organic acids), and

plant extracts including phytogenics which are secondary metabolites

including essential oils.

The benefits of using the above alternatives to bird health are generally linked

to a healthy GI tract microbiota (Maki et al., 2019). Consequently, much of the

literature on feed alternatives focuses on improving understanding around the

modulation of poultry GI tract microbiota and the effects of microbiota on

physiology, morphology, digestion, nutrient utilisation, immunity and reducing

GI tract pathogens. The use of alternative poultry feeds is also premised on

establishing and maintaining a balanced healthy microbiota in birds to improve

bird performance and health. This is also dependent on other factors such as

the chemical composition and concentration of the bioactive components in

their diet, amount of feed consumed, interaction within the GI tract, rate of

absorption, nutrient utilisation and excretion (Acamovic and Brooker, 2005).

However, while there is substantial research which highlights the benefits of

using alternatives to antibiotics in poultry feed the results are often inconsistent

(Gadde et al., 2017; Lillehoj et al., 2018).

Alternative poultry feed products should be safe for animal and human

consumption and comply with national regulations such as those imposed in

the EU by the European Food and Safety Agency (EFSA) and in the USA by

the Food and Drug Administration. For products to be considered as viable

alternatives to AGPs the potential alternative must be as effective or more

effective than antibiotics in terms of production and animal health (Collett and

Literature Review

28

Dawson, 2002; Sillanpää, 2015). One of the main advantages of using

alternative feed products is that they are typically less likely to be affected by

antibiotic resistance and they can be effective against MDR pathogens (PEW,

2016). Additionally, their effectiveness needs to be balanced against the

adequacy of farm management practices (Allen et al., 2014). Table 1.1.

provides a comparison of AGPs with alternative feed products against criteria.

Literature Review

29

Table 1.1. Comparison of AGPs with alternative poultry feed additives

Criteria AGPs Probiotics Prebiotics Essential

Oils

Enzymes Acidifiers Plant Extracts/

Phytochemicals

Direct antibacterial activity Yes No No Yes Yes Yes Yes

Immunomodulation Yes

Stimulation of beneficial bacteria

No Yes Yes Yes Yes Yes Yes

Reduction of pathogens Yes

Nutrient absorption Increased due to thinning of epithelial wall

Increased due to induction of digestive enzymes

Increased due to increased intestinal membrane permeability

Increased due to higher villi surface area

Increased due to breakdown of indigestible bonds

Increased due to gut acidification

Increased due to induction of digestive enzymes

Resistance development Common No, but can encourage transmission of resistance

No Uncommon No Uncommon No - Difficult due to multiple modes of action

Stability Stable

Ease of production Difficult - industrial fermentation and synthetic procedures

Medium - encapsulation

Medium - encapsulation

Easy – powdered plants, then water or solvent extraction

Difficult - genetic engineering or solid-state fermentation

Easy – calculate boiling temperature, use liquid or dry form

Easy – powdered plants, then water or solvent extraction

Ease of application Easy – Mixed in feed (Patterson and Burkholder, 2003; Alloui et al., 2013; Kumar et al., 2014; Valenzuela-Grijalva et al., 2017; Micciche et al., 2019; Rebello et al., 2019; Yadav

and Jha, 2019; Pearlin et al., 2020). Table 1.1. comparison of the effects of AGPS with different alternatives to AGPS that have been researched and used as

alternatives to AGPS in poultry feed.

Literature Review

30

1.1.4.1. Prebiotics

Prebiotics have been defined as compounds that can be selectively used by

microorganisms in the host to result in a health benefit (Gibson et al., 2017).

Prebiotics are indigestible by host enzymes which selectively stimulate the

activity and or growth of one or more bacteria in the poultry gut (Patterson and

Burkholder, 2003). Commonly used prebiotics in poultry feed include fructo-

oligosaccharides, mannan-oligosaccharides, xylo-oligosaccharides, galacto-

oligosaccharides, and β-glucans (Gaggìa et al., 2010; Hernandez-Patlan et al.,

2016; Teng et al., 2018).

Various studies have examined the effects of prebiotics in poultry feed on

modulating poultry gut microbiota and improving performance and growth

(Gadde et al., 2017). The beneficial effects reported include improved feed

conversion efficiency, increased body weight (Rosen, 2007; Benites et al.,

2008; Hooge and Connolly, 2011; Biswas et al., 2019), increased disease

resistance, increased nutrient availability (Ganguly, 2015), increased intestinal

villi height (Yang et al., 2007), and reduced pathogen counts (Jung et al.,

2008a; Shang et al., 2015; Yousaf et al., 2017). Despite these studies

highlighting potential beneficial effects of prebiotic supplementation on

performance and health there is no consensus over the potential beneficial

effects of prebiotics (Hwang et al., 2020). Furthermore, there are no approved

health claims for prebiotics (EFSA, 2018; FSAI, 2020). Research into the

effects of prebiotics is also limited and lacks depth of information such as mode

of action, and most studies use prebiotics that are genera specific not species

specific (Pourabedin and Zhao, 2015).

Literature Review

31

Although, further research is necessary to fully understand the mechanism of

action of prebiotics (Gibson et al., 2017) the following mechanisms have been

proposed. Prebiotics act as substrates for Lactobacillus and Bifidobacterium

to use for the production of antimicrobial compounds upon fermentation. They

modulate GI tract microbiota by reaching the lower intestine and stimulating

growth of beneficial indigenous microbiota (Ricke, 2018). This leads to the

production of short chain fatty acids (SCFAs) which lowers the intestinal pH

and increases enzyme digestive activity and the production of vitamins while

decreasing levels of cholesterol and triglycerides. This strengthens the poultry

immune system and helps to slow the growth of pathogens such as

Salmonella, Campylobacter, and E. coli (Patterson and Burkholder, 2003;

Teng and Kim, 2018). SCFAs may inhibit pathogen colonisation of the GI tract

by goblet cells raising mucin production creating a physical barrier for

pathogens (Tellez et al., 2006).

Another mechanism of prebiotics is competitive exclusion. This is when

beneficial organisms successfully compete and exclude bacterial pathogens

from accessing limited nutrients and mucosa attachment sites. Prebiotics also

produce bacteriocins like bifidocin, curvacin, helveticin, lactocin and nisin

which can inhibit intestinal pathogens (Chen et al., 2007). These mechanisms

make it more complex for pathogens to develop resistance mechanisms

against them (Suresh et al., 2018).

1.1.4.2. Probiotics

Probiotics are defined by the FAO and WHO as living microorganisms that

result in a health benefit to the host when they are consumed in appropriate

Literature Review

32

amounts (FAO and WHO, 2006). While there are a range of probiotic

microorganisms the most frequently used in poultry feed are bacterial including

Lactobacillus Bifidobacterium, and Enterococcus (FAOUN, 2016).

The effects of the addition of probiotics to poultry feed, include increased

numbers of beneficial bacteria, reduced GI colonisation of pathogens

(Nakphaichit et al., 2011; Yang et al., 2012; Jeong and Kim, 2014), improved

immune response (Bai et al., 2013; Salim et al., 2013; Ahmed et al., 2014),

improved feed conversion ratio, increased growth perfromance (Nakphaichit

et al., 2011; Salim et al., 2013; Mookiah et al., 2014; Park and Kim, 2014;

Ramlucken et al., 2020), and increased villus height and crypt depth (Salim et

al., 2013).

Although most studies demonstrate the beneficial effects of probiotics some

others have shown probiotics to have limited or no effects on poultry growth

and performance (O’Dea et al., 2006; Lee et al., 2010; Waititu et al., 2014).

The variation in study findings may be due to differences in processing, time,

and duration of administration of the probiotic, diet composition and variances

in the type and amount of the probiotic strain used (Gadde et al., 2017).

Additionally, some probiotic bacteria have antibiotic resistance genes and can

transfer antibiotic resistance, and the presence of enterotoxins in probiotic

bacteria may cause infections. Probiotics could therefore add to the burden of

the antibiotic resistant crisis by increasing the transfer of antibiotic resistant

genes (Marteau and Shanahan, 2003; EFSA, 2012).

Literature Review

33

While the mechanism of action of probiotics is not clearly understood two of

the main mechanisms include regulating and balancing the immune system

and the gut microbiota (Suresh et al., 2018). Probiotics assist in creating the

conditions for growth of beneficial microorganisms and to reduce colonisation

of bacterial pathogens through competitive exclusion and producing

bacteriocins and lactic acid (Gadde et al., 2017). Probiotics can alter the

intestinal epithelium in poultry leading to the increased production of mucins

by goblet cells. This helps prevent adhesion and colonisation of the intestinal

epithelium by pathogens (Smirnov et al., 2005). Studies have shown probiotics

can reduce the intestinal colonisation and spread of zoonotic pathogens such

as Salmonella and C. jejuni (FAOUN, 2016).

1.1.4.3. Synbiotics

Synbiotics are feed additives comprised of prebiotics and probiotics. The use

of synbiotics in poultry feed is founded on the idea that a combination of

probiotics and prebiotics will improve the survival of probiotic microorganisms

and the selective promotion of beneficial bacteria in the gastrointestinal (GI)

tract (Gibson and Roberfroid, 1995). Synbiotics can be independently chosen

for their activity on the host to stimulate changes in the host microbiota or the

prebiotic can be chosen to support the activity of the probiotic (Dunislawska et

al., 2017).

While there is limited information on the activity of synbiotics as a poultry feed

supplement (Gadde et al., 2017) some studies have shown synbiotics to

enhance GI tract microbiota, increase intestinal mucosa (Awad et al., 2009;

Literature Review

34

Sohail et al., 2015), improve feed conversion rate, and increase body weight

(Ashayerizadeh et al., 2009; Awad et al., 2009; Mookiah et al., 2014).

However, other studies noted no effect of performance (Willis et al., 2007; Jung

et al., 2008). Their mechanism of action depends on the combination of the

probiotics and prebiotics used in the poultry feed.

1.1.4.4. Enzymes

Enzymes are proteins with complex 3D molecular structures. Enzymes

function as biological catalysts with high substrate-specificity. Substrates in

poultry feed release nutrients during digestion. Different classes of enzymes

used as a poultry feed supplement include carbohydrases (cellulase, α-

galactosidase, α-amylase, β-mannanase, and pectinase xylanase), proteases,

and phytase (Gadde et al., 2017). The poultry GI tract naturally produces

endogenous enzymes to help digest nutrients, but they may not produce

enough enzymes to completely digest the nutrients. Exogenous enzymes are

added to poultry feed to help improve digestion by supplementing enzymes

birds may be deficient in such, as amylases and proteases, or to provide

enzymes which are not synthesised by the birds such as cellulases, and to

counter antinutritive effects (phytate, β-glucans, pentosans) (Fischer et al.,

2002). Phytases are targeted at all plant-derived ingredients, proteases are

targeted for all protein sources of plants, β-glucanases are targeted at barley,

oats, and rye while xylanases are targeted at wheat, barley, rye, triticale, and

fibrous plant materials (Ravindran, 2013).

Multiple enzymes are also used as supplemented in poultry feed as together

they may have a synergistic effect on improving digestibility and nutrient

Literature Review

35

utilisation (Juanpere et al., 2005; Cowieson and Kluenter, 2019). The benefits

of using enzymes as a poultry feed additive include, improved digestibility and

feed efficiency, increased performance and growth and enhanced immunity

(Adeola and Cowieson, 2011; Woyengo and Nyachoti, 2011; Kiarie, Romero

and Nyachoti, 2013; Alagawany, Elnesr and Farag, 2018; Cowieson and

Kluenter, 2019). However, the results of using enzymes as feed additives can

be inconsistent due to the source, type and amount of enzyme used, the

composition of poultry diet and genetic variations in the host (Son and

Ravindran, 2012; Cheng et al., 2014).

Proposed modes of action include increasing the abundance of beneficial GI

tract microbiota, producing SCFAs, increasing the digestibility of non-

degradable nutrients by host enzymes, utilising cell wall polysaccharides,

increasing the availability of minerals, starches and amino acids, reducing

viscosity in the intestine, and enhancing caecal fermentation of nutrients

(Choct, 2009; Bedford and Cowieson, 2012; Kiarie et al., 2013).

1.1.4.5. Organic acids

Organic acids are carboxylic acids with the particular chemical structure, R-

COOH, with intrinsic acidic properties. Organic acids used in poultry feed

include monocarboxylic acids (acetic, formic, propionic and butyric acids), or

carboxylic acids with the hydroxyl group (lactic, citric, malic, tartaric acids) or

short-chain carboxylic acids with double bonds (fumaric and sorbic acids)

(Khan and Iqbal, 2016).

Literature Review

36

Organic acids are used as supplements in the bird’s water and or as acidifiers

as supplements in feed. They are also produced as salts such as, calcium,

potassium, and sodium. The reported effects and activity of organic acids as a

supplement to poultry feed include, increased beneficial bacteria, reduced GI

colonisation of pathogens (Adhikari et al., 2019; Sabour et al., 2019), improved

immune response (Sabour et al., 2019), increased weight gain, and increased

feed efficency (Adil et al., 2010; Banday et al., 2015). However, results on the

use of organic acids to improve performance in poultry are often inconsistent.

This may be due to variation in rates of inclusion, the buffering capacity of other

feed ingredients, and the source of organic acids (Dibner and Buttin, 2002;

Kim, et al., 2015). In addition to the lack of consensus on the beneficial effects

of organic acids, studies have also found that bacterial pathogens can develop

resistance to organic acids, they can also decrease their internal pH which

protects them from organic acids their bactericidal effects (Dittoe et al., 2018).

This highlights the requirement to discover alternatives to antibiotics that do

not promote the development of resistance.

The mechanisms of action of organic acids are not completely understood but

four proposed mechanisms include, being able to permeate the cell wall of pH

sensitive bacteria and decrease their pH causing disruption of cell functions.

This can decrease the levels of some pathogens in the bird GI tract of birds.

This may also improve nutritional uptake by regulating the bacteria competing

for nutrients (Kim, et al., 2015). Another mechanism of action of organic acids

is to reduce the pH of the crop and poultry GI tract. This can improve

digestibility by increasing pepsin activity and proteolysis of proteins and by

stimulating the increased production of pancreatic juice (Dibner and Buttin,

Literature Review

37

2002). They may also slow down the passage of feed through the intestine

allowing more time for nutrient absorption (Centeno et al., 2007). Organic acids

can also enhance health of the gut by increasing the growth of epithelial cells

(Khan and Iqbal, 2016).

1.1.4.6. Essential oils

Currently, there are around 3000 known essential oils (EOs) (Krishan and

Narang, 2014). EOs are volatile, aromatic liquids attained from different parts

of plants such as their buds, flowers, leaves, seeds, herb, wood, fruits, roots

and twigs (Başer and Demirci, 2007). EOs are essentially comprised of two

types of organic compounds, including terpenes and phenylpropenes (Zhai et

al., 2018). However, two or three constituents usually contribute to up to 85%

of the mixture and therefore to the overall property (Miguel, 2010).

While numerous studies of EOs including blends of thymol and

cinnamaldehyde (Gadde et al., 2017). Tiihonen et al., 2010), combinations of

clove and cinnamaldehyde (Chalghoumi et al., 2013), blends of clove and

thymol (Kim et al., 2016), oregano (Hashemipour et al., 2013), coriander

demonstrate their positive effects on poultry health and performance, other

studies have found EOs to have little or no beneficial effects (Hernández et al.,

2004; Jang et al., 2007). The differences in findings may be due to variations

in the origin, type and composition of the EOs used and trial conditions (Franz

et al., 2010). The first commercial blend of phytonutrients approved by EU was

comprised of carvacrol, capsicum, cinnamaldehyde and oleoresin. A meta-

analysis of using this commercial blend as a poultry feed supplement found

Literature Review

38

the blend improved feed conversion ratio, reduced mortality, and increased

body weight (Bravo and Ionescu, 2008)

1.1.5. Alternative poultry feed supplements: plant extracts and

phytochemicals

1.1.5.1. Plant extracts and phytochemicals

Phytochemicals (phytogenics) and plant extracts represent a diverse group of

natural compounds. Of >100,000 naturally derived compounds that have been

described, >80,000 of these are plant derived (Hashemi and Davoodi, 2011).

Many of these plants have antibacterial properties against a range of bacterial

pathogens. They have been used in traditional herbal medicine for thousands

of years by many ancient civilisations including Babylon, China, Egypt, India,

Greece, Mesopotamia, and Rome to prevent and treat infections in animals

and humans (Aboelsoud, 2010; Srivastava, 2019). The use of numerous

medicinal plants for treating a range of infectious diseases, illnesses and

wounds have been cited in many ancient texts (Petrovska, 2012). Some of

these inlcude aloe, castor oil plant, cinnamon, clove, ginger, gentian violet,

garlic, ginseng, juniper, mandrake, nutmeg, oregano, parsley, pepper,

pomegrantite, poppy, wormwood (Petrovska, 2012). In more recent times

artemisinin from the Qinghao plant and quinine from the cinchona tree have

proved to be effective for the treatment of malaria (Subramani et al., 2017).

There is a lot of current potential for drug discovery from plant sources

(Patridge et al., 2016). Plant extract availability, accessibility, and abundance

in nature makes them suitable targets for investigation as antibiotic

Literature Review

39

alternatives. Plant extracts and phytochemicals extracted from plants have

been used as safe and effective feed alternatives in poultry diet to improve

productivity and bird health. They include a broad range of herbs and spices.

Their products and can be used in dried, solid, powder (ground form) or as

crude or concentrated extract form (Gadde et al., 2017). The main difference

between the two plant-based alternatives is that plant extracts refer to the

crude mixture of chemical compounds contained within plant tissue, whereas

phytochemicals usually specifically refer secondary metabolites which are

chemicals that protect plants from consumers such as grazing animals

(Acamovic and Brooker, 2005).

Plant tissue is comprised of range of chemical constituents (Kris-Etherton et

al., 2002). These result from a range of metabolic and biochemical processes

during plant growth and include alkaloids, glycosides, hormones, organic

acids, resins, volatile oils, sugars, amino acids, proteins and enzymes, tannins,

pigments, oils and waxes, and microminerals (Akyildiz and Denli, 2016). By

comparison with synthetic antibiotics, plant extracts and phytochemicals are

natural, less toxic, and free of residues (Hashemi et al., 2008).

Phytochemicals are grouped according to their chemical structure and function

and include terpenes (e.g. geraniol, humulene, cafestol, squalene) phenolics

(e.g. coumarin, lignin, flavonoids, tannins), nitrogen containing compounds

(e.g. alkaloids, glucosides) and sulphur containing compounds (e.g.

glutathione, glucosinolates, allinin) (Jamwal et al., 2018).

Literature Review

40

Plant secondary metabolites are produced and regulated through biosynthesis

often triggered by environmental or seasonal changes. Biosynthesis occurs in

compartmentalised cell sites in different parts of the plant such as the

chloroplast (e.g. alkaloids, quinolizidines, and some terpenes), endoplasmic

reticulum (e.g. phenolics, flavonoids, tannins), mitochondria (e.g. coniine and

some amines), vacuole (tannins), vesicles (e.g. alkaloids), leaves and seeds

(e.g. glucosinolates) (Acamovic and Brooker, 2005). The concentration of

secondary metabolites in different plant parts can vary depending on the stage

of development of the plant. The initial synthesis of secondary metabolites is

usually confined to one part of the plant such as the leaves, fruit, or roots. They

are then transported around the plant and stored in various plant tissues. For

example, terpenes are stored on the cuticle or in thylakoid membranes, resin

ducts and glandular hairs while some compounds produced for defence such

as flavonoids, alkaloids and glycosides are stored in the epidermis (Wiermann,

1981).

1.1.5.2. Plant extracts and phytochemicals: Modes of action

There is an extensive volume of research on plant extracts and

phytochemicals which demonstrates their antibacterial effects against a range

of pathogens (Gadde et al., 2017; Lillehoj et al., 2018).

While it is commonly agreed that plant extracts have multiple mechanisms of

action and their antibacterial activity is due to their chemical composition and

secondary metabolites, there is limited understanding around their

Literature Review

41

mechanisms of action. Five different mechanisms of action have been

proposed:

1. disruption of the structure and function of the cell membrane and

modification of the plasma membrane/cell wall

2. disruption of the synthesis of DNA and RNA and alteration of the function

of the cell

3. interference with intermediary metabolic processes and altered metabolic

processes

4. introduction of coagulation of the cytoplasmic constituent and

modification of the cytosolic environment

5. interruption of usual cell communication and disruption of quorum

sensing (Radulovic et al., 2013; Omojate et al., 2014; Górniak et al.,

2019).

1.1.5.3. Synergistic effects: Combinations of plant extracts and plant extracts

with antibiotics

Combinations of plant extracts may also provide possible alternatives to using

antibiotics in poultry farming while combinations of plant extracts with

antibiotics can result in decreasing the quantity of antibiotic additives used in

poultry feed. Research illustrates how chemical interactions between the

bioactive components of these combinations can result in a synergistic effect

on their overall antibacterial activity against pathogens including E. coli, S.

enteritidis, C. jejuni and L. monocytogenes (Poe, 1976; Natchimuthu et al.,

2012; Van Vuuren and Viljoen, 2012; Stefanović, 2018; Hossain et al., 2020).

Synergistic effects can increase the efficacy of the antibiotic by modulating its

Literature Review

42

antibacterial activity and increasing its ability to inhibit pathogens. This means

the antibiotic is more effective at lower concentrations therefore decreasing

the amount of antibiotic required. It is also possible that the antibacterial effect

of the above combinations may be antagonistic. This results in the combination

having a reduced ability to inhibit the growth of the above pathogens. Two

other effects of combinations are also possible. The combination may have an

additive effect. This when the overall antibacterial activity of the combination

is equal to the sum of the antibacterial effects of the individual constituents.

The combination may have an indifferent effect. This occurs when the

antibacterial activity is equal to that produced by the most potent individual

constituent activity (Poe, 1976; Abreu et al., 2012; Bhardwaj et al., 2016).

1.1.5.4. Synergistic effects of plant combinations: Modes of action

The complex nature of the mixtures of the chemical compositions of combined

plant extracts makes it difficult to develop a full understanding of their modes

of action (Caesar and Cech, 2019). However, in general the proposed

mechanisms of action of combined plant extracts included, multi-target effects

when individual constituents of a combination of plant extracts affect more than

one target causing an antagonistic or synergistic effect (Wagner and Ulrich-

Merzenich, 2009). Targets include proteins, receptors, DNA/RNA, substrates,

transport proteins, ion channels, and monoclonal antibodies. Plant secondary

metabolites such as polyphenols can bind to the cell walls of bacteria and

disrupt protein regulation and inhibit microbial enzymes while terpenoids

because of their high lipophilicity can penetrate the bacterial cell walls (Lin et

al., 2004; Navarro et al., 2015). Both mechanisms can cause damage to the

Literature Review

43

cell walls of bacteria. Flavonoids can disrupt the synthesis of cell walls, cell

membranes and the metabolism of energy. Compounds such as polyphenols

and saponins exhibit pharmacokinetic effects. Their presence can increase the

solubility, distribution, resorption rate, and metabolism of major plant extract

bioactive constituents. This enhances the bioavailability of bioactive

constituents and increases the efficacy of the plant extract combination against

bacterial pathogens (Caesar and Cech, 2019). In addition, the complex

bioactive chemical components and multiple mechanisms of action makes it

difficult for bacteria to develop resistance (Silver and Bostian, 1993).

1.1.5.5. Synergistic Effects of Plant and Antibiotic Combinations: Modes of

Action

While the mechanisms of synergistic antibacterial activity are not entirely

understood it has been proposed the secondary metabolites of the plant alter

and inhibit the bacteria’s mechanism to acquire resistance to the antibiotic

(Abreu et al., 2012; Stefanović, 2018; Khameneh et al., 2019). In general,

synergistic mechanisms of plant extracts and antibiotics combined include the

alteration of active sties on the bacterial cell. β-lactam antibiotics such as

ampicillin can disrupt and slow down the metabolism of peptidoglycan by

binding to penicillin-binding proteins (PBPs) which weakens the integrity of the

bacterial cell wall. Bacteria can develop resistance to the antibiotic by

decreasing its affinity of PBPs or by decreasing production of these proteins.

Combinations of plant extracts with β-lactam antibiotics has been shown to

significantly decrease the MIC of β-lactams against pathogens such as MRSA.

Examples include polyphenol corilagin from Arctostaphylos uva-ursi (Shimizu

Literature Review

44

et al., 2001), flavonoid baicalin from Scutellaria amoena (Liu et al., 2000),

epigallocatechin gallate from green tea (Zhao et al., 2001), and tellimagrandin

and rugosin B from wild rose (Shiota et al., 2000). Another synergistic

mechanism which plant extracts contribute to is the inhibition of enzymes

which can degrade or alter antibiotics. Synergistic interactions of

epigallocatechin gallate in green tea and ampicillin was found to inhibit β-

lactamases and increase the activity of the ampicillin against ampicillin

resistant S. aureus (Zhao et al., 2001). Secondary metabolites such as

phenolic compounds and terpenes in plant extracts can change function and

structure of the cell membrane of bacteria and increases its permeability. This

allows more of the antibiotic to access the bacterial cell and interact with

intercellular targets (Stefanović, 2018). Another way plants can improve the

effects of antibiotics is by disrupting bacterial efflux pumps. For example,

Lycopus europaeus in combination with erythromycin was found to double the

intensity of antibiotic activity against S. aureus strains by inhibiting the

bacteria’s multidrug efflux mechanisms msr(A) (macrolide resistance) and

tet(K) (tetracycline resistance) (Gibbons et al., 2003).

1.1.6. Effects of plant extracts and phytochemicals on intestinal bacterial

pathogens and microbiota in poultry

A wide range of plant extracts and phytochemical have been studied in in vitro

and in vivo studies to examine their antibacterial activity and potential use in

feed in the poultry diet (Sethiya, 2016). In vitro assays are commonly used to

screen plant extracts for antibacterial activity. Their effects on intestinal

Literature Review

45

bacterial pathogens and microbiota can then be further tested in in vivo poultry

trials, as in this study.

While in some in vitro assays some plant extracts may demonstrate

antibacterial activity against selected pathogens, they may not in in vivo poultry

trials. This is because in vivo studies can account for the complex interactions

between plant bioactive compounds and the biochemical processes which

occur in a living organism. Consequently, they provide more information, for

example on their effects on gut microbiota and changes in physiology and

morphology and metabolic functions.

1.1.6.1. In vitro studies

Many in vitro studies of plant extracts and phytochemicals have demonstrated

antibacterial activity against the pathogens tested in this study, including E.

coli, S. enteritidis, C. jejuni, and L. monocytogenes. Plant extracts and

phytochemicals which have demonstrated antibacterial activity against MDR

strains of E. coli and Salmonella include, cinnamon stick, cinnamon oil, and

cinnamaldehyde from Cinnamomum cassia Blume (minimum inhibitory

concentration/MIC range from 75 mg/L to 600 mg/L) and Oxalis corniculate

(MIC of 20mg/L) (Ooi et al., 2006; Shan et al., 2007; Manandhar et al., 2019;

Yang et al., 2019). An MIC below 1000 mg/L is considered to demonstrate

significant antibacterial activity that is worth investigating further (Ríos and

Recio, 2005). This in vitro research therefore indicates that these extracts

demonstrate significant antibacterial effects in vitro. An in vivo study

demonstrated that cinnamon essential oils combined with bamboo leaf

flavonoids inhibit E. coli populations in the GI tract of chickens (Yang et al.,

2019).

Literature Review

46

Other studies that have used the disc diffusion method to determine the

diameter of an area of bacteria on an agar plate that has been inhibited. These

studies found several plants that are effective against E. coli and Salmonella

including: Oregano, rosemary, tulsi, bryophyllum, thyme, and lemon grass.

These values were all lower and the extracts were therefore more effective in

comparison to the antibiotic controls, imepenem, and vancomycin (Dahiya and

Purkayastha, 2012). Furthermore, oregano essential oil was found to decrease

E. coli in the GI tract of chickens (Mohiti-Asli and Ghanaatparast-Rashti, 2017).

This verifies the results from the in vitro study of the antibacterial activity of

oregano and demonstrates that the in vitro results may be used to justify further

in vivo work.

Other plant extracts that inhibited S. enteritidis and E. coli investigated in

studies include: Mangifera indica Julie cultivar leaf; and Euadenia trifoliata

stem bark (Abiala et al., 2016). These values were significantly lower than

the control and were therefore more effective in comparison to the antibiotic

control meropenem.

Other studies have used the broth microdilution method to demonstrate the

antibacterial activity of plant extracts in vitro against C. jejuni. In vitro studies

of extracts of Chinese Leek have demonstrated weak antibacterial activity

against C. jejuni (Lee et al., 2004).

Other studies have used the broth microdilution method and the disc diffusion

method to demonstrate the significant antibacterial activity of plant extracts in

vitro against L. monocytogenes including Cinnamomum javanicum (Yuan et

al., 2017), Sophorae radix, and Psoraleae semen (Yoon and Choi, 2012).

Literature Review

47

Generally, in vitro studies do not have published in vivo studies that specifically

look at the antibacterial activity of these plant extracts against E. coli, L.

monocytogenes, C. jejuni, or Salmonella. This highlights a gap in the literature,

these studies lacked verification of the results from the lab in a follow up in vivo

study.

Various in vitro studies have also demonstrated the synergistic effects of

combined plant extracts and combinations of plant extracts with antibiotics.

Combinations of extracts of Alchornea cordifolia and Pterocarpus santalinoide

showed synergistic activity against Salmonella and E. coli (Obaji et al., 2020).

Salvia chamelaeagnea and Leonotis leonurus in combination reported a

synergistic effect against E. coli (Kamatou et al., 2006). Combinations of

oregano and thyme oregano and cinnamon bark were found to demonstrate

increased inhibition against C. jejuni, in comparison to individual plant extracts

(Navarro et al., 2015). Combinations of oregano and cranberry demonstrated

synergistic effects against L. monocytogenes (Lin et al., 2004). Extracts of

Green tea in combination with gentamicin and amikacin was found to enhance

the antibacterial effect and increased the inhibition of E. coli (Neyestani et al.,

2007). Combinations of rosemary with amoxicillin, gentamicin and difloxacin

and combinations of dill and doxycycline, majorana and gentamicin, and neem

with amoxicillin and doxycycline demonstrated synergistic effects against

Salmonella (Ashraf et al., 2018). Another study found that phenolic

compounds commonly found in plants significantly increased the susceptibility

of C. jejuni to ciprofloxacin and erythromycin in several human and poultry

isolates (Oh and Jeon, 2015). Cocos nucifera in combinations with penicillin,

Literature Review

48

chloramphenicol, ampicillin, ciprofloxacin, and tetracycline demonstrated

synergistic effects against L. monocytogenes (Akinyele et al., 2017).

1.1.5.6. In vivo studies

Many in vivo studies of plant extracts and phytochemicals as a feed

supplement indicate they modulate poultry GI microbiota leading to higher

quantities of beneficial bacteria such as Lactobacillus. Plant extracts also

reduce the abundance of detrimental bacteria such as E. coli, S. enteritidis, C.

jejuni and L. monocytogenes and toxic microbial metabolites due to their

antibacterial effect on intestinal pathogens (Gadde et al., 2017). The

modifications in microbiota are commonly associated with improved

digestibility, modulating the immune response, and inhibiting pathogens which

can lead to improved growth and performance.

Various studies of herbs and spices indicate that the inclusion of plant extracts

and phytochemicals in poultry diet can increase beneficial bacteria such as

Lactobacillus in different parts of the poultry gut (Hashemi and Davoodi, 2010;

Yang et al., 2019). Citrus extract in poultry feed increased the abundance of

Lactobacillus in the caecum (Yu et al., 2019). These beneficial bacteria have

a role in the modulation of the immune response of poultry as they reduce

colonisation of the GI tract by pathogens through competitive exclusion

mechanisms and secretion of organic acids and bacteriocins which lower the

pH of the GI tract of poultry to suppress pathogen growth (Yadav and Jha,

2019).

Literature Review

49

Citrus extract also decreased ammonia concentrations in the caecum in

comparison to the antibiotic and the control with no additive. Exposure to

ammonia can stimulate oxidative stress, inflammation, and decrease

microtubule activity (Wang et al., 2019). Decreasing ammonia concentrations

in the caecum can also lead to improved absorption of nutrients (Bogucka et

al., 2019). Citrus extract also significantly decreased total amines, indole, p-

cresol, methylamine, spermidine, and tyramine in comparison to the antibiotic

control (Yu et al., 2019). High concentrations of these fermentation products

can promote cancers (Davila et al., 2013). Therefore, citrus extract has

anticancer properties.

A range of plant extracts and phytochemicals in feed have demonstrated

antibacterial activity against intestinal pathogens in chickens. The control of

intestinal pathogens relieves immune defence stress in poultry by reducing

competition for essential nutrients and bacterial toxins (Hashemi and Davoodi,

2010). The combination of cinnamon essential oil and bamboo leaf flavonoid

against E. coli decreased E. coli populations in the chicken caecum in

comparison to the control group (Yang et al., 2019). Decreasing bacterial

pathogens such as E. coli and Salmonella in the GI tract of chickens can

prevent infectious disease and damage to the GI tract morphology and

physiology and therefore improve bird health, digestion, and nutrient utilisation

which can lead to enhanced performance (Dibner and Richards, 2005; Lee et

al., 2012). Oregano oil (Roofchaee et al., 2011; Mohiti-Asli and Ghanaatparast-

Rashti, 2017), Forsythia suspensa (Han et al., 2012), and Rosemary herb

powders (Norouzi et al., 2015) reduced the abundance of E. coli in the caecum

of chickens. Curcuma longa and Scutellaria baicalensis (Varmuzova et al.,

Literature Review

50

2015), Flos lonicerae with Baikal skullcap (W. Wang et al., 2019), Thymol, and

Carvol (Arsi et al., 2014) have demonstrated antibacterial activity against

Salmonella in poultry trials.

1.2. Aims and objectives

Aim

The thesis aim is to develop natural plant supplements (phytogenics) that can

be used in animal feeds as sustainable alternatives to conventional antibiotics

to promote animal health and performance.

Objectives

The objectives of this thesis were to:

1. To test the in vitro antibacterial activity of specific plant extracts in

reducing pathogenic bacteria.

2. To develop a screening process to select suitable plants for in

vivo studies.

3. To develop an in vitro model to predict the effect of plants within the

non-ruminant digestive system through the use of digesta collected

from poultry.

4. To select plants based on optimum in vitro effects and to examine their

efficacy in vivo on broiler performance and on the microbial profile of

the digestive system.

Chapter 2

51

2. Chapter 2

Evaluation of antibacterial activity of medical plants against

Campylobacter jejuni, Listeria monocytogenes, Escherichia coli and

Salmonella enterica subsp, enterica serovar Enteritidis

2.1. Introduction

The use and misuse of antibiotics in animal farming is commonly cited in the

research literature as one of the main causes of the global antibiotic crisis

(O’Neill, 2016; CDC, 2019; HM Government, 2019; IACG, 2019). This presents

a significant threat to animal and public health as it reduces the effectiveness

of many antibiotics used to treat infections and is likely to lead to increased

rates of global mortality and morbidity. Two of the ways of helping to address

the antibiotic crisis is to decrease the amount of antibiotics used in animal

farming and to research alternatives to using antibiotics WHO, 2015).

Therefore, this study focuses on poultry farming and the potential of using plant

extracts as alternatives to antibiotics to improve performance and the overall

bird health. There is a significant volume of research which supports potential

use of plant extracts as alternatives to antibiotics in poultry farming. (Sethiya,

2016; Lillehoj et al., 2018). One focus of the literature demonstrates the

antibacterial activity of a wide variety of plant extracts against a range of

bacterial pathogens (Gadde et al., 2017). Many of the findings are from in vitro

studies which are often used to screen the antibacterial activities of plant

extracts against certain pathogens. Plant extracts which exhibit antibacterial

activity above a certain MIC value can then be identified for further

investigation in in vivo studies. The antibacterial activity exhibited by plant

Chapter 2

52

extracts against a range of pathogens in this study, including E. coli,

Salmonella, C. jejuni, and L. monocytogenes has been demonstrated in

numerous in vitro studies (Table 2.1). Hence the purpose of this in vitro study

was to screen the antibacterial activity of various plant extracts against C.

jejuni, E. coli, S. enteritidis, and L. monocytogenes and to identify those with

the highest levels of antibacterial activity for further investigation and potential

use as a feed supplement in a poultry trial.

2.2. Materials and methods

2.2.1. Selection of plant extracts

In this in vitro study 40 plant extracts which are frequently used in traditional

Chinese medicine were screened for antibacterial activity against E. coli, S.

enteritidis, C. jejuni, and L. monocytogenes (Table 2.1). Most of the plants (32

of 40) were selected because they were identified in the literature review as

having antibacterial activity against several bacteria such as, Clostridium

difficile, Helicobacter, MRSA, Klebsiella pneumoniae, and Staphylococcus

aureus.

Twenty-two of these plant extracts demonstrated antibacterial activity against

one or more of E. coli, Salmonella, C. jejuni, and L. monocytogenes. Eight

plant extracts were randomly selected from a range of plants used in traditional

Chinese medicine that did not appear to be cited in the literature around plants,

phytochemicals, and antibacterial activity. This would help to address the gap

in the literature by providing new information about the antibacterial properties

of these plants and their potential use as alternatives in poultry feed to

decrease pathogenic bacteria.

Chapter 2

53

Table 2.1. Plant extracts used in the in vitro study

No Plant Species Common Name Commercial Name

In vitro antibacterial activity against:

1. *Scutellaria barbata D. Don (herb)

Barbed skullcap 半枝莲 Salmonella and E. coli (Yu et al., 2004); MRSA (Sato et al., 2000) Helicobacter pylori (Ma et al., 2010); Acinetobacter baumanni (Miyasaki et al., 2010); Pseudomonas aeruginosa (Zhang et al., 2013)

2. Gentiana scabra Bunge (Rhizome)

Gentian 龙胆草 H. pylori (Ma et al., 2010)

3. Epimedium brevicornum Maxim (Leaf)

Horny Goat Weed

淫羊藿 A. baumanni (Miyasaki et al., 2010)

4. Agrimonia pilosa Ledeb (herb)

Hairy agrimony 仙鹤草 A. baumanni (Miyasaki et al., 2010); E. coli and L. monocytogenes (S. Kim et al., 2011a); E. coli, L. monocytogenes and S enteritidis (McMurray et al., 2020); MRSA (Kim et al., 2020)

5. Allium macrostemon Bunge (bulb)

Long stamen chive

薤白 E. coli (Ji-Hyun and Mee-Aae, 2005)

6. Oldenlandia diffusa (Willd.) Roxb (herb)

Snake needle grass

蛇舌草

7. Lysimachia christinae Hance (herb)

Coin herb 金钱草

8. Taxillus chinensis (D.C.) Danser (stem and branch)

Mulberry mistletoe

桑寄生

9. Platycladus orientalis (L.) Franco (Leaf and twig)

Chinese thuja 侧柏叶 Salmonella typhimurium and Proteus vulgaris (Zazharskyi et al., 2019); C. difficile and Clostridium perfringens (Kim and Lee, 2015)

10. Aloe barbadensis Mill (root and leaf)

Chinese aloe 龙舌兰 MRSA (Pratiwi et al., 2015); S. aureus and Streptococcus pyogenes (Kambai, Jekada and Audu, 2019); E .coli and B. subtilis (Irshad et al., 2011); Agrobacterium tumefaciens, Proteus mirabilis, Bacillus cereus, S. aureus, and S.

Chapter 2

54

pyogenes (Subramanian et al., 2006).

11. Smilax glabra Roxb (tuber)

Cat greenbrier 菝契 H. pylori (Ma et al., 2010); A. baumannii and P. aeruginosa (Zhang et al., 2013); K. pneumoniae, (Franzblau and Cross, 1986); E. coli, L. monocytogenes and S enteritidis (McMurray et al., 2020)

12. Forsythia suspensa (Thunb.) Vahl (fruit)

Weeping forsythia

连翘 L. monocytogenes and Vibrio parahemolyticus (Wu et al., 2018); E. coli, S. aureus, and Salmonella enterica (Han et al., 2012);K. pneumoniae, P. vulgaris, and S. aureus (Franzblau and Cross, 1986)

13. Lsatis tinctoria L. (root and leaf)

Dyer’s Woad 板蓝根 H. pylori (Ma et al., 2010); MRSA (Kim et al., 2020)

14. Rheum palmatum L. (root)

Rhubarb 大黄 Enterococcus faecium, L. monocytogenes, Candida albicans, B. subtilis, Pseudomonas fluorescens, Enterococcus durans, P. aeruginosa, Listeria innocua, S. aureus, Staphylococcus epidermidis, Enterobacter aerogenes, E. coli, K. pneumonia, S. enteritidis, S. typhimurium, Salmonella infantis, and Salmonella kentucky (Canli et al., 2016)

15. Sargentodoxa cuneata (Oliv.) Rehder and E. H. Wilson (vine and stem)

Sargent Glory Vine

红藤

16. Lonicera japonica Thunb. (anthodium)

Honeysuckle 金银花 H. pylori (Ma et al., 2010); L. monocytogenes, S. enteritidis, and E. coli (Rahman and Kang, 2009); E. coli and S. aureus (Xiong et al., 2013); K. pneumoniae (Franzblau and Cross, 1986)

17. Corydalis bungeana Turcz (leaf)

Crested lark 黑彈樹

18. Glycyrrhiza uralensis Fisch (root)

Chinese liquorice

甘草 H. pylori (Li et al., 2005); MRSA (Lee et al., 2009); S. aureus, Streptococcus agalactiae, E. coli, and S. enterica (Sun et al., 2020)

19. Salvia miltiorrhiza Bunge (root and rhizome)

Red sage 丹参 MRSA (Kim et al., 2020);

Chapter 2

55

Bacillus coagulans, Staphylococcus albus and S. aureus (Li et al., 2011)

20. Ophiopogon japonicus (Thunb.) Ker-Gawl (root)

Monkey grass 麦冬 E. coli (Wang et al., 2016).

21. Arctium lappa L. (fruit)

Greater burdock 牛蒡子 H. pylori (Ma et al., 2010); E. coli and Salmonella abony (Ionescu et al., 2013).

22. Paeonia suffructicosa Andrews (root)

Cortex moutan 牡丹皮 H. pylori (Ma et al., 2010); MDR A. baumanni (Miyasaki et al., 2010); A. baumanii, P. aeruginosa, Ornithine resistant S. aureus, MRSA, S. aureus, and E. coli (Yang et al., 2013)

23. Reynoutria japonica Houtt (root)

Asian knotweed 虎杖 B. cereus, L. monocytogenes, S. aureus, E. coli, and Salmonella anatum (Shan et al., 2008); B. cereus, L. monocytogenes, S. aureus, E. coli, and S. anatum (Shan et al., 2007)

24. Areca catechu L. (pericarp)

Areca palm 大腹皮 B. cereus, L. monocytogenes, S. aureus, E. coli, and S. anatum (Shan et al., 2007); E. coli and Staphylococcus (Faden, 2018); B. subtilis and S. aureus (Rahman et al., 2014).

25. Astragalus propinquus Schischkin (root)

Mongolian milkvetch

黄芪 Alcaligenes faecalis, Aeromonas hydrophila, B. cereus, Salmonella newport, and Shigella sonnei (Lai et al., 2018)

26. Coptis chinensis Franch (root)

Chinese goldthread

黄莲 H. pylori (Ma et al., 2010); MRSA (Kim et al., 2020); A. baumanii, P. aeruginosa, Ornithine resistant S. aureus, MRSA, S. aureus, and E. coli (Yang et al., 2013); Aggregatibacter actinomycetemcomitans and Prevotella intermedia (Lee and Kim, 2019); K. pneumoniae, P. vulgaris, and S. aureus (Franzblau and Cross, 1986)

27. Paeonia lactiflora Pall (root)

Red peony root 赤芍 H. pylori (Ma et al., 2010); Streptococcus ratti, Streptococcus sobrinus, Streptococcus sanguinis,

Chapter 2

56

Streptococcus parasanguinis, Streptococcus criceti, Streptococcus gordonii, Streptococcus anginosus, Streptococcus downei, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum (Lee et al., 2018); Bacteroides fragilis, Bacteroides thetaiotaomicron, C. difficile, C. perfringens, Clostridium paraputrificum, E. coli, S. typhimurium, S. aureus, and K. pneumoniae (Luong et al., 2012)

28. Sophora tonkinensis Gagnep (root and rhizome)

Sophora 山豆根

29. Coix lacryma-jobi L. (bark)

Job’s tears 苡仁 A. baumannii and P. aeruginosa (Zhang et al., 2013); S. epidermis, S. aureus, B. cereus, B. subtilis, E. coli, P. aeruginosa, V. cholerae, Shigella dysenteriae, K. pneumoniae (Das et al., 2017); E. coli, S. aureus, and B. subtilis (Setya et al., 2020)

30. Chrysanthemum morifolium Ramat (seed)

Chrysanthemum 野菊花 H. pylori (Ma et al., 2010); B. cereus, L. monocytogenes, S. aureus, E. coli, and S. anatum (Shan et al., 2007)

31. Lobelia chinensis Lour (anthodium)

Asian lobelia 半還题 K. pneumoniae, S. aureus and B. cereus. (Leong, 2013).

32. Aucklandia lappa Decne (herb)

Costus 木香 S. aureus, S. albus, B. subtilis, S. typhimurium, S. dysenteriae, E. coli (Liu et al., 2014)

33. Iris domestica (L.) Goldblatt and Mabb (root)

Blackberry lily 射干 MRSA (Joung et al., 2014); A. actinomycetemcomitans and P. intermedia (Lee and Kim, 2019); E. coli, L. monocytogenes and S enteritidis (McMurray et al., 2020)

34. Pteris cretica var. nervosa (Thunb.) Ching and S.H. Wu (rhizome)

Cretan brake fern

凤尾草

35. Portulaca oleracea L. (rhizome)

Common purslane

马齿苋 E. coli, S. aureus, Shigella flexneri, and S. typhi (Lei et al., 2015);

Chapter 2

57

E. coli, S. aureus, Streptococcus agalactiae, Streptococcus dysgalactiae (Yu et al., 2014)

36. Cyrtomium falcatum (L.F.) C. Presl (herb)

Holly fern 贯众

37. Anemone chinensis Bunge (rhizome)

Pulsatilla chinensis

白头翁 H. pylori (Ma et al., 2010); K. pneumoniae, P. vulgaris, and S. aureus (Franzblau and Cross, 1986); E. coli, L. monocytogenes and S enteritidis (McMurray et al., 2020)

38. Lithospermum erythrorhizon Siebold and Zucc (root)

Purple gromwell 紫草 E. coli, P. aeruginosa, S. typhimurium, S. enteritidis, S. sonnei, S. dysenteriae and S. flexneri (Bae, 2004)

39. Spirodela polyrhiza (L.) Schleid (root)

Common duckweed

浮萍 S. polyrrhiza, A. hydrophila, Psedomonas putida, P. aeruginosa, P. fluorescens, V. parahaemolyticus, V. Alginolyticus, E. coli, and S. aureus (Das et al., 2012)

40. Fraxinus chinensis Roxb (herb)

Chinese ash bark

秦皮 H. pylori (Ma et al., 2010)

2.2.2. Bacterial isolates and reference strains used for screening

Clinical isolates of L. monocytogenes, S. enteritidis, C. jejuni and E. coli, and

reference strains Streptococcus pneumoniae and S. aureus were sourced as

frozen stocks from the National Collection of Type Cultures (NCTC), American

Type Culture Collection (ATCC), and collected as frozen stocks from the Agri-

Food and Biosciences Institute (AFBI), and Queen’s University Belfast (QUB)

from a range of retail and clinical sources (Table 2.2). Bacteria from AFBI were

previously identified at AFBI through phenotypical and serological methods

and bacteria from QUB were previously identified through 16S PCR. Once

obtained 16S PCR, Sanger sequencing, and BLAST analysis were used to

identify the genus of each bacterium (Table 2.2) according to the

Chapter 2

58

manufacturer’s instructions (MyTaq™ Red Mix, Bioline, UK) (Bioline, 2019).

Gram staining was also used to affirm whether the bacteria were Gram-

positive or Gram-negative (Principi and Esposito, 2016).

Table 2.2. Reference strains, clinical isolates, and their sources.

Species Isolate Source

C. jejuni (Gram-negative) NCTC 11322 Reference (NCTC)

RC152 Retail pack of raw chicken (AFBI)



L. monocytogenes (Gram-positive)

NCTC 11994 Reference (QUB)

LS12519 Retail ready to eat sliced meat (AFBI)

OT11230 Retail chopping block (AFBI)

CP102 Retail cheese and ham (AFBI)

CP1132 Retail cooked chicken breast (AFBI)

QA1018 Quality assurance sample (QAS) (AFBI)

S. enteritidis (Gram-negative)

NCTC 0074 Reference (QUB)

1F6144 QAS (AFBI)

LE103 Egg filter (AFBI)

QA04/19 QAS (AFBI)

E. coli (Gram-negative) ATCC 25922 Reference (QUB)

UM004 Urinary Tract Infection (UTI) (QUB)

UM011 UTI (QUB)

UM012 UTI (QUB)

S. pneumoniae (Gram-positive)

ATCC 49619 Reference (QUB)

S. aureus (Gram- positive) ATCC 29213 Reference (QUB)

2.2.3. Preparation of plant extracts: Ultrasound assisted extraction

The ultrasonic assisted extraction method (Toma et al., 1999; Azwanida, 2015)

was used with water as a solvent to obtain crude extracts from 40 dried plants

(Tong Ren Tang; Beijing) (Table 2.1). Water was chosen as a solvent to obtain

crude extracts as opposed to other solvents such as alcohols because it

dissolves a wider range of compounds (Abubakar and Haque, 2020), it is cost-

effective, and reduces the risk of solvent toxicity (Truong et al., 2019). The

Plant List (2013) was used to confirm the accepted names of these plants to

Chapter 2

59

avoid the use of synonyms. The rotary ball mill (Retch PM 100 planetary ball

mill, QUB) was used to powder 10mg of each plant. The sun wheel speed was

set at 600/min. This was done in accordance with the instructions of the

manufacturer. The yield obtained was 9 ± 0.8 mg of each plant. Two milligrams

of dried plant powder was dissolved with 1 ml deionised water, then fixed in an

ultrasonic bath (VMR Ultrasonic cleaning bath, QUB) at 80 °C for 15 minutes.

The samples were then removed and placed into a water bath to boil at 100°C

for 20 minutes. The extraction temperatures applied were based on the boiling

temperature of the solvent. Previous papers demonstrated that an extraction

temperature of 100 °C using water as a solvent may result in potent

antibacterial activity at lower concentrations (Onyebuchi and Kavaz, 2020) and

improved extraction yields (Matshediso et al., 2015). The increased mass

transfer rate and extraction yield demonstrated at higher temperatures, such

as 100 °C could be a due to improved solute desorption from the active sites

in the cell matrix (Co et al., 2012). The time of extraction was taken into

consideration because if plants are subjected to boiling temperatures for long

periods of time, for example over 6 hours, this can lead to denaturation (Wang

and Weller, 2006). However, the plants were powdered to increase the surface

area of the plant material and increase the rate of extraction (Pandey and

Tripathi, 2014). The plants were also subjected to ultrasound which caused

acoustic cavitation and increased the surface contact between the water and

plants, and the cell wall permeability. This increased the rate of extraction as

it enhanced the transport of water into the plant cells and the release of

compounds (Toma et al., 1999). Consequently, this reduced the time for

Chapter 2

60

extraction and the possibility of denaturation (Ebrahim et al., 2013; Azwanida,

2015). Prior to analysis the resulting solutions were stored at < 4°C.

2.2.4. Antibacterial testing: Broth microdilution method

Plant extracts were screened for their antibacterial effects using a

standardised broth microdilution method (CLSI, 2009) to determine their

minimum inhibitory concentration (MIC) and minimum bactericidal

concentration (MBC) against L. monocytogenes, S. enteritidis, C. jejuni and E.

coli (CLSI, 2009; Balouiri et al., 2016). Pure bacteria cultures were incubated

overnight under conditions optimised for their growth (Table 2.3). Two-fold

serial dilutions were constructed of individual antibiotics and plant extracts.

One hundred microlitres of each individual concentration (2000 mg/L to

0.06mg/L) of antibiotic and plant extract was individually added to single wells

of a 96 well plate (Figure 2.1). These concentrations refer to the dried weight

of the dried plants and the dried weight of the dry antibiotics. The culture of

bacteria was adjusted to an optical density which was equivalent to 1x108

CFU/mL then diluted to 1x106 CFU/mL. 100 µL of the resulting bacterial

solution was added to each individual well. The dilutions were set up in

replicates of three. In this study the detectable limit for the antibacterial activity

of plant extracts was set at MIC or MBC cut off values of 1000 mg/L. This cut

off value was based on research which suggests MICs or MBCs at or below

1000 mg/L indicates promising levels of antibacterial activity worth

investigation (Ríos and Recio, 2005). The MIC was the well with the lowest

concentration of antibacterial which had no visible growth. Visible growth was

defined by the observation of turbid wells, if there was no visible growth the

wells were clear to the naked eye and resembled the positive antibiotic control.

Chapter 2

61

Ten microlitres from each well was plated one concentration below and every

concentration above the MIC to the highest concentration onto agar and

incubated for 48 hours under conditions optimised for growth of bacteria (Table

2.3). The MBC was the lowest concentration where no growth occurred on the

plate at 24 hours and 48 hours. The range of bacteria used in this study grow

in different physiological conditions and at different rates. MIC and MBC values

were confirmed after 48 hours to account for different growth rates of bacteria,

particularly when investigating the susceptibility of Campylobacter spp. as

isolates of this bacteria may take longer than 24 hours to grow. Ten microlitres

from the growth control well was also plated onto agar and incubated for 48

hours under conditions optimised for growth of bacteria (Table 2.3). The

inclusion of the growth control in broth during MIC determination and on agar

during MBC determination allowed for a comparison with the test results to

ensure any inhibition in growth was due to the plant extracts and not due to

differences in bacterial growth in the broth and agar used. For each of the

above pathogens tested against each plant extract the negative control

included testing a corresponding pathogen sample in broth only. The positive

controls included testing the antibacterial activity of ampicillin against each

isolate of L. monocytogenes, S. enteritidis, and E. coli; and erythromycin

against each isolate of C. jejuni (EUCAST, 2018). A further quality control to

validate the method was implemented. When L. monocytogenes, S. enteritidis,

and E. coli were tested, ampicillin was simultaneously tested against S.

pneumoniae ATCC 49619. When C. jejuni was tested, erythromycin was

simultaneously tested against S. aureus ATCC 29213 (EUCAST, 2018).

Chapter 2

62

Table 2.3. Media and incubation conditions used for bacteria for broth microdilution.

Bacteria Broth Agar Incubation

conditions

C. jejuni Mueller Hinton

fastidious (MH-F)

MH-F 41 ± 1 °C;

microaerobic

L. monocytogenes Tryptone soy and

5% lysed horse

blood

Tryptone soy and

5% lysed horse

blood

35 ± 1 °C; 5% CO2

E. coli Mueller Hinton

broth (MHB)

Mueller Hinton agar

(MHA)

35 ± 1 °C; ambient air

S. enteritidis MHB MHA 35 ± 1 °C; ambient air

S. pneumoniae MH-F MH-F 35 ± 1 °C; 5% CO2

S. aureus MHB MHA 35 ± 1 °C; ambient air

Agar and broths were obtained from Oxoid, UK and lysed horse blood was obtained from TCS

Biosciences Ltd, UK. Anaeropacks and microaerophilic gas sachets were obtained from

BioMerieux, UK to maintain microaerophilic conditions to incubate Campylobacter.

2.2.5. Statistical analysis

MIC and MBC values were expressed as a value rounded to the nearest well

(Figure 2.1 to 2.3; Tables 2.4 to 2.7). Traditionally the SEM and SD values are

not expressed for this method (CLSI, 2009; EUCAST, 2018). The SEM and

SD was not included as this method was based on a qualitative reading which

was taken only when the 100% of the wells (three wells) changed colour and

the concentration of plant extract was inferred from this reading, therefore the

SEM and SD values of the mean of these readings would be 0. This method

is standardised to reduce error however there is still an associated systematic

and random error of mode ± 1 dilution range (EUCAST, 2018). ANOVA was

carried out to test significance of difference between MIC and MBC values of

different plant extracts (P <0.001) using Prism 5.0 (Prism 5.0 software; QUB).

Chapter 2

63

2.3. Results

2.3.1. Identification of bacterial isolates and reference strains used for

screening

The similarity of the 16S rRNA gene analysis indicated that 4 isolates belonged

to C. jejuni (Table 2.4. to 2.7.), 6 isolates to L. monocytogenes (Table 2.8. to

2.13.), 4 isolates to E. coli (Table 2.14. to 2.17.), and 4 isolates to S. enterica

(Table 2.18. to 2.21.). Sequence similarities ranged from 94.57% to 100%.

Table 2.4. 16S ribosomal RNA, partial sequence of C. jejuni

cttctagcttgctagaagtggattagtggcgcacgggtgagtaaggtatagttaatctgccctacacaagaggacaacagttg

gaaacgactgctaatactctatactcctgcttaacacaagttgagtagggaaagtttttcggtgtaggatgagactatatagtatc

agctagttggtaaggtaatggcttaccaaggctatgacgcttaactggtctgagaggatgatcagtcacactggaactgagac

acggtccagactcctacgggaggcagcagtagggaatattgcgcaatgggggaaaccctgacgcagcaacgccgcgtg

gaggatgacacttttcggagcgtaaactccttttcttagggaagaattctgacggtacctaaggaataagcaccggctaactcc

gtgccagcagccgcggtaatacggagggtgcaagcgttactcggaatcactgggcgtaaagggcgcgtaggcggattatc

aagtctcttgtgaaatctaatggcttaaccattaaactgcttgggaaactgatagtctagagtgagggagaggcagatggaatt

ggtggtgtaggggtaaaatccgtagatatcaccaagaatacccattgcgaaggcgatctgctggaactcaactgacgctaag

gcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccctaaacgatatgagggtgctagcttgctag

aacttagagacaggtgctgcacggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccac

gtatttagttgctaacggttcggccgagcactctaaatagactgccttcgtaaggaggaggaaggtgtggacgacgtcaagtc

atcatggcccttatgcccagggcgacacacgtgctacaatggcatatacaatgagacgcaataccgcgaggtggagcaaa

tctataaaatatgtcccagttcggattgttctctgcaactcgagagcatgaagccggaatcgctagtaatcgtagatcagccatg

ctacggtgaatacgttcccgggtcttgtactcaccgcccgtcacaccatgggagttga

Table 2.4. Partial sequence length of 1124 base pairs of 16S ribosomal RNA of C. jejuni

demonstrated a 100% similarity to C. jejuni (accession number NR_118520.1), query cover of

100%, and a significant E value of 0 using BLAST analysis.

Chapter 2

64


gtatagttaatctgccctacacnagaggacaacagttggaaacgactgctaatactctatactcctgcttaacacaagttgagt

agggaaagtttttcggtgtaggatgagactatatagtatcagctagttggtaaggtaatggcttaccaaggctatgacgcttaac

tggtctgagaggangatcagtcacactggaactgagacacggtccagactcctacgggaggcagcagtagggaatattgc

gcaatgggggaaaccctgacgcagcaacgccgcgtggaggatgacacttttcggagcgtaaactccttttcttagggaaga

attctgacggtacctaaggaataagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttactc

ggaatcactgggcgtaaagggcgcgtaggcggattatcaagtctcttgtgaaatctaatggcttaaccattaaactgcttggga

aactgatagtctagagtgagggagaggcagatggaattggtggtgtaggggtaaaatccgtagatatcaccaagaataccc

attgcgaaggcgatctgctggaactcaactgacgctaaggcgcgaaagcgtggggagcaaacaggattagataccctggt

agtccacgccctaaacgatgtacactagttgttagggtgctagcttgctagaacttagagacaggtgctgcacggctgtcgtca

gctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccacgtatttagttgctaacggttcggccgagcactcta

aatagactgccttcgtaaggaggaggaaggtgtggacgacgtcaagtcatcatggcccttatgcccagggcgacacacgtg

ctacaatggcatatacaatgagacgcaataccgcgaggtggagcaaatctataaaatatgtcccagttcggattgttctctgca

actcgagagcatgaagccggaatcgctagtaatcgtagatcagccatgctacggtgaatacgttcccgggtcttgta


demonstrated a 99.57% similarity to C. jejuni (accession number NR_118520.1), query cover

of 100%, and a significant E value of 0 using BLAST analysis.


agtggcgcacgggtgagtaaggtatagttaatctgccctacacaagaggacaacagttggaaacgatgctaatactctatac

tcctgcttaacacaagttgagtagggaaagtttttcggtgtaggatgagactatatagtatcagctagttggtaaggtaatggctt

accaaggctatgacgcttaactggtctgagaggangancagtcacactggaactgagacacggtccagactcctacggga

ggcagcagtagggaatattgcgcaatgggggaaaccctgacgcancaacgccgcgtggaggangacacttttcggagcg

taaactccttttcttagggaagaattctgacggtacctaaggaataagcaccggctaactccgtgccagcagccgcggtaata

cggagggtgcaagcgttactcggaatcactgggcgtaaagggcgcgtaggcggattatcaagtctcttgtgaaatctaatgg

cttaaccattaaactgcttgggaaactgatagtctagagtgagggagaggcagatggaattggtggtgtaggggtaaaatcc

gtagatatcaccaagaatacccattgcgaaggcgatctgctggaactcaactgacgctaaggcgcgaaagcgtggggagc

aaacaggattagataccctggtagtccacgccctaaacgatgtacactagttgttggggtggagcatgtggtttaattcgaaga

tacgcgaagaacnttacctgggcttgatatcntaagaacctttnagagataagagggtgctagcttgctagaacttagagaca

ggtgctgcacggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccacgtatttagttgcta

acggttcggccgagcactctaaatagactgccttcgtaaggaggaggaaggtgggacgatgtcaagtcatcatggcccttat

gcccagggcgacacacgtgctacaatggcatatacaatgagacgcaataccgcgaggtggagcaaatctataaaatatgt

cccagttcggattgttctctgcaactcgagagcatgaagccggaatcgctagtaatcgtagatcagccatgctacggtgaata

cgttcccgggtcttgtactcaccgcccgtcacacc




Chapter 2

65

Table 2.7. 16S ribosomal RNA sequence of C. jejuni

ctngctagaagtggattagtggcgcacgggtgantaaggtatagttaatctgccctacacaagaggacaacagttggaaac

gactgctaatactctatactcctgcttaacacaagttgagtagggaaagtttttcggtgtaggatgagactatatagtatcagcta

gttggtaaggtaatggcttaccaaggctatgacgcttaactggtctgagaggatgatcagtcacactggaactgagacacggt

ccagactcctacgggaggcagcagtagggaatattgcgcaatgggggaaaccctgacgcagcaacgccgcgtggagga

tgacacttttcggagcgtaaactccttttcttagggaagaattctgacggtacctaaggaataagcaccggctaactccgtgcc

agcagccgcggtaatacggagggtgcaagcgttactcggaatcactgggcgtaaagggcgcgtaggcggattatcaagtct

cttgtgaaatctaatggcttaaccattaaactgcttgggaaactgatagtctagagtgagggagaggcagatggaattggtggt

gtaggggtaaaatccgtagatatcaccaagaatacccattgcgaaggcgatctgctggaactcaactgacgctaaggcgcg

aaagcgtggggagcaaacaggattagataccctggtagtccacgccctaaacgatgtacactagttgttggggtgctagtcat

ctcagtaatgcagctaacgcattaagtgtaccnnnggggagtacggtcgcaagattaaaactcaaaggaatagacgggga

cccgcacaagcggtggagcatgtggtttaattcgaagatacgcgaagaaccttacctgggcttgatatcntaagaaccttttag

agataagagggtgctagcttgctagaacttagagacaggtgctgcacggctgtcgtcagctcgtgtcgtgagatgttgggttaa

gtcccgcaacgagcgcaacccacgtatttagttgctaacggttcggccgagcactctaaatagactgccttcgtaaggagga

ggaaggtgtggacgacgtcaagtcatcatggcccttatgcccagggcgacacacgtgctacaatggcatatacaatgagac

gcaataccgcgaggtggagcaaatctataaaatatgtcccagttcggattgttctctgcaactcgagagcatgaagccggaat

cgctagtaatcgtagatcagccatgctacggtgaatacgttcccgggtcttgtactcnccgcccgtcacnccntgggagttgatt

tcact




Chapter 2

66

Table 2.8. 16S ribosomal RNA, partial sequence of L. monocytogenes

gagagcttgctcttccaaagttagtggcggacgggtgagtaacacgtgggcaacctgcctgtaagttggggataactccggg

aaaccggggctaataccgaatgataaagtgtggcgcatgccacgcttttgaaagatggtttcggctatcgcttacagatgggc

ccgcggtgcattagctagttggtagggtaatggcctaccaaggcaacgatgcatagccgacctgagagggtgatcggccac

actgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacgga

ncaacgccgcgtgtactgttgttagagaagaacaaggataagagtaactgcttgtcccttgacggtatctaaccagaaannc

acggctaactacgtgncagcagccgcggtaatacgtaggtggcaagcgttgtccggatttattgggcgtaaagcgcgcgca

ggcggtcttttaagtctgatgtgaaagcccccggcttaaccggggagggtcattggaaactggaagactggagtgcagaag

aggagagtggaattccacgtgtagcggtgaaatgcgtagatatgtggaggaacaccagtggcgaaggcgactctctggtct

gtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgagtg

ctaagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatttt

agttgccagcatttagttgggcactctaaagtgactgccggtgcaagccggaggaaggtggggatgacgtcaaatcatcatg

ccccttatgacctgggctacacacgtgctacaatggatagtacaaagggtcgcgaagccgcgaggtggagctaatcccata

aaactattctcagttcggattgtaggctgcaactcgcctacatgaagccggaatcgctagtaatcgtggatcagcatgccacg

gtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtagggta

Table 2.8. Partial sequence length of 1138 base pairs of 16S ribosomal RNA of L.

monocytogenes demonstrated a 99.26% similarity to L. monocytogenes (accession number

NR_044823.1), query cover of 99%, and a significant E value of 0 using BLAST analysis.


gttagtggcggacgggtgagtaacacgtgggcaacctgcctgtaagttggggataactccgggaaaccggggctaataccg

aatgataaaatgtggcgcatgccacgcttttgaaagatggtttcggctatcgcttacagatgggcccgcggtgcattagctagtt

ggtagggtaatggcctaccaaggcaacgatgcatagccgacctgagagggtgatcggccacactgggactgagacacgg

cccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgtatga

agaaggttttcggatcgtaaagtactgttgttagagaagaacaaggataagagtaactgcttgtcccttgacggtatctaacca

gaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggatttattgggcgtaaagc

gcgcgcaggcggtcttttaagtctgatgtgaaagcccccggcttaaccggggagggtcattggaaactggaagactggagtg

cagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagatatgtggaggaacaccagtggcgaaggcgactct

ctggtctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacg

atgagtgctaagtgttagggggttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacgaccgcaa

ggttgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaacct

taccaggtcttgacatcctttgaccactctggagacagagctttcccttcggggacaaagtgacaggtggtgcatggttgtcgtc

agctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgattttagttgccagcatttagttgggcactctaaa

gtgactgccggtgcaagccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgct

acaatggatagtacaaagggtcgcgaagccgcgaggtggagctaatcccataaaactattctcagttcggattgtaggctgc

Chapter 2

67

aactcgcctacatgaagccggaatcgctagtaatcgtggatcagcatgccacggtgaatacgttcccgggccttgtacacac

cgcccgtcacaccacgagagtttgtaacacccgaagtcggtagggta





ggggacaaagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacc

cttgattttagttgccagcatttagttgggcactctaaagtgactgccggtgcaagccggaggaaggtggggatgacgtcaaat

catcatgccccttatgacctgggctacacacgtgctacaatggatagtacaaagggtcgcgaagccgcgaggtggagctaa

tcccataaaactattctcagttcggattgtaggctgcaactcgcctacatgaagccggaatcgctagtaatcgtggatcagcat

gccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtggcgg

acgggtgagtaacacgtgggcaacctgcctgtaagttggggataactccgggaaaccggggctaataccgaatgataaag

tgtggcgcatgccacgcttttgaaagatggtttcggctatcgcttacagatgggcccgcggtgcattagctagttggtagggtaat

ggcctaccaaggcaacgatgcatagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcct

acgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgtatgaagaaggttttcg

gatcgtaaagtactgttgttagagaagaacaaggataagagtaactgcttgtcccttgacggtatctaaccagaaagccacg

gctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggatttattgggcgtaaagcgcgcgcaggc

ggtcttttaagtctgatgtgaaagcccccggcttaaccggggagggtcattggaaactggaagactggagtgcagaagagg

agagtggaattccacgtgtagcggtgaaatgcgtagatatgtggaggaacaccagtggcgaaggcgactctctggtctgtaa

ctgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaa

gtgttagggggtttccgccccttagtgctgcagctaac




Chapter 2

68


gagagcttgctcttccaaagttagtggcggacgggtgagtaacacgtgggcaacctgcctgtaagttggggataactccggg

aaaccggggctaataccgaatgataaagtgtggcgcatgccacgcttttgaaagatggtttcggctatcgcttacagatgggc

ccgcggtgcattagctagttggtagggtaatggcctaccaaggcaacgatgcatagccgacctgagagggtgatcggccac

actgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacgga

gcaacgccgcgtgtatgaagaaggttttcggatcgtaaagtactgttgttagagaagaacaaggataagagtaactgcttgtc

ccttgacggtatctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccg

gatttattgggcgtaaagcgcgcgcaggcggtcttttaagtctgatgtgaaagcccccggcttaaccggggagggtcattgga

aactggaagactggagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagatatgtggaggaacac

cagtggcgaaggcgactctctggtctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctg

gtagtccacgccgtaaacgatgagtgctaagtgttagggggttccgccccttagtggaaactcaaaggaattgacgggggcc

cgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctttgaccactctgga

gacagagctttcccttcggggacaaagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccg

caacgagcgcaacccttgattttagttgccagcatttagttgggcactctaaagtgactgccggtgcaagccggaggaaggtg

gggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatagtacaaagggtcgcgaagcc

gcgaggtggagctaatcccataaaactattctcagttcggattgtaggctgcaactcgcctacatgaagccggaatcgctagt

aatcgtggatcagcatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacc

cgaagtcggtagggta




Chapter 2

69


agtgctgcagctaacgcattaagcactccgcctggggagtacgaccgcaaggttgaaactcaaaggaattgacgggggcc

cgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctttgaccactctgga

gacagagctttcccttcggggacaaagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccg

caacgagcgcaacccttgattttagttgccagcatttagttgggcactctaaagtgactgccggtgcaagccggaggaaggtg

gggatgacgtcaatcatcatgccccttatgacctgggctacacacgtgctacaatggatagtacaaagggtcgcgaagccgc

gaggtggagctaatcccataaaactattctcagttcggattgtaggctgcaactcgcctacatgaagccggaatcgctagtaat

cgtggatcagcatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccga

agtcggtggcggacgggtgagtaacacgtgggcaacctgcctgtaagttggggataactccgggaaaccggggctaatac

cgaatgataaagtgtggcgcatgccangcttttgaaagatggtttcggctatcgcttacagatgggcccgcggtgcattagcta

gttggtagggtaatggcctaccaaggcaacgatgcatagccgacctgagagggtgatcggccacactgggactgagacac

ggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgtat

gaagaaggttttcggatcgtaaagtactgttgttagagaagaacaaggataagagtaactgcttgtcccttgacggtatctaac

cagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggatttattgggcgtaaa

gcgcgcgcaggcggtcttttaagtctgatgtgaaagcccccggcttaaccggggagggtcattggaaactggaagactgga

gtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagatatgtggaggaacaccagtggcgaaggcga

ctctctggtctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaa

acgatgagt




Chapter 2

70


gagtacgaccgcaaggttgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagc

aacgcgaagaaccttaccaggtcttgacatcctttgaccactctggagacagagctttcccttcggggacaaagtgacaggtg

gtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgattttagttgccagcattta

gttgggcactctaaagtgactgccggtgcaagccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgg

gctacacacgtgctacaatggatagtacaaagggtcgcgaagccgcgaggtggagctaatcccataaaactattctcagttc

ggattgtaggctgcaactcgcctacatgaagccggaatcgctagtaatcgtggatcagcatgccacggtgaatacgttcccg

ggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtggcggacgggtgagtaacacgtggg

caacctgcctgtaagttggggataactccgggaaaccggggctaataccgaatgataaaatgtggcgcatgccacgcttttg

aaagatggtttcggctatcgcttacagatgggcccgcggtgcattagctagttggtagggtaatggcctaccaaggcaacgat

gcatagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagg

gaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgtatgaagaaggttttcggatcgtaaagtactgttgtta

gagaagaacaaggataagagtaactgcttgtcccttgacggtatctaaccagaaagccacggctaactacgtgccagcag

ccgcggtaatacgtaggtggcaagcgttgtccggatttattgggcgtaaagcgcgcgcaggcggtcttttaagtctgatgtgaa

agcccccggcttaaccggggagggtcattggaaactggaagactggagtgcagaagaggagagtggaattccacgtgta

gcggtgaaatgcgtagatatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggcgcgaa

agcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggttccgccc

cttagtgctgcagctaacgc




Chapter 2

71

Table 2.14. 16S ribosomal RNA, partial sequence of S. enteritidis

cttgctgcttcgctgacgagtggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacg

gtggctaataccgcataacgtcgcaagaccaaagagggggaccttcgggcctcttgccatcagatgtgcccagatgggatt

agcttgttggtgaggtaacggctcaccaaggcgacgatccctagctggtctgagaggatgaccagccacactggaactgag

acacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtg

tatgaagaaggccttcgggttgtaaagtactttcagcggggaggaaggtgttgtggttaataaccgcagcaattgacgttaccc

gcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggc

gtaaagcgcacgcaggcggtctgtcaagtcggatgtgaantccccgggctcaacctgggaactgcattcgaaactggcag

gcttgagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaag

gcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctggtagtccacg

ccgtaaacgatgtctacttggaggttgtgccctgaggcgtggctcaagcggtggagcatgtggtttaattcgatgcaacgcgaa

gaaccttacctggtcttgacatccacagannnntccagagatggattggtgccttcgggaactgtgagacaggtgctgcatgg

ctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcgattaggtcggg

aactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggct

acacacgtgctacaatggcgcatacaaagagaagcgacctcgcgagagcaagcggacctcataaagtgcgtcgtagtcc

ggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatcagaatgccacggtgaatacgttcccgg

gccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaa

Table 2.14. Partial sequence length of 1270 base pairs of 16S ribosomal RNA of S. enterica

demonstrated a 99.74% similarity to S. enterica (accession number NR_104709.1), query

cover of 100%, and a significant E value of 0 using BLAST analysis.


tcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggttaggccgggaactcaaag

gagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtg

ctacaatggcgcatacaaagagaagcgacctcgcgagagcaagcggacctcataaagtgcgtcgtagtccggattggagt

ctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatcagaatgccacggtgaatacgttcccgggccttgtaca

caccgcccgtcacaccatgggagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccg

gtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctg

gtagtccacgccgtaaacgatgtctacttggaggttgtgcccttgaggcgtggcggacgggtgagtaatgtctgggaaactgc

ctgatggagggggataactactggaaacggtggctaataccgcataacgtcgcaagaccaaagagggggaccttcgggc

ctcttgccatcagatgtgcccagatgggattagcttgttggtgaggtaacggctcaccaaggcgacgatccctagctggtctga

gaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgg

gcgcaagcctgatgcagccatgccgcgtgtatgaagaaggccttcgggttgtaaagtactttcagcggggaggaaggtgttg

tggttaat

Chapter 2

72


demonstrated a 99.46% similarity to S. enterica (accession number NR_104709.1), query

cover of 100%, and a significant E value of 0 using BLAST analysis.


taatgtctgggaaactgcctgatggagggggataactactggaaacggtggctaataccgcataacgtcgcaagaccaaa

gagggggaccttcgggcctcttgccatcagatgtgcccagatgggattagcttgttggtgaggtaacggctcaccaaggaga

ctgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctaca

atggcgcatacaaagagaagcgacctcgcgagagcaagcggacctcataaagtgcgtcgtagtccggattggagtctgca

actcgactccatgaagtcggaatcgctagtaatcgtggatcagaatgccacggtgaatacgttcccgggccttgtacacaccg

cccgtcacaccatgggag


demonstrated a 100% similarity to S. enterica (accession number NR_104709.1), query cover

of 100%, and a significant E value of 2e-140 using BLAST analysis.


tgttgccagcgattaggtcgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatc

atggcccttacgaccagggctacacacgtgctacaatggcgcatacaaagagaagcgacctcgcgagagcaagcggac

ctcataaagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatcagaatg

ccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaagaccaaagagggggaccttcgggcctcttgccatc

agatgtgcccagatgggattagcttgttggtgaggtaacggctcaccaaggcgacgatcccta


demonstrated a 100% similarity to S. enterica (accession number NR_104709.1), query cover

of 100%, and a significant E value of 4e-151 using BLAST analysis.

Chapter 2

73

Table 2.18. 16S ribosomal RNA, partial sequence of E. coli

agggtagaaattaccacagcatgcagtcgtagaagggtctagtaatgtttggacatgtctaagggggggaggctggaaagg

ctgagggggtgattatgaagtgcaaagtagaaaaacgcaaacgccgacgcgaaggagggggattcgggctcttggcaac

gagagtctcatgagggcttatgtggtggggggggaacgtgtcaacttgggggcgattgcaaggggggcggagaggtgacg

agcaccttggtgtttaccggcatagaatcttcgggggggagtgttggcaatatggttaaggggccgaggtatggaagctacgg

cgggaatgaggagtttttgggtgaaaagaattagtggggggagggggggaaagttaaatcttttctttttgcgtttccccgaaaa

aaaggtctggtaaattccggcccggggcccgggaaaaacggggggggggttttcttattgcgaaatacttggcccaaaggg

gcccccgctgggttggtaacccaaaaatggaattccccggcaccccccgggaattcctcctaggactggcaattttgggttcg

gagggggggggaaaaattccagggtaaacctggaaaagcgaaaggttttgggggaaaactgggggaaaacgaccccct

ttggaaaaattactttcaaggtgaaagaagtggagaaaaagggggtagaaatcccggaggctcccctccaaacgtattcga

tttgggagtgtccccttgaaggcgggctccccgaggaaacaccgttaagccgacccccggggagtccccgggaggttaaa

aattcaaaaaattttagtggggcccccccatgggggggggaagggggttaatccgtgaaaccggaaaaaacatcacaac

accctagtgtactgctagatgatattggcagctcccagcacgtctatgaaaagtactcgcgaccgcgcccctaaatagaattg

atgtaaacagccctctcttgggaaacctactagagactatatctatgcgcctacgtggcaagaccattgtcggggactttgggc

tctgacatcggatgagacgaggatgggactcagctagttgtggggtaaacgctcaactaggcgacgatccgtagctcgtccg

agaagatgaccagccacactggtactgagacacggtccagactcctacgtgacgcagcagtggggaatattgcacaatgg

gcgcaagcctgatgcagccatgccgcgtgtatgaagaagcgcttcggggttgtaaagtactttcagcggggaggaaggga

gtaaagttaatacctttgctcattgacgctacccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacgg

agggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgcgctc

aacctgggaactgcatctgatactggcaagcttgagtctcgtagaggggggtagaattccaggtgtagcggtgaaatgcgta

gagatctggaggaataccggtggcgaaggcggccccctggacgaagactgacgctcaggtgcgaaagcgtggggagca

aacaggattagataccctggtagtccacgccgtaaacgatgtcgacttggaggttgtgcctgaagcgtgctcccgagctaac

gcgttaagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacggggggccgcacaagcggtgg

agcatgtggtttaattcgatgcaacgcgaagaaccttacctggtcttgacatccacggaagttttcagagatgagaatgtgcttc

gggaaccgtgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtccccgcaacgagcgcaac

ccttatcctttgttgccagcggtccggccgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgt

caagtcatcatggcccttacgaccagggctacacacgtgctacaatggcgcatacaaagagaagcgacctcgcgagagc

aagcggacctcataaagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtgg

atcagaatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggtgcaaaagaagtat

agagcagagctgcgctcagctcagcactgtccgccgca

Table 2.18. Partial sequence length of 2314 base pairs of 16S ribosomal RNA of E. coli

demonstrated a 96.51% similarity to E. coli (accession number NR_114042.1), query cover of


Chapter 2

74


tctccggcttactgcagcatgatgtcgtacagggtctatactgtgtgggcatgcttggggggggtgctggaatgctgaagggcg

gttattagggggggaagtaaaacgcaatcgcgttggcgaggggggactctggctctggcatgaagtgtcagaggctgagct

agagggaggcaggctctctgtgggcgttcctaggtgggctgagagggacagcacctggacttttagcaggtccgcctttaag

ggaggcaataacggggaatatgcacaagggcgggcggattgctgcaagcgcggttttggaggaggtttgggttgtttggaat

aatattgggaggaggggaggaagaaaaatgtttgcctttgtcattatccggaaagaaagccgggtaattcctcttgcggggg

gggaatacgggggggttgtggtaatggtataattgcggaaggtacgcggtggtggtaagccaatgaaatcccccttgtcccc

ctgggaatgcttctagacggggaatttttggctcggagggggggaagactttcagggtaacccttaaaagggatatgttgggg

ggatacctggggggaacgggcccctgtgagaagactttctctccgagtcaaaagttggttgcgtaaaggataagaaacccc

ggaggccccccccaaaaacatgtccacttggaggttgccccttgagggcacgctcccctaagaaacgcgtaaacccacag

cctgggagtaccgcccgaggtaaaaaatcaaaaaaatttgagtgggccccccacaggtggggtattgggttaatttgataaa

agcggaaaatttatggtagcaacactcataccatcacctaagttgtattctgccaattgaagctgcgaagcctaaatcatgacg

tatataaaaaaatagtgtccgaccgcgccgcccccaggcgactatttactggaaactgcccgcccgatggggacaacactg

gaagactgtaatctaatgccgcactacgtggcaagattattgtggcggaccttctggcatcttggcattcggatgtggacaagat

gggattagctagtttttgggagtaacggctcatctaggcgacgatccgtagctgctctgagaggatgaccagccacactggaa

ctgagacacggtccagactctacgggacgcagcagtgggaaatattgcacaatgggcgcaagcctgatgcagccatgccg

cgtgtatgaagaagggcttcgggttgtaaagtactttcagcggggaggaagggagtaaagttaatatctttgctcattgacgtta

cccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgg

gcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgggctcaacctgggaactgcatctgatactggcaa

gcttgagtctcgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaatacaggtggcgaag

gcggccccctggacgaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctgtagtcacgcc

gtaaacgatgtcgactggaggttgtgcctgagcgtggctcagagctaacgtgcgcgttaagtcgaccgcctggggagtacgc

cgcaaggttaaaactcaaatgaattgacgggggccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaaga

accttacctggtcttgacatccacggaagttttcagagatgagaatgtgccttcgggaaccgtgagacaggtactgcatggctg

tcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggccgggaa

ctcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctac

acacgtgctacaatggcgcatacaaagagaagcgacctcgcgagagcaagcggacctcataaagtgcgtcgtagtccgg

attggagtctgcaactcgactccatgaagtcggaatcgctagtaatagtggatcagaatgccacggtgaatacgttcccgggc

cttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtatagagcagactgatcgctcagctagagcactg

tcgacccgcga




Chapter 2

75


aggggcggcatgtactgcagcatgatgtcgtacattgctgtctgtaagtggtgcgaatgcctcggggggggggatggtggaa

agggcgaggggtcgctagtggggcaaaactgatgaacgcaaaactcgtagagagagaggggcctttcggctctttcatcg

atgtgtcatgaggctgagttggtggtaggtaccgcttcttgaagcgcgttcctagctggtgtggccctttctagcctcctggatcat

ggccgggtccgcattataggaggcggcgttggagtatggttgaggggcggggggatgctgcattgagcggggattgttgagt

ttttggtttctggtcctttattgggtggaagaggtgaggatatagtgtatttttctgttttggcgagaaaaggattgtctctcatgcactg

ggtgtaaaacgggggagttcgttatttttattttgagggagagtgacctcgtttttggttcacgtaattaagtgctggaatcggggg

ccgggctctgagaggggaattagcactccgcccccccccccaccccctatagtttgttactagctcaaattgaaagctggcgg

caagcctaaacatcaagtgaacgtattattatatatttgtttcaaatccggcccgccccccaggagtaatttctggaaaccgcca

gccgtgtgggataacacccgaaacggtagttattccgcatacgtggtaagacctatgtgggggaccttctggcctcttggcatc

tgatgagccgaagatgggattagctagtttgttggagtaacggctcaactaggcgacgattctagctggtccgagaggatgac

cagcacacttgaactgagaccccgtccagactcctacgggagcagcagtgggaatattgcacaatgggcgcaagcctgat

gcagccatccgcgcgtatgaagaaaggcctccgggttgtaaagtactttcagcggggaggaagagagtaaagataatatct

ttgctcattgacgtcccccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgtt

aatcggaattactgggcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgggctcaacctgggaactgc

atctgatactggcaagcttgagtctcgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaa

taccggtggcgaaggcggccccctggacgaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagata

ccctggtagtccacgccgtaaacgatgtcgacttggaggttgtccttgaggcgtggctctagagctaacgcgcaaggtcgacc

gcctgggagtacggccgcaaggttaaacctcaaatgaattgacgggggccgcacaagcggtggagcatgtggtttaatttcg

atgcaacgcgaagaaccttacctggtcttgacatccacggaagttttcagagatgagaatgtgcttcgggaaccgtgagaca

ggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatccctttgttgccag

cggtccggccgggaactcaaaggagactgccagtgataacctggaggaaggtggggatgacgtcaagtcatcatggccct

tacgaccagggctacacacgtgctacaatggcgcatacaaagagaagcgacctcgcgagagcaagcggacctcataaa

gtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatcagaatgccacggt

gaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggtgcaaaagaagtagagagaggagcgatc

gagcgtgctagtgcagctgtccacctggga




Chapter 2

76


ccttatatattttgttatctctctagatgaacgctggcggcaggcctacacatgcaatcgacggttactagatagcagcttgcgcttt

gcggcgagtggcggacgggtaagtaatttcttgggaaccgcccgttgagggggataactacctgaaacggtagcctatacc

gcatacgtcgcaagaccaaagagggggacttcgggcctcttgccatcggatgtgcccagatgggattagctagtaggtggg

gtaacggctcacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaactgagacacggtccagac

cctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtatgaagaaggctt

cgggttgtaaagtactttcagcggggaggaagggagtaaagttaatacctttgctcattgacgttacccgcagaagaagcac

cggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttatcggaattactgggcgtaaagggcacgcag

ggcggtttgttaagtcagatgtgaaatccccgggctcaaccctggggaactgcatctgatactggcaagcttgagtctcgtaga

ggggggtagaattcccaggtgtagcggtgaaatgcgtagagatctggagaaataccggtggcgaagggcggccccctgg

acgaagactgacgctcaggtggaaaagcgtggggagcaaaacaggattagataccggtagtccacgccgtaaacgatgt

cgactggaggttgtccctgagcgttatatatgagttatcgctgttagatctgcagttctcacttcaccatgctgtcgtaacaagtac

ataactgtctctacatggttgggggtcgggtggaacgctgggggggtgattctgcgaaaagacaaaaacgcaaacggcga

agagaagggaggggcttttgggatccggcttcggtgtgttcagaggtatgagtgatctgggggggtacggctcattgagggg

cgattcctagctgggctgagaagttacgagcacccttgatgtgttgacggtccagctcttactgagggagtcgcgggagtacgt

ctagggcggcggtatcggtggaagaggggttgaggggtttttggtgttaaaaccttatttggggggagaggaaggtgaaatttt

gtttggatgtccggaaaaaagttcggtatctaatcttccgggccgggaaaaacgggggtggattttttattggaatttaatggacc

aaatgcccccccgggtgggtttggccagcccaaaatttaaatcccccgtccccccggggaaattcccccccagacggggga

aatttgagtctttcagaggggggcaaaatttcaggggaacccctaaaaagcgaaatgtttttgggggaaaaccggggggga

aaacggcccctgtcccaaacaggctccccgggcgaaaagttgtgaggaaaaagggtagattccccgggggtcccccccc

aaaacaatgctgattttgaagtttgccccttgagcgggcctttccgaagaaactcgtttaagccaacccctgggggaatttcccc

cccaggtaaaaaatataaaaaatttaatgggccccccgccagggggggggtttgtggttatattttcacgcgagcggtgttagt

cagatgtaatcctcgataacctgactgcatggtctgcagctgagttcgtggggaggtgaatcagctaccgtgaatgctaagatc

tgagatccgtgggagcgtcctgacgaagactgatgtcagtgagaagcgtggagcaacagattagatacctcgtagtcacgc

cgtaaacatgtcgacttgaggttgtgccttaagcgtgcttccggactaacgcgttaatcgaccgcctgggagtacggccgcaa

ggttaaaactcatatgaattgacgggggccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttac

ctggtcttgacatccacggaagttttcagagatgagaatgtcccttcgggaaccgtgagacaggtgctgcatggctgtcgtcag

ctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggccgggaactcaaa

ggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgacccagggctacacacg

tgctacaatggcgcatacaaagagacccgacctcgcgagagcaagcggacctcataaagtgcgtcgtagtccggattgga

gtctgcaactcgactccatgaagtcggaatagctagtaatcgtggatcagaatgccacggtgaatacgttcccgggccttgta

cacaccgcccgtccaccatgggagtgggcgcaaacgacttagagtagatcatgctcagatgaacgctgtcagtgca




Chapter 2

77

2.3.2. Antibacterial efficacy of plant extracts against pathogens

Plant extract MIC ranged from 7.81 mg/L to 1000 mg/L (Figure 2.1). Plant

extract MBC ranged from 31.25 mg/L to 1000 mg/L (Figure 2.2) against Gram-

positive bacteria such as L. monocytogenes and Gram-negative bacteria

including S. enteritidis, E. coli, and C. jejuni. A large proportion of plant extracts

inhibited growth (29 of 40; 72.5%) (Figure 2.1) of and killed (12 of 29; 72.5%)

(Figure 2.2) at least one of the following, L. monocytogenes, S. enteritidis, E.

coli, and C. jejuni with an MIC and MBC of ≤1000mg/L. The majority of plant

extracts therefore exhibited effective antibacterial activity against at least one

test pathogen. Furthermore, broad spectrum antibacterial activity was

indicated by 20 of 40 (50%) plant extracts through the inhibition of growth of

C. jejuni, E. coli, L. monocytogenes, and S. enteritidis at an MIC ≤1000 mg/L.

These included A. pilosa Ledeb; A. macrostemon Bunge; S. glabra Roxb; A.

chinensis Bunge; and I. domestica (L.) Goldblatt and Mabb. Eleven of these

plant extracts exhibited an MBC four-fold in comparison to their MIC and are

therefore bactericidal as they reduce any detectable amount of pathogen

strains at concentrations close to their MIC. Broad spectrum bactericidal

antibacterial activity was indicated by 7 of 40 (17.5%) plant extracts. These

plant extracts exhibited bactericidal activity against C. jejuni, E. coli, L.

monocytogenes and S. enteritidis isolates at ≤1000 mg/L. These plant extracts

included: A. pilosa Ledeb; S. glabra Roxb; A. chinensis Bunge; I. domestica

Chapter 2

78

(L.) Goldblatt and Mabb; O. diffusa (Willd.) Roxb; F. chinensis Roxb; and L.

erythrorhizon Siebold and Zucc.

The majority of plant extracts (25 of 40; 62.5%) inhibited C. jejuni at MIC ≤1000

mg/L. A significant proportion of plant extracts inhibited S. enteritidis; L.

monocytogenes, and E. coli, including: 23 of 40 (57.5%); 22 of 40 (55%); and

21 of 40 (52.5%), respectively (Figure 2.1). The majority of plant extracts (10

of 40; 25%) resulted in no detectable E. coli at MBC ≤1000 mg/L. A significant

proportion of plant extracts resulted in no detectable L. monocytogenes, C.

jejuni, and S. enteritidis, including: 9 of 40 (22.5%); 7 of 40 (17.5%); and 7 of

40 (17.5%), respectively (Figure 2.2). There was a lower proportion of plant

extracts with MBC ≤1000 mg/L compared to plant extracts with MIC ≤1000

mg/L (Figure 2.3).

A relatively small proportion of plant extracts (11 out of 40; 27.5%) did not

inhibit C. jejuni, E. coli, L. monocytogenes, and S. enteritidis isolates at ≤1000

mg/L. This was beyond the detectable limit for antibacterial activity in this

study. Overall, the plant extracts which exhibited the most potent significant

antibacterial activity against C. jejuni, L. monocytogenes, S. enteritidis, and E.

coli are A. chinensis Bunge, I. domestica (L.) Goldblatt and Mabb, A. pilosa

Ledeb and S. glabra Roxb (P <0.001) (Tables 2.22 to 2.25).

MIC and MBC of quality control of ampicillin against S. pneumoniae ATCC

49619 was 0.06 mg/L and 0.24 mg/L, respectively. MIC and MBC of quality

control of erythromycin against S. aureus ATCC 29213 was 0.5 and 4 mg/L,

respectively. These values are in accordance with (EUCAST, 2018). This

validates the method showing that it was done in accordance with EUCAST

Chapter 2

79

(2018) standards. The MIC values of ampicillin were: 0.25 mg/L to 1 mg/L

against L. monocytogenes; 2 mg/L to 8 mg/L against S. enteritidis; and 4 mg/L

to 8 mg/L against E. coli. The MIC values of erythromycin were 2 mg/L to 4

mg/L against C. jejuni

Figure 2.1. The proportion of plant extracts which exhibit an MIC within categories ranging

from 7.81 mg/L to >1000 mg/L against at least one isolate of C. jejuni, L. monocytogenes, S.

enteritidis and E. coli.

C. jejuni L. monocytogenes S. enteritidis E. coli

Figure 2.2. The proportion of plant extracts which exhibit an MBC within categories ranging

from 7.81 mg/L to >1000 mg/L against at least one isolate of C. jejuni, L. monocytogenes, S.

enteritidis and E. coli.

0

2

4

6

8

10

12

14

16

18

20

7.81 31.25 62.5 125 250 500 1000 >1000

Qu

an

tity

of

pla

nt

extr

acts

MIC (mg/L)

Chapter 2

80

C. jejuni L. monocytogenes S. enteritidis E. coli

Figure 2.3. The proportion of plant extracts which exhibit an MIC and MBC ranging from 7.81

mg/L to >1000 mg/L against at least one isolate of C. jejuni, L. monocytogenes, S. enteritidis

and E. coli.

0

5

10

15

20

25

30

35

7.81 31.25 62.5 125 250 500 1000 >1000

Qu

an

tity

of

pla

nt

extr

acts

MBC (mg/L)

0

5

10

15

20

25

30

35

7.81 31.25 62.5 125 250 500 1000 >1000

Quantitiy o

f pla

nt

extr

acts

MIC and MBC (mg/L)

C. jejuni MIC L. monocytogenes MIC S. enteritidis MIC E. coli

MIC

C. jejuni MBC L. monocytogenes MBC S. enteritidis MBC E. coli

MBC

Chapter 2

81

2.3.2.1. Antibacterial efficacy of plant extracts against C. jejuni

The plant extracts which exhibited MIC and MBC values ≤1000 mg/L against

C. jejuni are presented (Table 2.22). A. chinensis Bunge exhibited significant

antibacterial activity against every C. jejuni isolate (P <0.001) with the lowest

MIC (62.5 mg/L) and MBC (250 mg/L). I. domestica (L.) Goldblatt and Mabb

and A. pilosa Ledeb also exhibited significant antibacterial activity against

every C. jejuni isolate (P <0.001) with a comparatively low MIC range (31.25

mg/L to 500 mg/L) and MBC (500 mg/L to >1000 mg/L) (Table 2.22).

Chapter 2

82

Table 2.22. MIC (mg/L) and MBC (mg/L) of plant extracts against C. jejuni

Extract C. jejuni

NCTC 11322 RC152 RC104 RC179

MIC MBC MIC MBC MIC MBC MIC MBC

Erythromycin 4a 12a 2a 8a 2a 8a 4a 12a

A. pilosa Ledeb 31.25a 500b 31.25a 500b 125b >1000d 500d 500b

A. macrostemon Bunge 125bc >1000d 125bc >1000d 250c >1000d 125b >1000d

S. glabra Roxb 250c 1000c 250c 1000c 250c 1000c 250c 1000c

A. chinensis Bunge 62.5b 250b 62.5b 250b 62.5b 250b 62.5b 250b

I. domestica (L.) Goldblatt and Mabb 125bc 500b 125bc 500b 125b 500b 125b 500b

O. diffusa (Willd) Roxb 500d 1000c 500d 1000c 500d >1000d 500d 1000c

S. cuneata (Oliv.) Rehder and E. H. Wilson 500d >1000d 500d >1000d 500d >1000d 1000e >1000d

P. suffructicosa Andrews 500d >1000d 500d >1000d 500d >1000d 1000e >1000d

C. moriflorum Ramat 250cd 1000c 250cd 1000c 250c 1000c 250c 1000c

S. polyrrhiza (L.) Schleid 250cd 1000c 250cd 1000c 250c 1000c 250c 1000c

L. erythrorhizon Siebold and Zucc 250cd 1000c 250cd 1000c 250c 1000c 250c 1000c

O. japonicus (Thunb) Ker-Gawl 500d >1000d 500d >1000d 500d >1000d 500d >1000d

G. scabra Bunge 500d >1000d 500d >1000d 500d >1000d 500d >1000d

L. japonica Thunb 1000e >1000d 1000e >1000d >1000f >1000d >1000f >1000d

P. nervosa (Thunb.) 500d >1000d 500d >1000d 500d >1000d 500d >1000d

L. tinctoria L 1000e >1000d 1000e >1000d 1000e >1000d 1000e >1000d

L. christinae Hance 1000e >1000d 1000e >1000d >1000f >1000d 500d >1000d

P. lactiflora Pall 500d >1000d 250cd >1000d >1000f >1000d 500d >1000d

A. propinquus Schischkin >1000f >1000d 1000e >1000d 1000e >1000d >1000f >1000d

C. chinensis Franch 1000e >1000d >1000f >1000d 1000e >1000d 1000e >1000d

C. falcatum (L.F.) C. Presl 1000e >1000d 1000e >1000d 1000e >1000d 1000e >1000d

S. tonkinensis Gagnep >1000f >1000d 500d >1000d >1000f >1000d >1000f >1000d

R. palmatum L 1000e >1000d 1000e >1000d 1000e >1000d 1000e >1000d

C. lacryma-jobi L 1000e >1000d 1000e >1000d 1000e >1000d 1000e >1000d F. chinensis Roxb 1000e >1000d 1000e >1000d 1000e >1000d 1000e >1000d

Values are the mean rounded to the nearest well of one experiment in triplicate. Data with the same letter are not significantly different from each other

(P <0.001) within one column. For example, MIC 4a is not significantly different to 31.25a and 125bc is not significantly different to 250c, and 62.

Chapter 2

83

2.3.2.2. Antibacterial efficacy of plant extracts against L. monocytogenes


L. monocytogenes are presented (Table 2.23). The most potent and effective

antibacterial activity against L. monocytogenes of all antibacterial plant

extracts was demonstrated by S. glabra Roxb and A. pilosa Ledeb (P <0.001).

S. glabra Roxb and A. pilosa Ledeb exhibited significant antibacterial activity

against every L. monocytogenes isolate (P <0.001) with the lowest MIC range

of 31.25 mg/L to 250 mg/L and MBC of 250 mg/L to 1000 mg/L (Table 2.23).

Chapter 2

84

Table 2.23. MIC (mg/L) and MBC (mg/L) of plant extracts against L. monocytogenes

Extract L. monocytogenes

NCTC 11994 LS12519 OT11230 CP102 CP1132 QA1018

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

Ampicillin 0.25a 2a 0.5a 4a 0.5a 4a 0.25a 2a 0.25a 2a 1a 4a

A. pilosa Ledeb 31.25ab 250b 31.25a 250b 62.5b 1000c 31.25ab 250b 125b 1000d 31.25a 250b

A. macrostemon Bunge 62.5bcd >1000d 62.5b >1000e 62.5b >1000d 62.5b >1000e 1000e 1000d 31.25a >1000d

S. glabra Roxb 62.5bcd 250b 31.25a 250b 125c 1000c 31.25ab 250b 31.25a 250b 31.25a 250b

A. chinensis Bunge 125cd 500bc 125c 500c 125c 500b 125c 500c 125b 500c 125b 500b

I. domestica (L.) Goldblatt and Mabb 125cd 500bc 125c 500c 125c 500b 125c 500c 125b 500c 125b 500b

O. diffusa (Willd) Roxb 125cd 500bc 125c 500c 500e 1000c 125c 500c 125b 500c 125b 500b

S. cuneata (Oliv.) Rehder and E. H. Wilson 125cd >1000d >1000g >1000e >1000g >1000d 62.5b >1000e 250c >1000e >1000 >1000d

P. suffructicosa Andrews 62.5bcd >1000d 125c >1000e 250d >1000d 250d >1000e 500d >1000e 250c >1000d

C. moriflorum Ramat 125cd 500bc 125c 500c 125c 500b 125c 500c 125b 500c 125b 500b

S. polyrrhiza (L.) Schleid 125cd 1000c 125c 1000d 125c 1000c 125c 1000d 125b 1000d 125b 1000c

L. erythrorhizon Siebold. and Zucc 125cd 500bc 125c 500c 125c 1000c 125c 500c 125b 500c 125b 500b

E. brevicornum Maxim 125cd >1000d 250d >1000e 125c >1000d 125c >1000e 125b >1000e 500d >1000d

O. japonicus (Thunb) Ker-Gawl 125cd 1000c 125c 1000d 250d 1000c 250d 1000d 250c 1000d 125b 1000c

G. scabra Bunge 500f >1000d 500e >1000e 1000f >1000d 500e >1000e 500d >1000e 500d >1000d

L. japonica Thunb 250e >1000d 250d >1000e 250d >1000d >1000g >1000e 250c >1000e >1000f >1000d

P. nervosa (Thunb.) 500f >1000d 500e >1000e 1000f >1000d 500e >1000e 500d >1000e 500d >1000d

L. tinctoria L 250e >1000d 250d >1000e 500e >1000d >1000g >1000e 500d >1000e 250c >1000d

L. christinae Hance 250e >1000d 250d >1000e 500e >1000d >1000g >1000e >1000f >1000e >1000f >1000d

A. propinquus Schischkin 250e >1000d 1000f >1000e 1000f >1000d 500e >1000e >1000f >1000e 500d >1000d

C. falcatum (L.F.) C. Presl 500f >1000d 500e >1000e 500e >1000d 500e >1000e 500d >1000e 500d >1000d

S. tonkinensis Gagnep 1000g >1000d 1000f >1000e 500e >1000d 1000f >1000e 1000e >1000e 1000e >1000d

C. lacryma-jobi L 1000g >1000d 1000f >1000e >1000g >1000d 1000f >1000e 1000e >1000e 1000e >1000d

F. chinensis Roxb >1000h >1000d >1000g >1000e 1000f >1000d 1000f >1000e 1000e >1000e 1000e >1000d

Values are the mean rounded to the nearest well of one experiment in triplicate. Data with the same letter are not significantly different among each other

(P <0.001) for one column. For example, MIC 0.25a is not significantly different to 31.25ab.

Chapter 2

85

2.3.2.3. Antibacterial efficacy of plant extracts against S. enteritidis

Plant extracts which exhibited MIC and MBC ≤1000 mg/L against S. enteritidis

are presented (Table 2.24). A. chinensis Bunge exhibited significant

antibacterial activity against every tested S. enteritidis isolate (P <0.001) with

the lowest MIC of 62.5 mg/L and MBC of 250 mg/L. O. diffusa (Willd) Roxb, I.

domestica (L.) Goldblatt and Mabb, and S. glabra Roxb exhibited significant

antibacterial activity against the majority of tested S. enteritidis isolates

(P <0.001) with a low MIC range of 62.5 mg/L to 250 mg/L and an MBC range

of 500 mg/L to >1000 mg/L (Table 2.24).

Chapter 2

86

Table 2.24. MIC (mg/L) and MBC (mg/L) of plant extracts against S. enteritidis

Extract S. enteritidis

NCTC 0074 IF6144 LE103 QA0419


Ampicillin 4a 16a 2a 8a 4a 16a 8a 32a

A. pilosa Ledeb 500d 1000c 125b 1000d 125b 1000c 125b 1000c

A. macrostemon Bunge 62.5a >1000d 62.5b >1000e 62.5ab >1000d 62.5ab >1000d

S. glabra Roxb 250c >1000d 125b 1000d 125b 1000c 125b 1000c

A. chinensis Bunge 62.5a 250b 62.5b 250b 62.5ab 250b 62.5a 250b

I. domestica (L.) Goldblatt and Mabb 250c 1000c 125b 500c 125b 500b 125b 500b

O. diffusa (Willd) Roxb 62.5a >1000d 125b 500c 125b 500b 125b 500b

S. cuneata (Oliv.) Rehder and E. H. Wilson 500d >1000d 500d >1000e 500d >1000d 500d >1000d

P. suffructicosa Andrews 250c >1000d 250c >1000e 250c >1000d 250c >1000d

C. moriflorum Ramat 500d 1000c 250c 1000d 250c 1000c 250c 1000c

S. polyrrhiza (L.) Schleid 500d >1000d 500d >1000e 500d >1000d 500d >1000b

L. erythrorhizon Siebold. and Zucc 125b 500b 125b 500c 125b 500b 250c 1000c

E. brevicornum Maxim 1000e >1000d 1000e >1000e 1000e >1000d 1000e >1000d

O. japonicus (Thunb) Ker-Gawl 1000e >1000d 1000e >1000e 1000e >1000d 1000e >1000d

G. scabra Bunge 500d >1000d 500d >1000e 500d >1000d 500d >1000d

L. japonica Thunb >1000f >1000d 1000e >1000e 1000e >1000d 1000e >1000d

P. nervosa (Thunb.) 500d >1000d 500d >1000e 500d >1000d 500d >1000d

L. tinctoria L 1000e >1000d 1000e >1000e 1000e >1000d 1000e >1000d

L. christinae Hance >1000f >1000d >1000f >1000e >1000f >1000d >1000f >1000d

G. uralensis Fisch 250c >1000d >1000f >1000e >1000f >1000d >1000f >1000d

C. falcatum (L.F.) C. Presl 500d >1000d 500d >1000e 500d >1000d 500d >1000d

S. tonkinensis Gagnep 500d >1000d 500d >1000e 500d >1000d 500d >1000d

C. lacryma-jobi L 1000e >1000d 1000e >1000e 1000e >1000d 1000e >1000d


(P <0.001) for one column. For example, MIC 4a is not significantly different to 62.5a.

Chapter 2

87

2.3.2.4. Antibacterial efficacy of plant extracts against E. coli


E. coli are presented (Table 2.25). A. pilosa Ledeb exhibited significant

antibacterial activity against every tested E. coli isolate (P <0.001) with the

lowest MIC of 7.81 mg/L and MBC of 31.25mg/L. A. chinensis Bunge and I.

domestica (L.) Goldblatt and Mabb exhibited potent significant antibacterial

activity against every tested E. coli isolate (P <0.001) with comparatively low

MIC and MBC values ranging from MIC of 62.5 mg/L to 125 mg/L and MBC of

500 mg/L (Table 2.25).

Chapter 2

88

Table 2.25. MIC (mg/L) and MBC (mg/L) of plant extracts against E. coli

Extract Pathogen

E. coli

ATCC 25922 UM004 UM011 UM012


Ampicillin 8a 16a 4a 16a 4a 16a 4a 16a

A. pilosa Ledeb 7.81a 31.25a 7.81a 31.25a 7.81a 31.25a 7.81a 31.25a

A. macrostemon Bunge 31.25a 500c 31.25a >1000e 31.25a >1000d 31.25a >1000d

S. glabra Roxb 125ab 500c 125ab 500c 125ab 500b 125ab 500b

A. chinensis Bunge 125ab 500c 125ab 500c 125ab 500b 125ab 500b

I. domestica (L.) Goldblatt and Mabb 62.5a 500c 62.5a 500c 62.5a 500b 62.5a 500b

O. diffusa (Willd) Roxb 250bc 250b 250b 250b 250bc 500b 250bc 500b

S. cuneata (Oliv.) Rehder and E. H. Wilson 250bc >1000e 250b >1000e 250bc >1000d 250bc >1000d

P. suffructicosa Andrews 250bc >1000e 250b >1000e 250bc >1000d 250bc >1000d

C. moriflorum Ramat 125ab 500c 125ab 500c 125a 500b 125ab 500b

S. polyrrhiza (L.) Schleid 500c >1000e 500c >1000d 500c >1000d 500c >1000d

L. erythrorhizon Siebold. and Zucc 125ab 500c 125ab 500c 125ab 500b 125ab 500b

E. brevicornum Maxim 250bc 1000d 250b 1000d 250bc 1000c 250bc 1000c

O. japonicus (Thunb) Ker-Gawl 250bc 1000d 250b 1000d 250bc 1000c 250bc 1000c

G. scabra Bunge 250bc >1000e 500c >1000e 500c >1000d 500c >1000d

L. japonica Thunb 500c >1000e 500c >1000e 500c >1000d 500c >1000d

P. nervosa (Thunb.) 250bc >1000e 250b >1000e 250bc >1000d 250bc >1000d

L. tinctoria L 500c 1000d 500c 1000d 500c 1000c 500c 1000c

L. christinae Hance 500c >1000e >1000 >1000e >1000e >1000d >1000e >1000d

P. lactiflora Pall 1000d >1000e 1000d >1000e 1000d >1000d 1000d >1000d

C. chinensis Franch 500c >1000e 500c >1000e 500c >1000d 500c >1000d

C. falcatum (L.F.) C. Presl 1000d >1000e 1000d >1000e 1000d >1000d 1000d >1000d

S. tonkinensis Gagnep 1000d >1000e 1000d >1000e 1000d >1000d 1000d >1000d

C. lacryma-jobi L 1000d >1000e 1000d >1000e 1000d >1000d 1000d >1000d


(P <0.001) for one column. For example, MIC 8a is not significantly different to 7.81a, 31.25a, 62.5a, and 125ab.

Chapter 2

89

2.4. Discussion

2.4.1. Purpose of study and methods

The purpose of this in vitro study was to search and identify plant extracts with

potent antibacterial properties against isolates of L. monocytogenes (Gram-

positive), E. coli, C. jejuni, and S. enteritidis (Gram-negative).

In this study most of the plant extracts were selected because they had

demonstrated antibacterial activity against several human pathogens including

L. monocytogenes, S. enteritidis, C. jejuni, and E. coli (Table 2.1). Therefore,

it was anticipated that a number of the plant extracts in this in vitro study would

demonstrate antibacterial activity against L. monocytogenes, S. enteritidis, C.

jejuni, and E. coli. This was the first study to investigate these crude plant

extracts against all four bacteria and some plants were specifically chosen to

address a gap in the scientific literature as there appeared to be no research

investigating their antibacterial properties.

The broth microdilution method was chosen because it was recommended in

the official guidelines (CLSI, 2009). The plant extracts were prepared by an

ultrasonic assisted boiling water extraction method. This method is frequently

used to extract plant compounds. Water was used as a solvent rather than

other organic solvents because it reduces the risk of solvent toxicity, while

potentially providing a comparable extraction yield to, for example methanol

(Truong et al., 2019).

Negative, positive, and method validation control experiments were conducted

in parallel with the plant extracts antibacterial testing. This enhanced

experimental validity and provided benchmarks for comparison with plant

Chapter 2

90

extracts results for antibacterial activity (EUCAST, 2018). Ampicillin and

erythromycin were selected and used as positive controls. The lowest MIC was

0.25 mg/L and 2 mg/L, respectively, which is within the expected range.

The results show using the MIC/MBC cut off value of ≤1000 mg/L that just over

one quarter of the plant extracts did not inhibit or kill C. jejuni, E. coli, L.

monocytogenes and S. enteritidis isolates. In one sense this was a limitation

of the method as 11 out of the 40 plant extracts may have exhibited

antibacterial activity at higher concentrations above the detectable limit.

However, this cut off value was necessary as increasing the concentration of

plant extracts above 1000 mg/L would result in having to increase ratio of dry

plant material in the poultry feed mix in the in vivo trial. This would also

increase the cost of the in vivo trial and possibly the poultry nutrient uptake

from replacing too much feed with carefully calculated nutrients with dry plant

material.

2.4.2. Plants that possess strong antibacterial properties

A. pilosa Ledeb, S. glabra Roxb, A. chinensis Bunge, and I. domestica (L.)

Goldblatt and Mabb demonstrated strong significant antibacterial activity

through low MIC and MBC values against Gram-negative bacteria (E. coli, and

S. enteritidis), and Gram-positive bacteria (L. monocytogenes). The most

effective antibacterial activity against E. coli was exhibited by A. pilosa Ledeb

(lowest MIC of 7.81 mg/L). This activity was comparable to the antibacterial

activity of ampicillin against E. coli (lowest MIC of 4 mg/L) and therefore

demonstrated potent antibacterial activity

Chapter 2

91

Following these, A. chinensis Bunge, I. domestica (L.) Goldblatt and Mabb,

and S. glabra Roxb all demonstrated strong antibacterial activity against E.

coli, L. monocytogenes, and C. jejuni. The most potent antibacterial activity

against S. enteritidis was exhibited by A. chinensis Bunge (lowest MIC of 62.5

mg/L). Following these plants, strong antibacterial activity against S. enteritidis

was exhibited by A. pilosa Ledeb, I. domestica (L.) Goldblatt and Mabb, and

S. glabra Roxb.

A review of the literature suggested crude extracts of A. pilosa Ledeb, S. glabra

Roxb, A. chinensis Bunge, and I. domestica (L.) Goldblatt and Mabb did not

appear to have been previously screened for antibacterial activity against C.

jejuni, S. enteritidis, L. monocytogenes or E. coli and therefore there are very

few results for comparison.

Two studies reported no antibacterial activity of A. pilosa (Franzblau and

Cross, 1986; Kim et al., 2011) or I. domestica (L.) Goldblatt and Mabb

(Franzblau and Cross, 1986). This contrasted with this research and the

difference in findings could be due to the different methods used. Both studies

used the disc diffusion method to screen for antibacterial activity whilst this

research used the broth microdilution method. The advantages of the broth

microdilution method over the diffusion method include improved sensitivity for

quantitative analysis (Nasir et al., 2015). The broth microdilution method also

produces accurate reliable quantitative results (Klančnik et al., 2010; Balouiri

et al., 2016). The improved sensitivity of the broth microdilution method used

in this study may have allowed for the detection of antibacterial activity in the

extracts of these plants. A large amount of antibacterial compounds are non-

polar and this reduces their ability to diffuse in aqueous agar which can lead

Chapter 2

92

to inconsistencies in the reproducibility of results in studies using the disc

diffusion method (Eloff, 2019). Another study by Xu et al., (2013) also used the

broth microdilution method to establish the antibacterial effect of solvent

extracts of S. glabra Roxb against E. coli. The study by Kim et al., (2011b)

used different solvents to obtain plant extracts. Franzblau and Cross, (1986)

used water extraction and lypophilised the extract. By contrast, in this study

plants extracts were boiled then subjected to ultrasonication. Different

extraction procedures can result in different bioactive chemical compositions

of crude extracts of plants (Seidel, 2012). Ultrasound causes acoustic

cavitation which increases the surface contact between the solvent, samples,

and cell wall permeability. This enhances the mass transport of the solvent in

to the plant cells and enhances the release of compounds (Toma et al., 1999;

Azwanida, 2015) and this may explain the potent antibacterial activity

demonstrated in this research.

The broad-spectrum antibacterial activity of A. pilosa Ledeb, S. glabra Roxb,

A. chinensis Bunge, and I. domestica (L.) Goldblatt and Mabb may be the

result of the synergistic action of multiple bioactive compounds. For example,

A. chinensis Bunge contains saponins and quercetin (Kumar et al., 2008) and

demonstrate antibacterial activity against several pathogens such as S.

enteritidis (N. K. Lee et al., 2016; Khan et al., 2018). The chemical composition

of A. pilosa Ledeb contains phenol derivatives and catechin. S. glabra Roxb is

also rich in catechins and I. domestica Goldblatt and Mabb is rich in phenol

derivatives. These secondary metabolites exhibit antibacterial activity against

S. aureus. (Yamaki et al., 1989; Kasai et al., 1992; Zhang et al., 2016; Hua et

al., 2018). These secondary metabolites were reported to exert antibacterial

Chapter 2

93

activity against L. monocytogenes and E. coli and may account for the

inhibition of these pathogens’ growth in this study (Skroza et al., 2019; Diarra

et al., 2020).

2.4.3. Plants that possess weak to moderate antibacterial properties

The current results demonstrated moderate antibacterial activity of Lonicera

japonica Thunb. and C. morifolium Ramat against L. monocytogenes and this

supported previous results (Shan et al., 2007; Rahman and Kang, 2009).

Moderate antibacterial activity demonstrated by G. uralensis Fisch, and L.

erythrorhizon Siebold and Zucc against S. enteritidis also supported previous

results (Bae, 2004; Sun et al., 2020). This study also highlighted the moderate

activity of A. macrostemon Bunge, L. erythrorhizon Siebold and Zucc, P.

suffructicosa Andrews, and the weak antibacterial activity of L. japonica Thunb,

C. chinensis Franch, P. lactiflora Pall, and S. polyrhiza (L.) against E. coli. This

supported and contributed to the verification of previous results (Franzblau and

Cross, 1986; Bae, 2004; Ji-Hyun and Mee-Aae, 2005; Rahman and Kang,

2009; Luong et al., 2012; Xiong et al., 2013; Yang et al., 2013).

Numerous studies above reported similar findings of plants studied. However,

given the lack of published research literature on many of the plant extracts it

did not seem reasonable to make claims about the consistency of results as

often there may be only one or two papers on the antibacterial activity of a

plant extract on a pathogen. This and the fact that there is no one standardised

method for screening plant extracts for antibacterial activity in general helps to

Chapter 2

94

explain inconsistent results in in vitro studies and in particular the differences

in the findings between this study and others.

The composition of secondary metabolites varies across plant species and in

separate plant parts within the same species and this may explain the range

of antibacterial activity of these different plant extracts. Several studies

investigated the chemical composition of the plant extracts and suggested they

are strongly associated with antibacterial activity and inhibition of pathogen

growth. For example, Lonicera japonica Thunb was found to have phenolic

and oxygenated sesquiterpene compounds (Rahman et al., 2014). C.

morifolium contained phenolic compounds (Shan et al., 2007). C. chinensis

Franch and P. suffructicosa Andrews contained flavonoid and phenolic

compounds (Yang et al., 2013) while S. polyrhiza (L.) Schleid had alkaloid and

steroid compounds (Das et al., 2012).

The modes of action of plant extracts against a particular bacterial pathogen

depends on their constituent proportions of secondary metabolites (Patra,

2012; Omojate et al., 2014). This could explain the differences in antibacterial

activity towards different bacteria. Plant extracts are also likely to exhibit

multiple modes of action against bacterial pathogens (Wink, 2015). It is

probable that the broad spectrum antibacterial activity of plant extracts is due

to synergistic effects of a combination of different secondary metabolites,

which may not be detected by analysing a single compound (Elston, 2009;

Wink, 2015). Unlike antibiotics which commonly consist of a single bioactive

agent with a single mode of action plant extracts have a complex composition

consisting of a collection of compounds with multiple modes of action and

multiple targets (Schmidt et al., 2010). Due to the multiple modes of action of

Chapter 2

95

plant extracts it has been proposed that fewer changes are needed for bacteria

to acquire resistance to antibiotics than to plant extracts (Silver and Bostian,

1993). These plants that possess antibacterial properties may be potential

candidates to be studied further as alternatives to antibiotics.

2.5. Conclusion

This study identified four plant extracts with strong broad-spectrum

antibacterial activity including, A. pilosa Ledeb, S. glabra Roxb, A. chinensis

Bunge, and I. domestica (L.) Goldblatt and Mabb. Furthermore, the

antibacterial activity of A. pilosa Ledeb was comparable to erythromycin and

ampicillin. This highlights the potent activity of these plants and the potential

benefits of investigating these plants further for their antibacterial properties

and suitability for their inclusion in poultry feed. Experiments that determine

the effect of A. pilosa Ledeb, S. glabra Roxb, A. chinensis Bunge, and I.

domestica (L.) Goldblatt and Mabb at different time points and concentrations

would be useful in quantifying their antibacterial activity further.

Chapter 3

96

3. Chapter 3

In vitro synergistic antibacterial activity of plant extracts and antibiotics

against Campylobacter jejuni, Listeria monocytogenes, Escherichia coli

and Salmonella enterica subsp. enterica serovar Enteritidis.

3.1. Introduction

This study builds on the findings from the in vitro assay reported in chapter 2

which evaluated the antibacterial activity of plant extracts against E. coli, S.

enteritidis, C. jejuni and L. monocytogenes. Previous studies of the

antibacterial activity of combinations of different plant extracts and in

combination with antibiotics have reported additive and synergistic effects.

These combinations could potentially exhibit a stronger inhibitory effect on

pathogens than any one the combination’s individual constituents (Poe, 1976;

Abreu et al., 2012). This potentially could lead to a reduction in the use of

antibiotics in feed supplements.

Poultry farming is one of the most rapidly growing global markets within

agriculture. The nature of poultry farming with its relatively short production

cycle, high feed conversion ratios and comparatively low costs per unit output

have contributed to its worldwide growth (Mottet and Tempio, 2017). The

demand for poultry meat is estimated to increase by 121% by 2050 compared

to 2005. Correspondingly, in developing countries, the annual growth for global

poultry meat production is estimated to increase from 2005 to 2050 by 2.4%

(Alexandratos and Bruinsma, 2012). It is also predicted that this global

increase in poultry production will contribute to one third of the overall 60%

increase in the global consumption of antibiotics in animals raised for food from

Chapter 3

97

around 63, 000 tons in 2010 to 104, 000 tons by 2030 (Van Boeckel et al.,

2015). Many countries continue to use antibiotics in poultry feed for therapeutic

use and to support growth (OIE, 2021), therefore the number of antibiotics

used is likely to increase with the increasing demand for poultry meat.

However, the increased use and misuse of antibiotics in poultry feed is one of

the main causes of the development and spread of antibiotic resistance

(Čižman, 2003; Morgan et al., 2011). This increment in antibiotic usage could

potentially cause increased bacterial resistance which is associated with the

emergence of new types of multi-resistant foodborne bacteria such as

Salmonella, Campylobacter spp., and Escherichia coli (O’Neill, 2016; Olaimat

et al., 2018). At a global level one of the main strategies to reduce the growing

threat of antibiotic resistance is to reduce and ultimately phase out the

unnecessary use of antibiotics in animal farming (WHO, 2015; O’Neill, 2016;

ICGA, 2019). Many countries continue to use antibiotics in poultry feed and

this antibiotic use is predicted to increase despite this strategy. Therefore,

although bacteria can acquire resistance when exposed to even low levels of

antibiotics (Cantón and Morosini, 2011), the gradual reduction of antibiotics in

poultry feed provides an intermediate step towards phasing out their use. This

could potentially be achieved if combinations of antibiotics and plant extracts

resulted in potent antibacterial activity. Reduced levels of antibiotics and their

antibacterial activity could be compensated for with the addition of plant

extracts and their antibacterial activity.

The purpose of this study was to conduct an in vitro assay to determine the

antibacterial efficacy of combinations of the plant extracts A. pilosa Ledeb, A.

chinensis Bunge, S. glabra Roxb and combinations of each of these plant

Chapter 3

98

extracts with ampicillin and erythromycin against E. coli, S. enteritidis, C. jejuni

and L. monocytogenes. These three plant extracts were selected because they

demonstrated the highest levels of antibacterial activity out of the four plant

extracts which exhibited strong broad-spectrum antibacterial activity including,

A. pilosa Ledeb, S. glabra Roxb, A. chinensis Bunge, and I. domestica (L.)

Goldblatt and Mabb in the in vitro assay discussed in chapter 2. Three plants

were chosen rather than four for practical considerations. If four plants extracts

were chosen for investigation in the synergy study as opposed to three plants

extracts this would have doubled the number of plant extract combinations to

test from 3 to 6, and the number of plant extract with antibiotic combinations

from 6 to 8. Therefore, these three plants were selected to provide sufficient

results to determine whether the plants should be tested individually or in

combination in further investigations. In addition, there did not appear to be

any previous research on the additive or synergistic, effects of the plant

extracts investigated in this study; A. pilosa Ledeb, A. chinensis Bunge, S.

glabra Roxb or in combination with ampicillin and erythromycin on E. coli, S.

enteritidis, C. jejuni and L. monocytogenes.

Chapter 3

99

3.1.1. Aim and objectives

The aim of this study was to evaluate the combinations of plant extracts and

combinations of plant extracts with antibiotics for possible synergistic

antibacterial activity against E. coli, S. enteritidis, C. jejuni and L.

monocytogenes. The objectives of the study were to:

• conduct an in vitro assay to assess the additive or synergistic antibacterial

activity of different combinations of extracts of A. pilosa Ledeb, A. chinensis

Bunge, S. glabra Roxb against E. coli, S. enteritidis, C. jejuni and L.

monocytogenes; and

• conduct an in vitro assay to assess the additive or synergistic antibacterial

activity of extracts of A. pilosa Ledeb, A. chinensis Bunge, S. glabra Roxb

each respectively combined with ampicillin and erythromycin against E. coli,

S. enteritidis, C. jejuni and L. monocytogenes.


3.2.1. Bacterial isolates used for screening

The procedure for sourcing and identification of bacterial isolates was the

same as in chapter 2.2.2.

3.2.2. Preparation of plant extracts

The preparation of the plant extracts was carried out in the same was as in

section 2.2.3 with one exception. The plants in this study included: the herb of

A. pilosa Ledeb; the tuber of S. glabra Roxb; and the root of A. chinensis

Bunge.

Chapter 3

100

3.2.3. Broth microdilution of individual and combined plant extracts

Extracts from A. pilosa Ledeb, A. chinensis Bunge, and S. glabra Roxb were

screened for their activity as individual extracts and in combinations using a

modified version of the broth microdilution (CLSI, 2009) against L.

monocytogenes, S. enteritidis, C. jejuni and E. coli (Figure 3.1). Pure bacteria

cultures were incubated overnight under conditions optimised for their growth

(Table 3.1). Two-fold serial dilutions were constructed of ampicillin,

erythromycin, and individual plant extracts. One hundred microlitres (50 µL +

50 µL in case of combination) (Al-Bayati, 2008) of each antibiotic concentration

(2000 mg/L to 0.06 mg/L) and concentration of plant extract (2000 mg/L to 0.98

mg/L) was individually added to single wells of a 96 well plate. The culture of

bacteria was adjusted to an optical density which was equivalent to 1x108

CFU/mL then diluted to 1x106 CFU/mL. The resulting bacterial solution (100

µL) was added to each individual well. For each of the above pathogens tested

against each plant extract the negative control included testing a

corresponding pathogen sample in broth only. The positive controls included

testing the antibacterial activity of ampicillin against each isolate of L.

monocytogenes, S. enteritidis, and E. coli, and erythromycin against each

isolate of C. jejuni (EUCAST, 2018). A further quality control to validate the

method was implemented. When L. monocytogenes, S. enteritidis, and E. coli

were tested, ampicillin was simultaneously tested against S. pneumoniae

ATCC 49619. When C. jejuni was tested, erythromycin was simultaneously

tested against S. aureus ATCC 29213 (EUCAST, 2018). The dilutions were

set up in replicates of three. The MIC was the well with the lowest

concentration of antibacterial which had no visible growth.

Chapter 3

101

Figure 3.1. Schematic diagram of the synergy assay procedure

PC was the positive control, this was ampicillin when L. monocytogenes, S. enteritidis, and E.

coli were being tested. The PC was erythromycin when C. jejuni was tested. GC was the

growth control which included broth inoculated with bacteria. 50 µL of one antimicrobial A, and

50 µL of a different antimicrobial B were individually added to single wells of a 96 well plate

with 100 µL of 1x106 CFU/mL bacterial solution.

Chapter 3

102

Table 3.1. Media and incubation conditions used for bacteria for broth microdilution


conditions

C. jejuni Mueller Hinton

fastidious (MH-F)

MH-F 41 ± 1 °C;

microaerobic

L. monocytogenes Tryptone soy and 5%

lysed horse blood

Tryptone soy and

5% lysed horse

blood

35 ± 1 °C ;5%

CO2

E. coli Mueller Hinton broth

(MHB)

Mueller Hinton

agar (MHA)

35 ± 1 °C;

ambient air

S. enteritidis MHB MHA 35 ± 1 °C;

ambient air

S. pneumoniae MH-F MH-F 35 ± 1 °C ;5%

CO2

S. aureus MHB MHA 35 ± 1 °C;

ambient air





The following formula was used to calculate the fractional inhibitory

concentration (FIC) and FIC index (FICI) for each plant extract and antibiotic

in each combination: FIC = FICA + FICB. In this formula FICA represents the

MIC of antibiotic or plant extract A in the combination divided by the MIC of

antibiotic or plant extract A, and FICB is the MIC of antibiotic or plant extract B

in the combination divided by MIC of antibiotic or plant extract B (Hall et al.,

1983).The combination of plant extracts and antibiotics was designated

synergistic when the FIC was <0.5. Additivity was determined by FIC of >0.5

to 1. Indifference was determined by an FIC of >1 to ≤4. Antagonism was

indicated when the FIC was >4 (Prinslo et al., 2008). The assays were set up

in replicates of three and when all MICs were the same value this was

considered as a statistically significant result.

Chapter 3

103

3.3. Results

3.3.1. Combined antibacterial activity of plant extracts

The combinations of extracts of A. pilosa Ledeb, A. chinensis Bunge, and S.

glabra Roxb exhibited indifferent or antagonistic effects against the majority of

bacteria isolates (Figure 1), with the following exceptions: extracts of A. pilosa

Ledeb and A. chinensis Bunge showed a synergistic effect against C. jejuni

(Table 3.2); extracts of A. chinensis Bunge and S. glabra Roxb exhibited a

synergistic effect against E. coli (Table 3.4); and A. pilosa Ledeb and S. glabra

Roxb showed an antagonistic effect against E. coli (Table 3.3).

MIC and MBC of quality control of ampicillin against S. pneumoniae ATCC

49619 was 0.06 mg/L and 0.24 mg/L, respectively. MIC and MBC of quality

control of erythromycin against S. aureus ATCC 29213 was 0.5 and 4 mg/L,

respectively. These values are in accordance with (EUCAST, 2018). This

validates the method verifying it was done in accordance with EUCAST (2018)

standards.

Chapter 3

104

Figure 3.2. The number of isolates of bacteria that were susceptible to different

combined plant extracts and their degree of susceptibility (FICI)

A. pilosa Ledeb with A. chinensis Bunge A. pilosa Ledeb with S. glabra Roxb A. chinensis Bunge with S. glabra Roxb

0

2

4

6

8

10

12

14

16

Synergy Additive Indifference Antagonism

Qu

an

titiy o

f b

acte

ria

isola

tes

Fractional inhibitory concentration index

Chapter 3

105

Table 3.2. MIC (mg/L), FIC index and interpretation (FICI) of individual and combinations of

plant extracts in the presence of C. jejuni, L. monocytogenes, S. enteritidis, and E. coli.

Pathogen Plant extract

A.

pilosa

Ledeb

A.

chinensis

Bunge

A. pilosa and A. chinensis

Bunge

MIC (mg/L) FIC FICI

C. jejuni

NCTC 11322 31.25 62.5 7.81 0.375 Synergy

RC152 31.25 62.5 7.81 0.375 Synergy

RC104 125 62.5 15.60 0.374 Synergy

RC179 500 62.5 7.81 0.375 Synergy

L. monocytogenes NCTC 11994 31.25 125 31.25 1.25 Indifference

LS12519 31.25 125 31.25 1.25 Indifference

OT11230 62.5 125 62.5 1.25 Indifference

CP102 31.25 125 31.25 1.25 Indifference

CP1132 125 125 62.5 1 Indifference

QA1018 31.25 125 31.25 1.25 Indifference

S. enteritidis NCTC 0074 500 62.5 125 2.25 Indifference

1F6144 125 62.5 62.5 1.5 Indifference

LE103 125 62.5 62.5 1.5 Indifference

QA04/19 125 62.5 62.5 1.5 Indifference

E. coli

ATCC 25922 7.81 125 31.25 4.25 Antagonism

UM004 7.81 125 31.25 4.25 Antagonism

UM011 7.81 125 31.25 4.25 Antagonism

UM012 7.81 125 31.25 4.25 Antagonism

Values are the mean rounded down to the nearest well of one experiment in triplicate

Chapter 3

106




A.

pilosa

Ledeb

S.

glabra

Roxb

A. pilosa and S. glabra Roxb

MIC (mg/L) FIC FICI

C. jejuni

NCTC 11322 31.25 250 31.25 1.125 Indifference

RC152 31.25 250 31.25 1.125 Indifference

RC104 125 250 125 1.5 Indifference

RC179 500 250 250 1.5 Indifference

L. monocytogenes

NCTC 11994 31.25 62.5 31.25 1.5 Indifference

LS12519 31.25 31.25 31.25 2 Indifference

OT11230 62.5 125 62.5 1.5 Indifference

CP102 31.25 31.25 31.25 2 Indifference

CP1132 125 31.25 31.25 1.25 Indifference

QA1018 31.25 31.25 31.25 2 Indifference

S. enteritidis

NCTC 0074 500 250 250 1.5 Indifference

1F6144 125 125 125 2 Indifference

LE103 125 125 125 2 Indifference

QA04/19 125 125 125 2 Indifference

E. coli

ATCC 25922 7.81 125 31.25 4.25 Antagonism

UM004 7.81 125 31.25 4.25 Antagonism

UM011 7.81 125 31.25 4.25 Antagonism

UM012 7.81 125 31.25 4.25 Antagonism


Chapter 3

107




A.

chinensis

Bunge

S.

glabra

Roxb

A. chinensis Bunge and

S. glabra Roxb

MIC (mg/L) FIC FICI

C. jejuni

NCTC 11322 62.5 250 62.5 1.25 Indifference

RC152 62.5 250 62.5 1.25 Indifference

RC104 62.5 250 62.5 1.25 Indifference

RC179 62.5 250 62.5 1.25 Indifference

L.

monocytogenes

NCTC 11994 125 62.5 62.5 1.25 Indifference

LS12519 125 31.25 31.25 1.25 Indifference

OT11230 125 125 62.5 1 Indifference

CP102 125 31.25 62.5 2.5 Indifference

CP1132 125 31.25 31.25 1.25 Indifference

QA1018 125 31.25 31.25 1.25 Indifference

S. enteritidis

NCTC 0074 62.5 250 125 2.5 Indifference

1F6144 62.5 125 62.5 1.5 Indifference

LE103 62.5 125 62.5 1.5 Indifference

QA04/19 62.5 125 62.5 1.5 Indifference

E. coli

ATCC 25922 125 125 62.5 0.5 Synergy

UM004 125 125 62.5 0.5 Synergy

UM011 125 125 62.5 0.5 Synergy

UM012 125 125 62.5 0.5 Synergy


Chapter 3

108

3.3.2. Combined antibacterial activity of plant extracts with antibiotics

The combinations of extracts of A. pilosa Ledeb, A. chinensis Bunge, S. glabra

Roxb with antibiotics including ampicillin and erythromycin exhibited additive

effects against all the tested bacteria isolates including C. jejuni, L.

monocytogenes, E. coli, and S. enteritidis (Tables 3.5 to 3.10).

Table 3.5. MIC (mg/L), FIC index and interpretation (FICI) of individual antibiotics and

individual plant extracts; and combinations of plant extracts with antibiotics in the presence of

C. jejuni, L. monocytogenes, S. enteritidis, and E. coli.

Pathogen Antimicrobial

A. pilosa

Ledeb

Ampicillin A. pilosa Ledeb and

Ampicillin

MIC (mg/L) FIC FICI

C. jejuni

NCTC 11322 31.25 16 8 0.756 Additive

RC152 31.25 16 8 0.756 Additive

RC104 125 16 8 0.564 Additive

RC179 500 16 8 0.516 Additive

L. monocytogenes

NCTC 11994 31.25 0.25 0.125 0.504 Additive

LS12519 31.25 0.5 0.25 0.508 Additive

OT11230 31.25 0.5 0.25 0.508 Additive

CP102 31.25 0.25 0.125 0.504 Additive

CP1132 62.5 0.25 0.125 0.502 Additive

QA1018 31.25 1 0.50 0.531 Additive

S. enteritidis

NCTC 0074 500 4 2 0.504 Additive

1F6144 125 2 1 0.508 Additive

LE103 125 4 2 0.516 Additive

QA04/19 125 8 4 0.532 Additive

E. coli

ATCC 25922 7.81 8 2 0.506 Additive

UM004 7.81 4 2 0.756 Additive

UM011 7.81 4 2 0.756 Additive

UM012 7.81 4 2 0.756 Additive


Chapter 3

109





A.

chinensis

Bunge

Ampicillin A. chinensis Bunge

and Ampicillin

MIC (mg/L) FIC FICI

C. jejuni

NCTC 11322 62.5 16 8 0.628 Additive

RC152 62.5 16 8 0.628 Additive

RC104 62.5 16 8 0.628 Additive

RC179 62.5 16 8 0.628 Additive

L. monocytogenes

NCTC 11994 125 0.5 0.25 0.502 Additive

LS12519 125 0.5 0.25 0.502 Additive

OT11230 125 0.5 0.25 0.502 Additive

CP102 125 0.25 0.125 0.501 Additive

CP1132 125 0.5 0.25 0.502 Additive

QA1018 125 1 0.5 0.504 Additive

S. enteritidis

NCTC 0074 62.5 4 2 0.532 Additive

1F6144 62.5 4 2 0.532 Additive

LE103 62.5 4 2 0.532 Additive

QA04/19 62.5 8 4 0.564 Additive

E. coli

ATCC 25922 125 4 2 0.516 Additive

UM004 125 4 2 0.516 Additive

UM011 125 4 2 0.516 Additive

UM012 125 4 2 0.516 Additive


Chapter 3

110


individual plant extracts; and combinations of plant extracts with antibiotics in the presence

of C. jejuni, L. monocytogenes, S. enteritidis, and E. coli.


S.

glabra

Roxb

Ampicillin S. glabra Roxb and

Ampicillin

MIC (mg/L) FIC FICI

C. jejuni

NCTC 11322 250 16 8 0.532 Additive

RC152 250 16 8 0.532 Additive

RC104 250 16 8 0.532 Additive

RC179 250 16 8 0.532 Additive

L. monocytogenes

NCTC 11994 62.5 0.25 0.125 0.502 Additive

LS12519 31.25 0.5 0.25 0.508 Additive

OT11230 125 0.5 0.25 0.502 Additive

CP102 31.25 0.25 0.125 0.504 Additive

CP1132 31.25 0.25 0.125 0.504 Additive

QA1018 31.25 1 0.5 0.516 Additive

S. enteritidis

NCTC 0074 250 4 2 0.508 Additive

1F6144 125 2 1 0.508 Additive

LE103 125 4 2 0.516 Additive

QA04/19 125 8 4 0.532 Additive

E. coli

ATCC 25922 125 8 2 0.516 Additive

UM004 125 4 2 0.516 Additive

UM011 125 4 2 0.516 Additive

UM012 125 4 2 0.516 Additive


Chapter 3

111





A.

pilosa

Ledeb

Erythromycin A. pilosa Ledeb and

Erythromycin

MIC (mg/L) FIC FICI

C. jejuni

NCTC 11322 31.25 2 1 0.532 Additive

RC152 31.25 2 1 0.532 Additive

RC104 125 2 1 0.508 Additive

RC179 500 4 2 0.504 Additive

L. monocytogenes

NCTC 11994 31.25 0.5 0.25 0.508 Additive

LS12519 31.25 2 1 0.532 Additive

OT11230 62.5 1 0.50 0.508 Additive

CP102 31.25 0.5 0.25 0.508 Additive

CP1132 125 0.5 0.25 0.502 Additive

QA1018 31.25 1 0.50 0.516 Additive

S. enteritidis

NCTC 0074 500 16 8 0.516 Additive

1F6144 125 16 8 0.564 Additive

LE103 125 16 8 0.564 Additive

QA04/19 125 16 8 0.564 Additive

E. coli

ATCC 25922 7.81 16 3.91 0.745 Additive

UM004 7.81 16 3.91 0.745 Additive

UM011 7.81 16 3.91 0.745 Additive

UM012 7.81 16 3.91 0.745 Additive


Chapter 3

112





A.

chinensis

Bunge

Erythromycin A. chinensis Bunge

and Erythromycin

MIC (mg/L) FIC FICI

C. jejuni

NCTC 11322 62.5 2 1 0.516 Additive

RC152 62.5 2 1 0.516 Additive

RC104 62.5 2 1 0.516 Additive

RC179 62.5 4 2 0.532 Additive

L.

monocytogenes

NCTC 11994 125 1 0.5 0.504 Additive

LS12519 125 2 1 0.508 Additive

OT11230 125 4 2 0.516 Additive

CP102 125 2 1 0.508 Additive

CP1132 125 2 1 0.508 Additive

QA1018 125 2 1 0.508 Additive

S. enteritidis

NCTC 0074 62.5 16 8 0.628 Additive

1F6144 62.5 16 8 0.628 Additive

LE103 62.5 16 8 0.628 Additive

QA04/19 62.5 16 8 0.628 Additive

E. coli

ATCC 25922 125 16 8 0.564 Additive

UM004 125 16 8 0.564 Additive

UM011 125 16 8 0.564 Additive

UM012 125 16 8 0.564 Additive


Chapter 3

113


individual plant extracts; and combinations of plant extracts with antibiotics in the presence

of C. jejuni, L. monocytogenes, S. enteritidis, and E. coli.


S.

glabra

Roxb

Erythromycin S. glabra Roxb and

Erythromycin

MIC (mg/L) FIC FICI

C. jejuni

NCTC 11322 250 2 1 0.504 Additive

RC152 250 2 1 0.504 Additive

RC104 250 2 1 0.504 Additive

RC179 250 4 2 0.508 Additive

L. monocytogenes

NCTC 11994 62.5 1 0.5 0.508 Additive

LS12519 31.25 2 1 0.532 Additive

OT11230 125 4 2 0.516 Additive

CP102 31.25 2 1 0.532 Additive

CP1132 31.25 2 1 0.532 Additive

QA1018 31.25 2 1 0.532 Additive

S. enteritidis

NCTC 0074 250 16 8 0.532 Additive

1F6144 125 16 8 0.564 Additive

LE103 125 16 8 0.564 Additive

QA04/19 125 16 8 0.564 Additive

E. coli

ATCC 25922 125 16 8 0.564 Additive

UM004 125 16 8 0.564 Additive

UM011 125 16 8 0.564 Additive

UM012 125 16 8 0.564 Additive


3.4. Discussion

Previous in vitro studies of combinations of plant extracts, and plant extracts

with antibiotics have indicated synergistic activity (Shimizu et al., 2001; Al-

Bayati, 2008). The findings from chapter 2, were used to identify plant

candidates with strong antibacterial properties for further studies. The

antibacterial activity of different combinations of A. pilosa Ledeb, A. chinensis

Bunge, and S. glabra Roxb extracts and with antibiotics were assessed against

C. jejuni, L. monocytogenes, E. coli, S. enteritidis using a broth microdilution

synergistic assay.

Chapter 3

114

A range of methods can be used to study the antibacterial properties of plant

and antibiotic concentrations. These include, checkerboard assays, time kill,

and E-test methods (White et al., 1996; Bonapace et al., 2002; Sopirala et al.,

2010). The present study used a modified broth microdilution (CLSI, 2009)

method as it is recommended in the guidelines.

3.4.2. Indifferent activity of combinations of plant extracts

Although, there appears to be a lack of references to synergistic assays of the

above combinations in the literature other studies of combinations of plant

extracts or solutions with antibiotics have shown that combinations of plant

extracts can modify the overall potency of their antibacterial activity on

pathogens (Abreu, McBain and Simões, 2012; Bhardwaj et al., 2016;

Cheesman et al., 2017).

In some cases, these types of combinations may have little or no effect on their

antibacterial activity, while in other cases the combination could present

antagonistic antibacterial activity which reduces the ability of the combination

to inhibit pathogen growth. Vuuren et al., (2011) noted that antagonistic and

indifferent interactions in synergy assays are commonly overlooked or rejected

by journals despite the abundance of adverse reactions with plants from

traditional herbal medicine. This appears to be the first study to report the

indifferent activity of combinations of A. pilosa Ledeb, A. chinensis Bunge, and

S. glabra Roxb against pathogens commonly found in the poultry GI tract.

Most combinations of the plant extracts when tested against C. jejuni, L.

monocytogenes, E. coli, S. enteritidis resulted in indifferent activity. In this

Chapter 3

115

study, combinations of extracts of A. pilosa Ledeb and A. chinensis Bunge

showed a synergistic effect against C. jejuni (Table 3.2); and combinations of

extracts of A. pilosa Ledeb and S. glabra Roxb, A. pilosa Ledeb and A.

chinensis Bunge, and A. chinensis Bunge, and S. glabra Roxb exhibited a

synergistic effect against E. coli (Tables 3.2 and 3.4).

These results highlight the potential synergistic interactions of plant extract

combinations. There appears to be no literature that investigates the

synergistic or additive effects of the above combinations against E. coli, or C.

jejuni for comparison. However, examples of studies of combinations of plant

extracts which have demonstrated similar synergistic effects against the

pathogens in this study include combinations of Alchornea cordifolia and

Pterocarpus santalinoide against Salmonella and E. coli (Obaji, Enweani and

Oli, 2020), Salvia chamelaeagnea and Leonotis leonurus against E. coli

(Kamatou et al., 2006), oregano, thyme and cinnamon bark against C. Jejuni

(Navarro et al., 2015), oregano and cranberry against L. monocytogenes (Lin,

Labbe and Shetty, 2004).

While in general synergistic mechanisms are well studied, they remain largely

unknown for specific combinations of plant extracts. This is partly because of

the complex nature of the mixtures of the chemical compositions of combined

plant extracts. Often, important constituents are unknown and are within a

complex mixture comprised of a plethora, often thousands of unique

components (Caesar and Cech, 2019). This makes it challenging to explain

indifferent, additive, synergistic, or antagonistic results. This was also reflected

in the literature which commonly lacked details on synergistic mechanisms of

plant extract combinations (Cheesman et al., 2017; Kamatou et al., 2006;

Chapter 3

116

Khameneh et al., 2019; Navarro et al., 2015; Osuntokun and Faniyi, 2020).

The large number of plant extracts would generate a significant number of

possible combinations. This will inherently result in a large gap in the literature

because it would seem unlikely that all these combinations can be researched.

3.4.3. Additive activity of plant extracts and antibiotics

Another type of synergism is when plant extracts are combined with certain

antibiotics to enhance the effectiveness of antibiotics as modifiers of resistance

(Caesar and Cech, 2019). While this is supported by a numerous in vitro

studies (Akinyele et al., 2017; Stefanović, 2018) there appeared to be a lack

of literature on the synergistic activities of the combinations of plant extracts

and antibiotics investigated in this study. While this study did not report

synergy, it did show an additive effect against C. jejuni, L. monocytogenes, E.

coli, S. enteritidis, when A. pilosa Ledeb, A. chinensis Bunge, and S. glabra

Roxb where each combined with ampicillin and erythromycin. This

demonstrated that the accumulation of the plant extract and antibiotic

decreased the total concentration of antibiotic required to achieve an

antibacterial effect. Other research showed that catechins, a phenolic

compound prevalent in A. pilosa Ledeb, A. chinensis Bunge, and S. glabra

Roxb (Hua et al., 2018; Kasai et al., 1992) may have in part contributed to

increasing the susceptibility of C. jejuni, L. monocytogenes, E. coli, and S.

enteritidis (Zhao et al., 2001; Esimone et al., 2006; Hemaiswarya et al., 2008;

Qin et al., 2013; Gomes et al., 2017; Reygaert, 2018b).

Chapter 3

117

However other studies provide examples of synergistic effects of combinations

of plant extracts with antibiotics. These include extracts of Green tea with

gentamicin and amikacin against E. coli (Neyestani et al, 2007), rosemary with

amoxicillin, gentamicin and difloxacin against Salmonella (Abdeltawab et al.,

2018), phenolic compounds with ciprofloxacin and erythromycin against C.

jejuni (Oh and Jeon, 2015), Cocos nucifera with ampicillin, chloramphenicol,

penicillin, ciprofloxacin, and tetracycline against L. monocytogenes (Akinyele

et al., 2017).

These studies investigated different plant extracts and antibiotics so it would

be difficult to draw meaningful comparisons on the results. However, in

contrast to the broth microdilution method these studies used the time kill

method, checkerboard, and disc diffusion methods. These methods were

criticised for being time, labour, and material intensive (Shikov et al., 2018).

Research also shows and that no two synergy methods produce comparable

results (Doern, 2014). Even using the same method can produce variable

results ranging from antagonistic to synergistic effects as they are dependent

on concentrations and combinations used (Smith et al., 2007; Coutinho et al.,

2009; Abreu et al, 2012). Furthermore, there is debate in the literature about

the interpretation of synergy and this could lead to variation in the results

reported from studies. The analysis of results in this study used an

interpretation of synergy recommended by Odds (2003) as FICI of <0.5. These

values minimise experimental error due to MIC methodology which has an

intrinsic range of three dilutions (mode ± 1 dilution) variability and is still widely

accepted as the synergistic cut off point. However, other studies by Bell, (2005)

and Prinsloo et al., (2008) included an additional FICI value for additivity where

Chapter 3

118

FIC values are within 0.5 to 1 as this provides further information which may

be of use when interpreting data (Bell, 2005). This study used this additive

category noting the proviso that the results are not interpreted and reported as

being synergistic. This variability in results and methods highlights the need

for a standardised method for synergy testing. Hence the reason for using the

broth microdilution method in this study.

3.5. Conclusion

A review of the literature suggested this may be the first study to explore and

record the antibacterial activity and synergistic effects of the above plant and

antibiotic combinations. The results indicated potential synergistic effects for

combinations of A. chinensis Bunge and A. pilosa Ledeb and A. chinensis

Bunge and S. glabra Roxb, respectively against C. jejuni and E. coli. The

results also indicated potential additive effects for each of the above plant

extracts and pathogens when combined with ampicillin and erythromycin.

While the results tend to support the use of specific plant extracts and plant

extracts and antibiotic combinations further research would be needed to

validate the results, especially before moving to an in vivo poultry trial. The

results of this study highlight the potential of plant extracts in combination with

each other or with antibiotics as a way of decreasing the use of antibiotics in

poultry feed.

Chapter 4

119

4. Chapter 4

Antibacterial activity of extracts from A. pilosa Ledeb, S. glabra Roxb, A.

chinensis Bunge, and I. domestica (L.) Goldblatt and Mabb against

Campylobacter jejuni, Listeria monocytogenes, Escherichia coli and

Salmonella enterica subsp. enterica serovar Enteritidis using a poultry

caecum model

4.1. Introduction

Initial screening methods from chapter 2 (McMurray et al., 2020) and chapter

3 indicated that the crude extracts of A. pilosa Ledeb, S. glabra Roxb, A.

chinensis Bunge, and I. domestica (L.) Goldblatt and Mabb exhibited potent

antibacterial activity in vitro against L. monocytogenes, S. enteritidis, C. jejuni

and E. coli. In vitro assays using broth and a poultry caecum model were used

to further investigate antibacterial activities of these plant extracts against the

above pathogens.

The caecum model is a recently emerging model and was used in this study

because it had been highlighted in other research as a way of modelling the

antibacterial activity of plant extracts against pathogens in an environment

which is closer to the natural conditions experienced by pathogens in a living

bird. The model may provide results which better approximate those found in

an in vivo poultry trial. (Solis de los Santos et al., 2008; Johny et al., 2009;

Solis de los santos et al., 2009; Skrivanova et al., 2016).

Chapter 4

120

In this study caecum content was chosen to determine the activity of plant

extracts because the poultry caecum acts as a primary colonisation site and

reservoir for the pathogens of study, E. coli, and S. enteritidis (Vasudevan et

al., 2005; Vaezirad et al., 2017; Tarabees et al., 2019) and can be colonised

by L. monocytogenes (Rothrock et al., 2017). Firmicutes such as lactic acid

bacteria primarily colonise the caecum and catalyse the breakdown of

indigestible starch cellulose (Clavijo and Florez, 2017). An increase in

pathogens such as L. monocytogenes, S. enteritidis, or E. coli could lead to

decreased feed conversion ratios and nutrient absorption. Therefore, the

poultry caecum was chosen to model the effects of these plant extracts in vitro

because if the plant extracts inhibit these pathogens, then they may have a

modulatory effect on the caecum microbiota in vivo and this could in turn

improve feed conversion ratios and performance. Crude extracts of A. pilosa

Ledeb, S. glabra Roxb, A. chinensis Bunge, and I. domestica (L.) Goldblatt

and Mabb have not been evaluated for their ability to inhibit S. enteritidis, C.

jejuni, E. coli and L. monocytogenes in poultry caecum content before.

4.1.2. Aims and objectives

The aim of this study was to determine the antibacterial properties of the plant

extracts of A. pilosa Ledeb, S. glabra Roxb, A. chinensis Bunge, and I.

domestica (L.) Goldblatt and Mabb against L. monocytogenes, S. enteritidis,

C. jejuni and E. coli in a poultry caecum model for potential use as poultry feed

additive. The objectives of this study were to:

Chapter 4

121

• Conduct an in vitro assay to assess the amount of bactericidal activity

over 24 hours in broth of the extracts of A. pilosa Ledeb, S. glabra Roxb,

A. chinensis Bunge, and I. domestica (L.) Goldblatt and Mabb

• Conduct an in vitro model assay using poultry caecum content to

assess the amount of bactericidal activity over 24 hours of the extracts

of A. pilosa Ledeb, S. glabra Roxb, A. chinensis Bunge, and I.

domestica (L.) Goldblatt and Mabb


4.2.1. Bacterial isolates used for screening

The procedure for sourcing and identification of bacterial isolates was the

same as in chapter 2.2.2 with one exception. The following strains were used:

S. enteritidis J116, QA76 and NCTC 0074; L. monocytogenes QA1018,

CM191 and NCTC 11994; E. coli ATCC 225922, UM004 and UM012; and C.

jejuni RC152, RC104 and NCTC 11322.

4.2.2. Preparation of plant extracts

The preparation of the plant extracts was carried out in the same was as in

section 2.2.3 with one exception. The plants in this study included: the herb of

A. pilosa Ledeb; the tuber of S. glabra Roxb; and the root of A. chinensis

Bunge; and the rhizome of I. domestica (L.) Goldblatt and Mabb.

4.2.3. Time kill assay in broth

Each bacteria isolate was incubated overnight under appropriate conditions in

broth prior to testing (Table 4.1). Plant extracts which exhibited the lowest MIC

Chapter 4

122

against pathogens in the broth microdilution study were chosen to be studied

using a modification of the time kill method (CLSI, 1999). To quantify the

bactericidal activity of plant extracts against E. coli, L. monocytogenes, S.

enteritidis, and C. jejuni time kill assays were used. Time kills curve analyses

for I. domestica (L.) Goldblatt and Mabb and A. pilosa Ledeb in the presence

of E. coli, A. pilosa Ledeb, and S. glabra Roxb in the presence of L.

monocytogenes, A. chinensis Bunge in the presence of S. enteritidis, and A.

chinensis Bunge in the presence of C. jejuni were conducted. Plant extracts

were tested against both clinical isolates and reference strains including: S.

enteritidis J116, QA76 and NCTC 0074; L. monocytogenes QA1018, CM191

and NCTC 11994; E. coli ATCC 225922, UM004 and UM012; and C. jejuni

RC152, RC104 and NCTC 11322. Volumes of plant extracts were added to

individual 1ml samples of broth resulting in final concentrations of 31.25, 62.5,

125 and 250 mg/L of I. domestica (L.) Goldblatt and Mabb against S.

enteritidis; 15.63, 31.25, 62.5, and 125 of A. pilosa Ledeb against L.

monocytogenes; 31.25, 62.5, 125 and 250 mg/L of A. chinensis Bunge against

C. jejuni; 7.81, 15.63, 31.25 and 62.5 mg/L of A. pilosa Ledeb against E. coli;

31.25, 62.5, 125 and 250 mg/L of A. chinensis Bunge; against S. enteritidis;

31.25, 62.5, 125 and 250 mg/L of S. glabra Roxb against L. monocytogenes.

These concentrations of 1/2, 1, 2 and 3xMIC were selected to provide a range

of concentrations to evaluate the bacteriostatic activity based on MIC values

from chapter 2 and the bactericidal activity based on MBC values from chapter

2. These values were chosen to examine bactericidal activity in broth and to

provide sufficient results to determine the concentrations required for testing

bactericidal activity in the in vitro caecum content model. Replicates (n=5) of

Chapter 4

123

bacteria with plant extract or antibiotic control dilutions were plated onto agar

under appropriate conditions (Table 4.1) at 0, 0.5, 1, 2, and 24 hours. Previous

research demonstrated that plant extracts exhibit rapid bactericidal activity

within less than 1 hour against E. coli, S. enterica, C. jejuni, and L.

monocytogenes (Friedman et al., 2002; Vasudevan et al., 2005; Johny et al.,

2010). Therefore, the time points of 0, 0.5, 1, and 2 hours were selected to

provide data about the initial bactericidal activity of the plant extracts. This

activity was followed by a plateau in the previous research investigating plant

extracts (Friedman et al., 2002; Vasudevan et al., 2005; Johny et al., 2010).

Therefore, two further time points, 2 hours and 24 hours were chosen to

provide data about the period that the bactericidal activity of the plant extracts

would last. All of these time points were selected to provide sufficient data for

analysis to choose plant extracts for further investigation in the poultry caecum

model. Individual colonies were observed and counted using a plate counter

(Stuart Scientific, UK) for each of the agar plates to determine the total viable

count of surviving bacteria. The total viable bacteria count was recorded.

Sterile broth was the negative control. Ampicillin was the positive control for E.

coli, L. monocytogenes, and S. enteritidis, erythromycin was the positive

control for C. jejuni (EUCAST, 2018). The concentrations of ampicillin used

were: 0.25, 0.5, 1, 2, and 4 mg/L against L. monocytogenes; and 4, 8, 16, 32,

and 64 mg/L against S. enteritidis and E. coli. These in turn, were added to

100 μL of the pathogen in broth to give final approximate inoculation of 5x105

CFU/mL. The control samples were incubated under appropriate conditions in

broth (Table 4.1). The positive and negative control samples and the test

sample populations of surviving pathogens were determined by plating onto

Chapter 4

124

agar under appropriate conditions (Table 4.1) and over time intervals of 0, 0.5,

1, 2, and 24 hours. Individual colonies were observed, counted, and recorded

for each of the positive, negative and control samples to determine the total

viable count of surviving bacteria.

Table 4.1. Media and incubation conditions used for bacteria


conditions

C. jejuni Mueller Hinton-F

(Oxoid, UK)

Mueller Hinton-F (Oxoid,

UK)

41 ± 1 °C;

microaerobic gas

sachets and box

(BioMerieux, UK).

L.

monocytogenes

Tryptone soya

broth and 5% lysed

horse blood (TCS

Biosciences Ltd,

UK)

Tryptone soya agar and

5% lysed horse blood

broth (TCS Biosciences

Ltd, UK)

35 ± 1 °C ;5% CO2

E. coli Mueller Hinton

Broth (MHB)

(Oxoid, UK)

Mueller Hinton Agar

(MHA) (Oxoid, UK)


S. enteritidis (MHB) (Oxoid, UK) (MHA) (Oxoid, UK) 35 ± 1 °C; ambient air




4.2.4. Time kill in an in vitro caecum model

The contents of the caecum were collected from 3-week male Ross 308 broiler

chickens (n=45). These chickens were offered a commercial cereal-based diet

(12.9 MJ/kg apparent metabolise energy and 200 g/kg crude protein) at AFBI,

UK, and were immediately stored at -80°C prior to analysis. Animal Welfare

Ethical Review Body at AFBI approved the trial and the trial was carried out

following the Animals Scientific Act 1986. One millilitre of caecum sample was

mixed with 1ml selective broth to kill and exclude bacteria that did not belong

to the genus being investigated. Previous studies mixed an equal amount of

Chapter 4

125

digest content with an equal amount of PBS or broth to make the caecum

contents more viscous, suitable for pipetting, and suitable for comparison with

a MacFarland Standard (Johny et al., 2010; Park et al., 2017; Skrivanova et

al., 2016; Vasudevan et al., 2005). This was incubated overnight under

appropriate conditions (Table 2). Time kill methods (CLSI, 1999) were altered

to appropriate them for use in an in vitro model using caecum content in place

of broth as the growth medium, the method with modifications is detailed as

follows. The bacteriostatic and bactericidal activity of E. coli, L.

monocytogenes, and S. enteritidis by extracts from A. pilosa Ledeb and A.

chinensis Bunge, I. domestica (L.) Goldblatt and Mabb, and Smilax glabra

Roxb was quantified using time kill assays. These plant extracts were chosen

to assay using the time kill method because they exhibited low MIC values

(≤62.5mg/L) in the previous time kill assay using broth. Each plant extract was

added to 1ml caecum content to produce 1/2, 1, 2, 3 and 4xMIC of each plant

extract. After analysis of the previous time kill in broth a further concentration

of 4xMIC was included in the in vitro caecum content model to provide

additional information about the bactericidal activity of the plant extracts at

higher concentrations. Separate mixtures of L. monocytogenes, S. enteritidis,

and E. coli inoculum were prepared. Each mixture contained three strains of

the same pathogen: E. coli strains UM004, UM012, and UM011; L.

monocytogenes strains QA1018, LS12519, and CP102; and S. enteritidis

strains QA60, LE103, and QA76. Five individual bacteria colonies were chosen

from each of three clinical isolates per genus. These were incubated overnight

in broth under appropriate conditions optimised for growth (Table 2). The three

isolates were each sedimented by centrifugation (3,600 x g for 15 mins at 4°C).

Chapter 4

126

The pellet was suspended in phosphate buffered saline for comparison against

a MacFarland Standard (Johny et al., 2010) to determine the concentration

required to achieve 1X108 CFU/mL. The bacterial inoculation was dilution 1 in

100 in selective broth to achieve 1x106 CFU/mL. Bacteria were mixed with

plant extracts in caecum solution to reach a final inoculation of 5x105 CFU/mL,

then incubated overnight under appropriate growth conditions. Replicates

(n=5) of bacteria with plant extract dilutions were plated onto agar at 0, 0.5, 1,

2, 4, 6, and 24 hours and incubated under appropriate conditions (Table 4.1).

The previous time kill assays revealed that the plants decreased the

percentage of detectable E. coli, C. jejuni, S. enteritidis, and L. monocytogenes

within 2 hours and exhibited bactericidal activity within 24 hours. Two further

time points including 4 hours and 6 hours were chosen to provide additional

information about the bactericidal activity of the plant extracts between 2 hours

and 24 hours. Individual colonies were observed and counted using a plate

counter (Stuart Scientific, UK) for each of the agar plates to determine the total

viable count of surviving bacteria. The total viable bacteria count was recorded.

The negative control was distilled water in place of plant extracts. Ampicillin

was the positive control for E. coli, L. monocytogenes, and S. enteritidis

(EUCAST, 2018).

Table 4.2. Media and incubation conditions used for three bacteria species

Bacteria Broth Incubation conditions

L. monocytogenes

PALCAM (Sigma, UK) 35 ± 1 °C ;5% CO2

E. coli MacConkey (Sigma, UK) 35 ± 1 °C; ambient air

S. enteritidis Tetrathionate Brilliant Green (Sigma, UK)


Chapter 4

127


Bactericidal activity was designated when there was a decrease in the

detection of ≥99.9% of the total viable count of bacteria and when there were

no visible colonies of bacteria on the agar plate after incubation (CLSI, 1999).

Bacteriostatic activity was designated when the original concentration of

inoculum was maintained or when there was a decrease in the detection of

<99.9% of the total viable count of bacteria. Data was log transformed. ANOVA

was carried out to test significance of differences in total viable cell count

between concentrations of plant extracts, ampicillin, and growth control (P

<0.001) using Prism 5.0 (Prism 5.0 software, QUB).

4.3. Results

4.3.1. Time kill

The total viable count of surviving bacteria populations in the presence of plant

extracts incubated in broth for 24 hours are presented in figures 4.1 to 4.4. The

values in figures 4.1 to 4.4 represent the mean of 3 experiments ± SEM. At

concentrations of 3xMIC all tested plant extracts exhibited bactericidal activity

and therefore decreased the detection of ≥99.9% of the total viable count of at

least one pathogen (Figure 4.1 to 4.4). For example, the extract of A. pilosa

Ledeb and the antibiotic ampicillin demonstrated bactericidal activity by 24

hours against L. monocytogenes and E. coli (Figures 4.1 and 4.2). The extract

of S. glabra Roxb and the antibiotic ampicillin demonstrated bactericidal

activity by 24 hours against L. monocytogenes (figure 4.1). The extract of I.

domestica (L.) Goldblatt and Mabb demonstrated bactericidal activity by 24

hours against E. coli (Figure 4.2). The extract of A. chinensis Bunge

Chapter 4

128

demonstrated bactericidal activity against both S. enteritidis and C. jejuni by

24 hours (Figures 4.3 and 4.4). The MIC of these plant extracts all

demonstrated detectable bacteriostatic activity against the surviving pathogen

populations throughout the duration of 24 hours (Figures 4.1 to 4.4). The

activity of A. pilosa Ledeb against E. coli was comparable to the positive

ampicillin control as comparatively low concentrations of each were required

to exhibit detectable bacteriostatic activity, 7.81 mg/L exhibited detectable

bacteriostatic activity E. coli over 24 hours and 4 mg/L ampicillin exhibited

detectable bacteriostatic activity E. coli over 24 hours. The growth control was

significantly different to all three antimicrobials at 2 and 24 hours (Figures 4.1

to 4.4; P <0.001). This highlights the antibacterial properties of these plant

extracts incubated in broth.

Figure 4.1 - Time kill of L. monocytogenes NCTC 11994, LS12519 and QA1018 incubated

with antimicrobials in broth over 24 hours.

Figure 4.1a. MIC

0.0 0.5 1.0 1.5 2.01.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

24

A. pilosa Ledeb

S. glabra Roxb

Ampicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Chapter 4

129

Figure 4.1b. 3xMIC

0.0 0.5 1.0 1.5 2.01.0×10-01

1.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

24

A. pilosa Ledeb

S. glabra Roxb

Ampicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Figure 4.1c. 1/2xMIC to 4xMIC

0 1 21.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

2 24

1/2xMIC MIC 2xMIC 3xMIC ]- A. pilosa Ledeb

1/2xMIC MIC 2xMIC 3xMIC ]- ampicillin

Growth control

1/2xMIC MIC 2xMIC 3xMIC ]- S. glabra Roxb

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Chapter 4

130

Figure 4.2. Time kill of E. coli ATCC 25922, UM004 and UM012 incubated with

antimicrobials in broth over 24 hours.

Figure 4.2a. MIC

0.0 0.5 1.0 1.5 2.01.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

24

A. pilosa Ledeb

S. glabra Roxb

Ampicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Figure 4.2b. 3xMIC

0.0 0.5 1.0 1.5 2.01.0×10-01

1.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

24

A. pilosa Ledeb

S. glabra Roxb

Ampicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Chapter 4

131


0 1 21.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

2 24

1/2xMIC MIC 2xMIC 3xMIC ]-A. pilosa Ledeb

Growth control

1/2xMIC MIC 2xMIC 3xMIC ]-S. glabra Roxb

1/2xMIC MIC 2xMIC 3xMIC ]-ampicillin

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Chapter 4

132

Figure 4.3. Time kill of S. enteritidis NCTC 0074, QA0419 and 1F6144 incubated with


Figure 4.3a. MIC and 3xMIC

0.0 0.5 1.0 1.5 2.01.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

24

A. chinensis Bunge MIC

Ampicillin MIC

A. chinensis Bunge 3xMIC

Growth control

Ampicillin 3xMIC

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/ml)

Figure 4.3b. 1/2xMIC to 4xMIC

0 1 21.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

2 24

1/2xMIC MIC 2xMIC 3xMIC A. chinensis Bunge

Growth control

1/2xMIC MIC 2xMIC 3xMIC ampicillin

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Chapter 4

133

Figure 4.4. Time kill of C. jejuni NCTC 11322, RC152 and RC179 incubated with


Figure 4.4a. MIC and 3xMIC

0 1 21.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

2 24

A. chinensis Bunge MIC

Erythromycin MIC

A. chinensis Bunge 3xMIC

Erythromycin 3xMIC

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Figure 4.4b. 1/2xMIC to 4xMIC

0 1 21.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

2 24

1/2xMIC MIC 2xMIC 3xMIC ]-A. chinensis Bunge

Growth control

1/2xMIC MIC 2xMIC 3xMIC ]-erythromycin

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Chapter 4

134

4.3.2. Bactericidal activity in an in vitro cecum model against L.

monocytogenes

All plant extracts and ampicillin at higher concentrations (250 mg/L to 1000

mg/L) decreased the detection of ≥ 99.9% of surviving viable L.

monocytogenes in 24 hours (Table 4.5 and figures 4.5b and 4.5c). All plant

extracts and ampicillin decreased the detection of (P <0.001) L.

monocytogenes inoculum in 0.5 hours (Table 4.5, figure 4.5b, and 4.5c). I.

domestica Goldblatt and Mabb demonstrated bactericidal activity at 24 hours

and demonstrated a significantly (P <0.001; Table 5 and figures 4.5b and 4.5c)

low percentage decrease in the detection of L. monocytogenes compared to

the other plant extracts from 0.5 hours to 6 hours. A. pilosa Ledeb was the

most effective against L. monocytogenes (Tables 4.4, 4.5 and Figures 4.5a,

4.5b, and 4.5c). Lower concentrations of (31.25 mg/L to 125 mg/L) I. domestica

(L.) Goldblatt and Mabb, A. chinensis Bunge, A. pilosa Ledeb, S. glabra Roxb,

and ampicillin (1mg/L) demonstrated detectable bacteriostatic activity against

L. monocytogenes throughout the duration of 24 hours compared to the growth

control (Table 4.4 and Figure 4.5a). The growth control was significantly

different to five antimicrobials at 24 hours (P <0.001; Figure 4.5a, 4.5b, and

4.5c).

Chapter 4

135

Table 4.4. Percentage kill of L. monocytogenes NCTC 11994, QA1018, LS12519, and CP102

incubated with MIC of plant extracts and ampicillin over 24 hours

Antimicrobial MIC (mg/L)

Time

(hours)

A.

pilosa

Ledeb

(31.25)

A.

chinensis

Bunge

(125)

S.

glabra

Roxb

(62.5)

I. domestica

(L.)

Goldblatt

and Mabb

(125)

Ampicillin

(1)

SEM SD

Percentage kill of L. monocytogenes

0 0 0 0 0 0 0 0

0.5 58.16d 41.41c 32.41b 8.27a 40.32c 0.004 0.018

1 90.37e 54.00d 37.76b 3.004a 40.96c 0.059 0.132

2 88.04d 56.27c 52.91b 2.264a 91.13e 0.007 0.037

4 76.66d 47.71b 54.68c 2.628a 90.76e 0.012 0.061

6 -189.44b 50.72d 59.30e -294.95a 5.12c 0.045 0.225

24 -365.20d 44.22e -4353b -5950a -464.10c 0.134 0.671

Negative numbers (-) indicate growth (no decrease in the detection of bacteria). a, b superscripts indicate significance.

Means without common superscripts are significantly different (P <0.001). For example, I. domestica (L.) Goldblatt

and Mabba killed a significantly lower percentage of L. monocytogenes at 0.5 hours compared to all other plants with

letters b, c, d.

Table 4.5. Percentage kill of L. monocytogenes NCTC 11994, QA1018, LS12519, and CP102

incubated with 4xMIC of plant extracts and ampicillin over 24 hours

Antimicrobial 4xMIC (mg/L)

Time

(hours)

A. pilosa

Ledeb

(250)

A.

chinensis

Bunge

(1000)

S.

glabra

Roxb

(500)

I. domestica

(L.)

Goldblatt

and Mabb

(1000)

Ampicillin

(8)

SEM SD

Percentage kill of L. monocytogenes

0 0 0 0 0 0 0 0

0.5 95.51b 96.20b 47.80a 93.70b 95.59b 0.123 0.616

1 96.07b 99.99c 43.82a 95.67c 95.60c 0.106 0.531

2 99.57b 99.99b 44.24a 99.99b 99.78b 0.218 1.088

4 99.99b 99.99b 42.63a 99.99b 99.80b 0.082 0.408

6 99.99b 99.99b 99.20a 99.99b 99.99b 0.025 0.123

24 99.99 99.99 99.99 99.99 99.99 0 0


Means without common superscripts are significantly different (P <0.001). For example, S. glabra Roxba decreased

a significantly lower percentage of the detection of L. monocytogenes at 0.5 hours compared to all other plants with

letter b.

Chapter 4

136

Total viable count of L. monocytogenes NCTC 11994 and QA1018, LS12519 and CP102

incubated with plant extracts and ampicillin over 24 hours

Figure 4.5a. MIC

0 1 2 3 4 5 61.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

24

A. pilosa Ledeb

S. glabra Roxb

A. chinensis Bunge

I. domestica Goldmatt and Mabb

Ampicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Figure 4.5b. 4xMIC

0 1 2 3 4 5 61.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

24

A. pilosa Ledeb

S. glabra Roxb

A. chinensis Bunge

I. domestica Goldmatt and MabbAmpicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Chapter 4

137


0 1 2 3 4 5 61.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

6 24

1/2MIC MIC 2xMIC 3xMIC 4xMIC ]- A. pilosa Ledeb

Growth controlMIC 2XMIC 3xMIC 4xMIC ]- ampicillin

1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- S. glabra Roxb

1/2xMIC

1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- A. chinensis Bunge

1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- I. domestica Goldblatt and Mabb

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

The values represent the means of two experiments with 5 replicates ± standard error of

the mean.

Chapter 4

138

4.3.3. Bactericidal activity in an in vitro cecum model against E. coli

All plant extracts at higher concentrations (62.5 mg/L to 1000 mg/L) decreased

the detection of ≥ 99.9% of surviving viable E. coli in ≤ 6 hours (Table 4.7,

figure 4.6b, and 4.6c). Ampicillin and A. pilosa Ledeb decreased the detection

of E. coli inoculum in 0.5 hours (P <0.001; Table 4.7, figure 4.6b, and 4.6c). I.

domestica Goldblatt and Mabb demonstrated bactericidal activity at 6 hours

and exhibited a significantly low decrease in the percentage of detectable E.

coli compared to the other plant extracts at two hours (P <0.001; Table 4.7,

figure 4.6b, and figure 4.6c). A. pilosa Ledeb was the most effective against E.

coli. The activity of A. pilosa Ledeb (7.81mg/L) was comparable to that of

ampicillin (4 mg/L), both compounds exhibited detectable bacteriostatic activity

against E. coli throughout 24 hours at a comparatively low concentration

(Table 4.6 and Figure 4.6a). The growth control was significantly different to

five antimicrobials at 6 hours and 24 hours (P <0.001).

Table 4.6. Percentage kill of E. coli ATCC 25922, UM004, UM011, and UM012 incubated with

MIC of plant extracts and ampicillin over 24 hours


Time

(hours)

A. pilosa Ledeb (7.81)

A.

chinensis

Bunge

(125)

S. glabra

Roxb

(125)

I. domestica

(L.)

Goldblatt

and Mabb

(62.5)

Ampicillin

(8)

SEM SD

Percentage kill of E. coli

0 0 0 0 0 0 0 0

0.5 30.30d 4.01b 1.68a 5.57c 40.01e 0.009 0.043

1 26.97d 6.90b 2.25a 9.09c 39.94e 0.009 0.045

2 30.68b 43.69c 42.44c 21.71a 94.52d 0.023 0.116

4 42.40b 44.02c 50.52d 27.94a 94.20e 0.024 0.118

6 34.53b 44.62c 51.16d 22.92a 93.95e 0.024 0.119

24 19.12b 46.15c 53.75d 20.14b -448.2a 0.073 0.364


Means without common superscripts are significantly different (P <0.001). For example, Ampicillinc decreased the

detection of a significantly higher percentage of E. coli at 0.5 hours compared to all other plants with letters b, c, d, or e.

Chapter 4

139

Table 4.7. Percentage kill of E. coli ATCC 25922, UM004, UM011, and UM012 incubated with

4xMIC of plant extracts and ampicillin over 24 hours


Time (hours)

A. pilosa Ledeb (62.5)

A. chinensis Bunge (1000)

S. glabra Roxb (1000)

I. domestica (L.) Goldblatt and Mabb (500)

Ampicillin (64)

SEM SD

Percentage kill of E. coli

0 0 0 0 0 0 0 0

0.5 96.24b 0.76a 0.25a 1.92a 95.10b 0.418 2.092

1 96.16b 0.76a 1.49a 2.31a 95.92b 0.400 1.997

2 99.60b 99.24b 99.27b 31.01a 99.66b 0.124 0.621

4 99.99a 99.62a 99.99a 56.59a 99.99a 0.073 0.367

6 99.99 99.99 99.99 99.99 99.99 0 0

24 99.99 99.99 99.99 99.99 99.99 0 0 Negative numbers (-) indicate growth (no decrease in the detection of bacteria). a, b superscripts indicate significance.

Means without common superscripts are significantly different (P <0.001). For example, A. pilosa Ledeba and

ampicillina decreased a significantly higher percentage of the detection of E. coli at 0.5 hours compared to all other

plants with letter b.

Figure 4.6. Total viable count of E. coli ATCC 25922 and UM004, UM011 and UM012

incubated with plant extracts and Ampicillin over 24 hours

Figure 6a. MIC

0 1 2 3 4 5 61.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

24

A. pilosa Ledeb

S. glabra RoxbA. chinensis Bunge

I. domestica Goldblatt and MabbAmpicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Chapter 4

140

Figure 4.6b. 4xMIC

0 2 4 61.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

24

A. pilosa Ledeb

S. glabra Roxb

A. chinensis Bunge

I. domestica Goldblatt and Mabb

Ampicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)


0 1 2 3 4 5 61.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

6 24

1/2xMIC MIC 2XMIC 3XMIC 4XMIC ]- A. pilosa Ledeb

Growth control

1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- A. chinensis Bunge1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- I. domestica Golblatt and Mabb


1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- ampicillin

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)


the mean.

Chapter 4

141

4.3.4. Bactericidal activity in an in vitro cecum model against S. enteritidis

At higher concentrations, 4xMIC (500 mg/L to 4000 mg/L) all the studied plant

extracts decreased the detection of S. enteritidis within two hours (P <0.001;

Table 4.9, Figure 4.7b). A. chinensis Bunge, I. domestica Goldblatt and Mabb,

and ampicillin exhibited bactericidal activity and decreased the detection of

surviving viable S. enteritidis population by ≥99.9%. A. pilosa Ledeb and S.

glabra Roxb demonstrated the lowest reduction in total viable count of S.

enteritidis (99.61% and 99.52% decrease) (P <0.001; Table 4.9, Figure 4.7b).

A. chinensis Bunge was the most effective against S. enteritidis (Table 4.8 and

4.9; Figures 4.7a and 4.7b). At lower concentrations, the MIC (62.5 mg/L to

500 mg/L) of I. domestica (L.) Goldblatt and Mabb, A. chinensis Bunge, A.

pilosa Ledeb, and S. glabra Roxb all demonstrated detectable bacteriostatic

activity against Salmonella enteritidis throughout the duration of 24 hours

compared to the growth control (Table 4.8 and Figure 4.7a). The growth control

was significantly different to five antimicrobials at 6 and 24 hours (P <0.001;

Figures 4.7a and 4.7b).

Chapter 4

142

Table 4.8. Percentage kill of S. enteritidis NCTC 0074, QA0419, LE103, and 1F6144 in the

presence of MIC of plant extracts and ampicillin over 24 hours


Time

(hours)

A. pilosa Ledeb (500)

A.

chinensis

Bunge

(62.5)

S. glabra

Roxb

(250)

I. domestica

(L.)

Goldblatt

and Mabb

(250)

Ampicillin

(4)

SEM SD

Percentage kill of S. enteritidis

0 0 0 0 0 0 0 0

0.5 -1.47a 3.92b 12.06d 5.67c 39.46e 0.011 0.055

1 2.39a 4.28b 12.77c 16.07d 41.08e 0.011 0.054

2 42.69d 36.12c 30.52b 27.53a 93.04e 0.012 0.058

4 36.65d 36.48c 31.41b 28.98a 93.59e 0.012 0.058

6 39.42d 37.33c 33.87b 25.26a 93.72e 0.013 0.063

24 43.11e 38.76d 33.87c 27.91b -525.2a 0.095 0.473


Means without common superscripts are significantly different (P <0.001). For example, ampicillinc decreased the

detection of a significantly higher percentage of S. enteritidis at 0.5 hours compared to all other plants with letter a, b,

c, or d.

Table 4.9. Percentage kill of Salmonella enteritidis NCTC 0074, QA0419, LE103, and 1F6144

in the presence of 4xMIC of plant extracts and ampicillin over 24 hours

Time (hours)


A. pilosa

Ledeb

(4000)

A. chinensis Bunge (500)

S. glabra

Roxb

(2000)

I.

domestica

(L.)

Goldblatt

and Mabb

(2000)

Ampicillin

(32)

SEM SD

Percentage kill of S. enteritidis

0 0 0 0 0 0 0 0

0.5 0.98a 0.37a 0.76a 1.15a 95.64b 0.278 1.391

1 1.62a 2.64a 0.37a 2.32a 96.03b 0.298 1.492

2 99.21b 99.25b 34.14a 99.22b 99.60b 0.453 2.267

4 99.27b 99.62b 61.24a 99.22b 99.80b 0.279 1.394

6 99.67ab 99.99b 99.99b 99.22a 99.99b 0.006 0.032

24 99.67b 99.99b 99.99b 99.61a 99.99b 0.006 0.032 Negative numbers (-) indicate growth (no decrease in the detection of bacteria). a, b superscripts indicate significance.

Means without common superscripts are significantly different (P <0.001). For example, ampicillinb decreased the

detection of a significantly higher percentage of S. enteritidis at 0.5 hours compared to all other plants with lettera.

Chapter 4

143

Figure 4.7. Total viable count of S. enteritidis NCTC 0074, and QA0419, LE103 and 1F6144

in plant extracts and ampicillin over 24 hours

Figure 4.7a. MIC

0 1 2 3 4 5 61.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

24

A. pilosa Ledeb

S. glabra Roxb

A. chinensis Bunge


Ampicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Figure 4.7b. 4xMIC

0 1 2 3 4 5 61.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

24

A. pilosa Ledeb

S. glabra Roxb

A. chinensis Bunge


Ampicillin

Growth control

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)

Chapter 4

144


0 1 2 3 4 5 61.0×1000

1.0×1001

1.0×1002

1.0×1003

1.0×1004

1.0×1005

1.0×1006

1.0×1007

1.0×1008

1.0×1009

1.0×1010

6 24

1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- A. pilosa Ledeb

Growth control

1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- A. chinensis Bunge


1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- ampicillin1/2xMIC MIC 2xMIC 3xMIC 4xMIC ]- I. domestica Golblatt and Mabb

Time (hours)

To

tal

via

ble

co

un

t (C

FU

/mL

)


the mean.

4.4. Discussion


In this chapter, the time kill assay (CLSI, 1999) was used to assess the

bactericidal activity of four plant extracts against L. monocytogenes, C. jejuni,

E. coli and S. enteritidis in an in vitro model using poultry caecum content. The

majority of research investigating the antibacterial properties of plant extracts

tends to focus on initial screening methods that use synthetic mediums, such

as broth or agar dilution, and disc diffusion (Liu et al., 2007; Fong, et al., 2016).

Chapter 4

145

These initial screening methods are valuable as they provide a discretised

value to show whether a plant extract exhibits antibacterial activity at one 24

hour time point or not and are used to identify plants with potent antibacterial

activity in vitro.

The in vitro caecum content model was used to provide further information

about the four plant extracts identified in chapter 2 with the highest levels of

antibacterial activity (McMurray et al., 2020). Although only three rather than

four plant extracts were used in the synergy study this was simply for practical

reasons as explained in chapter 3. Therefore, in the in vitro caecum content

model the antibacterial activity of A. pilosa Ledeb, S. glabra Roxb, A. chinensis

Bunge, and I. domestica (L.) Goldblatt and Mabb over time and different

concentrations against L. monocytogenes, E. coli, and S. enteritidis was

investigated.

4.4.2. Time kill in broth

The results from the time kill assay demonstrated that A. pilosa Ledeb, S.

glabra Roxb, A. chinensis Bunge, and I. domestica (L.) Goldblatt and Mabb all

demonstrated detectable bacteriostatic activity against L. monocytogenes, C.

jejuni, E. coli and S. enteritidis in broth from 0 hours to 24 hours. This justified

their use in the next experiment to determine the effect of these plants in the

inhibition of growth of L. monocytogenes, E. coli and S. enteritidis in chicken

caecum contents.

Chapter 4

146

4.4.3. In vitro caecum content model

The novel results from this in vitro model presented rapid bactericidal activity

against both inoculated bacteria cultures and endogenous bacteria. This

strongly supported and confirmed the rapid bactericidal activity found in the

time kill experiment using broth. These results were similar to previous

research which used in vitro poultry caecum content models to demonstrate

the bactericidal activity of other plant extracts (Vasudevan et al., 2005; Johny

et al., 2010; Rubinelli et al., 2017). Consequently, this suggests that these plant

extracts could relatively quickly reduce the bacterial pathogen count rate to low

numbers.

The results from this in vitro poultry caecum content model demonstrated that

the extracts of A. pilosa Ledeb had an inhibitory effect on E. coli and L.

monocytogenes at MIC 7.81 mg/L and MIC 31.25 mg/L. This antibacterial

activity is comparable to ampicillin which inhibits L. monocytogenes, and E.

coli at 5mg/L, and 4mg/L, respectively (Kim et al., 1984; Lemaire et al., 2005).

This highlights the potential of this extract for further study as a replacement

for antibiotics in poultry feed in an in vivo trial to inhibit populations of

pathogens in the GI tract of poultry.

S. glabra Roxb (2000mg/L), A. chinensis Bunge (500mg/L), and I. domestica

(L.) Goldblatt and Mabb (2000mg/L) decreased the detection of S. enteritidis

populations by ≥99.9% within 6 hours. Research by Vasudevan et al., (2005)

using a similar model demonstrated the bactericidal activity of caprylic acid

which is a medium chain fatty acid in autoclaved chicken caecum contents with

Chapter 4

147

complete inactivation of S. enteritidis within 24 hours. The use of this poultry

caecum content model to demonstrate the antibacterial activity of plant

extracts was later validated when further results showed that caprylic acid

could significantly reduce S. enteritidis in commercial broiler chickens when

mixed in feed (Solis De Los Santos et al., 2008; Johny et al., 2009; Solis de

los santos et al., 2009). Therefore, the results showing significant antibacterial

activity of plant extracts demonstrated with chicken caecum contents in this

study provide a valid basis for future studies with plant extracts in chickens.

Research by Johny et al., (2010) reported that essential oils including

carvacrol, trans-cinnamaldehyde, eugenol, and thymol reduced S. enteritidis

and C. jejuni by ≥99.9% within 8 hours in chicken caecum contents in vitro.

The more rapid antibacterial activity demonstrated in this study may be due to

differences in methods. In the study by Johny et al., (2010) contents from

poultry caecum were autoclaved to eradicate activity of all endogenous

bacteria. However, this study used the chicken caecum content model with a

significant modification. Selective broth was employed as a growth medium to

maintain the existing populations of endogenous bacteria in the poultry

caecum belonging to the genus being investigated. Autoclaving was avoided

to ensure the proteins in the poultry caecum were not denatured at high

temperatures. This is more representative of the natural live environment in

the chicken GI tract. This is the first study to use this model to assess the

antibacterial effect of the plant extracts of A. pilosa Ledeb, S. glabra Roxb, A.

chinensis Bunge, and I. domestica (L.) Goldblatt and Mabb.

Chapter 4

148

Research by (Rubinelli et al., 2017) reported the bactericidal effect of Calrose

rice bran cultivars in poultry caecum content on Salmonella typhimurium. An

increase in Firmicutes such as lactic acid bacteria was found. This highlights

a limitation to the methodology used in this study. The caecum content was

treated with selective broth so only the pathogen of interest could be studied

and in turn the Firmicutes and other bacterial population could not be studied

at the same time. It would be useful to investigate the effect of A. pilosa Ledeb,

S. glabra Roxb, A. chinensis Bunge, and I. domestica Goldblatt and Mabb on

the populations of beneficial bacteria, such as lactic acid bacteria in future

studies, such as a poultry trial.

4.5. Conclusion

Extracts of A. pilosa Ledeb, S. glabra Roxb, A. chinensis Bunge, and I.

domestica (L.) Goldblatt and Mabb demonstrated detectable bacteriostatic

activity against L. monocytogenes, E. coli, C. jejuni and S. enteritidis in broth

over 24 hours. Furthermore, A. pilosa Ledeb, S. glabra Roxb, and A. chinensis

Bunge decreased caecum colonisation of E. coli, L. monocytogenes, and S.

enteritidis. The results in this study justify further in vivo poultry trials

investigating the antibacterial activity of these plants in the GI tract of broiler

chickens. This would provide the opportunity to determine the effects of these

plants on poultry GI microbiota, digestibility, performance, and health.

Chapter 5

149

5. Chapter 5

The effect of A. pilosa Ledeb, A. chinensis Bunge, and S. glabra Roxb

on broiler performance, nutrient digestibility, and gastrointestinal tract

microbiota

5.1. Introduction

Antibiotics have been widely used in poultry farming since the 1940s for

therapeutic, prophylactic and growth promotion purposes (Collignon and

McEwen, 2019; Kirchhelle, 2018). The overall health of the bird depends on

maintaining the health of its GI tract. Antibiotics used as supplements in poultry

feed diets help to maintain bird GI tract health by selectively modifying

microbiota. Antibiotics inhibit colonisation by bacterial pathogens (Dibner and

Richards, 2005), improve poultry digestion, nutrient utilisation and feed

efficiency (Allen and Stanton, 2014; Lee et al., 2012).

However, the use of antibiotics in poultry farming has been identified as one of

the main causes of the global antibiotic crisis. Currently, high levels of antibiotic

and multidrug resistance across a range of bacterial pathogens have been

reported in all countries. Antibiotic resistant infections results in an estimated

33, 000 deaths yearly in the EU (ECDC, 2019) and 35, 000 deaths yearly in

the USA (CDC, 2020).

Therefore, finding an alternative to antibiotics that can be used to improve

performance and bird health without selecting for antibiotic resistance could

contribute to reducing the use of antibiotics in poultry farming. A significant

volume of research suggests plant extracts could provide a viable alternative

to using antibiotics as a supplement in poultry diets (Hashemi and Davoodi,

Chapter 5

150

2010; Yang et al., 2019). Studies of plant extracts used to supplement poultry

diets have shown they can provide similar benefits to antibiotics such as

inhibiting pathogens, increasing beneficial bacteria, and improving digestibility,

growth and performance (Gadde et al., 2017; Lillehoj et al., 2018).

The results of chapter two highlighted antibacterial properties of A. pilosa

Ledeb, A. chinensis Bunge, S. glabra Roxb against C. jejuni, L.

monocytogenes, E. coli and S. enteritidis. In chapter four these plant extracts

were found to demonstrate significant rapid bactericidal activity against the

bacterial pathogens L. monocytogenes, E. coli and Salmonella enteritidis in an

in vitro model using poultry caecum as a growth medium. A. pilosa Ledeb, A.

chinensis Bunge, S. glabra Roxb were therefore chosen for an in vivo trial

based on their antibacterial activity in the previous study.

The aim of this study was to establish the effect of plant supplementation to

broiler chicken diets on performance, nutrient digestibility, and GI tract

microbiota of broiler chickens. The objectives were to:

• determine the effect of plant dietary inclusion on production

performance of, and nutrient digestibility in, broiler chickens,

• establish the effect of plant dietary inclusion on intestinal pathogenic

bacteria Campylobacter spp. and E. coli, and on beneficial bacteria

including Lactic acid bacteria in the broiler caecum.

Chapter 5

151

5.2. Methods

5.2.1. Experimental diets

The experimental diets were formulated to exceed or meet the nutritional

recommendations for Male Ross 308 broilers (Aviagen, 2019). Ingredient

composition and formulation nutrient specification are presented in Table 5.1

and 5.2. Birds were offered the starter diet from day 0 to 14 followed by the

grower/finisher diet from 14 to 35 days. The root of A. chinensis Bunge, the

tuber of S. glabra Roxb, and the herb of A. pilosa Ledeb were sourced

commercially from Hutchison Whampoa Guangzhou Baiyunshan Chinese

Medicine Co. Ltd and purchased from Kuang Kua Qing Pharmacy as dried

plants. The accepted names of these plants are in accordance with The Plant

List (2013). These names were used to prevent the use of synonyms. Each

plant (4kg) was powdered through a 1mm screen using a hammer mill (Christie

and Norris, AFBI) according to manufacture instructions. The antibacterial

activity of these aqueous plant extracts was investigated in the previous in vitro

studies in chapters 2 to 4 using the broth microdilution method (CLSI, 2009)

and modifications of the time kill method (CLSI, 1999). This requires aqueous

solutions of plants and antibiotics for susceptibility testing. However, solid dried

plants in feed were chosen for the in vivo trial. This method was selected as

opposed to administering the aqueous plant extract in water solutions. The

main reason for this was the nutrient bioavailability in feed is likely to be higher

than that of water because the feed is retained within the digestive system for

a longer time. This allows nutrients and the compounds in the plants to be

absorbed and processed and allows time for the compounds to have an effect

on the microbiota. Whereas, the plant extracts in drinking water will pass

Chapter 5

152

through the urinary system more quickly (Lohakare et al., 2004). In addition,

the loss of water due to evaporation and spillages may lead to inaccuracies in

measuring the volume of water consumed by the birds.

There were six dietary treatments. Two dietary treatments were considered as

positive controls. One positive control contained a diet with reduced nutrient

specification and 40 mg/kg of amoxicillin (T1). Another positive control

consisted of a diet with the recommended nutrient specification (T2). One

dietary treatment was considered as the negative control. This was a diet with

reduced nutrient specification containing no antibiotic or plant (T3) (Table 5.1

and 5.2). A. pilosa Ledeb, A. chinensis Bunge, S. glabra Roxb (T4, T5, and

T6, respectively) were added at 20 g/kg inclusion rate to create the other

experimental treatment diets (Table 5.1). There was a significant gap in the

literature related to the effects of A. pilosa Ledeb, A. chinensis Bunge and S.

glabra Roxb in poultry feed. However, the inclusion rate of 20 g/kg of A.

chinensis Bunge was chosen based on previous research which demonstrated

that an inclusion rate of 20 g/kg of A. chinensis Bunge was the optimal

inclusion rate for improved FCR and increased body weight gain (Xiang et al.,

2009). The nutritional composition of these plants was analysed, and the diet

formulations modified accordingly to take account of the key nutrients they

contained (Table 5.3).

Chapter 5

153

Table 5.1. Composition (g/kg) of experimental treatment diets

Feed ingredient Starter (0-14d) Grower/finisher (14-35d)

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Wheat 640 650 640 619 619 619 6261 6776 6261 6116 6117 6116

Wheat Pollard 50 0 50 50 50 50 100 25 100 100 100 100

Rapeseed Meal 30 0 30 25 25 25 25 0 25 0 0 0

Soybean meal 48 214 252 214 220 220 220 180 180 180 195 195 195

Full fat soya 0 20 0 0 0 0 0 54 0 10 10 10

Soy oil 20 30 20 20 20 20 30 30 30 25 25 25

Salt 5 5 5 5 5 5 1 1 1 1 1 1

Sodium Bicarbonate 5 5 5 5 5 5 5 0 5 5 5 5

DL Methionine 2 2 2 2 2 2 1.8 1.9 1.8 1.8 1.8 1.8

Lysine HCl 3.7 3.0 3.7 3.6 3.6 3.6 3.2 2.7 3.2 2.9 2.8 2.9

Threonine 1.2 1.0 1.2 1.2 1.2 1.2 1.4 1.3 1.4 1.3 1.3 1.3

Limestone 2 4 2 2 2 2 1 1 1 1 1 1

Mono Dicalcium Phosphate 9.5 10 9.5 9.5 9.5 9.5 7.5 7.5 7.5 7.4 7.4 7.4

Vitamin premix 15 15 15 15 15 15 15 15 15 15 15 15

Titanium dioxide 3 3 3 3 3 3 3 3 3 3 3 3

Amoxicillin 10mg/kg 0.1 0 0 0 0 0 0.1 0 0 0 0 0

A. chinensis Bunge 0 0 0 0 20 0 0 0 0 0 20 0

S. glabra Roxb 0 0 0 0 0 20 0 0 0 0 0 20

A. pilosa Ledeb 0 0 0 20 0 0 0 0 0 20 0 0 Starter supplied per tonne of diet: vitamin A 10MIU, vitamin D3 5MIU, vitamin E 100g, vitamin K 4g, thiamine (B1) 3g, riboflavin (B2) 6g, pyridoxine 5g, vitamin B12 27.5mg, biotin (2.5%) 0.25g, calcium pantothenate 12.5g, nicotinic 45g, folic acid 1.5g, iodate-calcium 2g, selenite-sodium 0.25g, iron sulphate 45g, molybdate-sodium 0.5g, manganese oxide 90g, copper sulphate 15g, zinc oxide 90g, betaine 500g, optiphos 5000 25g, hostazyme 50g, Finisher supplied per tonne of diet: vitamin A 10MIU, vitamin D3 5MIU, vitamin E 75g, vitamin K 3g, thiamine (B1) 2g, riboflavin (B2) 4g, pyridoxine 4g, vitamin B12 22.5mg, biotin (2%) 0.15g, calcium

pantothenate 10g, nicotinic 40g, folic acid 1.5g, iodate-calcium 2g, selenite-sodium 0.25g, iron sulphate 40g, manganese oxide 90g, copper sulphate 15g, zinc oxide 90g, betaine 500g, optiphos 5000

25g, hostazyme 50g.

Chapter 5

154

Table 5.2. Calculated analysis of treatment diets

Nutrient (g/kg)

Starter (0-14d)

Finisher/grower (15-35d)

T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6

Formulated nutrient content

Crude protein 209 219 209 209 209 208 194 202 194 196 196 195

Metabolisable energy MJ/kg 12.84 13.17 12.84 12.89 12.87 12.88 13.17 13.56 13.17 13.30 13.29 13.30

Calcium 9.5 9.5 9.5 9.5 9.5 9.5 7.9 7.8 7.9 7.8 7.8 7.8

Phosphorus 6.9 7.0 6.9 6.8 6.8 6.8 6.3 6.3 6.3 6.2 6.2 6.2

Available Phosphorus 4.4 4.5 4.4 4.4 4.4 4.4 4.0 4.0 4.0 4.0 4.0 4.0

Fat 35.3 47.6 35.3 35.3 35.3 35.0 44.8 54.0 44.8 41.9 41.9 41.6

Fibre 27.1 25.0 27.1 33.3 31.9 31.4 26.1 25.7 26.1 31.0 29.7 29.2

Methionine 5.1 5.2 5.1 5.1 5.1 5.1 4.7 4.9 4.7 4.7 4.7 4.7

Cysteine 3.8 3.8 3.8 3.7 3.7 3.7 3.6 3.6 3.6 3.5 3.5 3.5

Methionine + Cysteine 9.0 9.1 9.0 9.0 9.0 8.9 8.4 8.6 8.4 8.3 8.3 8.3

Lysine 12.5 12.9 12.5 12.5 12.5 12.4 11.0 11.3 11.0 11.1 11.0 11.0

Histone 5.1 5.4 5.1 5.1 5.1 5.1 4.7 4.9 4.7 4.7 4.7 4.7

Tryptophan 2.5 2.7 2.5 2.5 2.5 2.5 2.3 2.4 2.3 2.3 2.3 2.3

Threonine 8.4 8.6 8.4 8.4 8.4 8.3 7.9 8.2 7.9 7.9 7.9 7.9

Arginine 12.6 13.6 12.6 12.6 12.6 12.6 11.4 12.2 11.4 11.7 11.7 11.7

Isoleucine 8.0 8.7 8.0 8.1 8.1 8.0 7.3 7.8 7.3 7.5 7.5 7.5

Leucine 14.7 15.7 14.7 14.7 14.7 14.6 13.5 14.3 13.5 13.8 13.8 13.7

Phenylalanine 9.6 10.3 9.6 9.6 9.6 9.6 8.8 9.5 8.8 9.0 9.0 9.0

Tyrosine 6.7 7.2 6.7 6.7 6.7 6.6 6.1 6.5 6.1 6.2 6.2 6.2

Valine 9.2 9.7 9.2 9.2 9.2 9.1 8.5 8.9 8.5 8.6 8.6 8.5

Glycine 8.4 8.8 8.4 8.4 8.4 8.3 7.7 8.1 7.7 7.8 7.8 7.7

Serine 9.4 10.1 9.4 9.4 9.4 9.4 8.6 9.2 8.6 8.8 8.8 8.8

Phenylalanine + Tyrosine 16.2 17.5 16.2 16.3 16.3 16.2 14.9 16.0 14.9 15.2 15.3 15.2

Chapter 5

155

5.2.2. Experimental design, birds, and management

A total of 420-day-old male Ross 308 broiler chicks, obtained from Moy Park

Hatchery were allocated randomly to 30 pens (size 1.5m x 2m). The chickens

were assigned to one of six dietary treatments (five pen replicates; 14 chicks

per pen) and raised under environmentally controlled conditions. These

conditions followed a standard temperature regimen that decreased gradually

from 32oC to 22oC by 0.5oC increments per day. A 18hrL:6hrD lighting

programme was followed according to management recommendations for

Ross 308 broilers (Aviagen, 2018). Poultry feed and water were provided ad

libitum. The trial was approved by the Animal Welfare Ethical Review Body at

AFBI and conducted under Animals Scientific Act 1986.

All birds were weighed immediately after their arrival to determine their initial

weight and assigned an individual leg-tie for individual identification throughout

the trial. Body weight was recorded weekly on a per pen, and on an individual

basis and feed intake was recorded weekly on a per pen basis. Feed intake

and body weight were used to subsequently calculate growth performance

parameters such as, weight gain, and feed conversion ratio (FCR). Mortality

and morbidity rates were recorded (Roofchaee et al., 2011).

Litter quality was assessed by measuring pH and moisture content. Ammonia

concentrations from each pen were measured on days 20, 27, and 34 to

provide an estimation of ammonia production per pen at each time period. One

15 litre clear plastic container (470mm x 300mm x 170mm) was placed in each

pen. One Gastec Passive Dosi-tube No. 3D for ammonia (SKC, UK) with a

measurement range of 2.5 to 1,000ppm, was held in place in each plastic

container with two level Diall M4 x 46mm hollow wall anchor cup hooks for 4

Chapter 5

156

hours to take an on-the-spot time-weighted average. This is a modification to

a previous method (Xu et al., 2017). The ammonia concentrations were

calculated using the following equation: NH3 (ppm) = (ppm-h)/t. ppm-h was the

Dosimeter tube reading in ppm h and t was the sampling time in hours as in

previous methods (Wang-Li et al., 2020). One NH3 monitor, the multi-gas

detector eco (International Gas Detectors ltd, UK) with a measurement range

of 0-100ppm was also placed in each plastic container for an on-the-spot

reading three separate times at days 27 and 34. The objective was to measure

ammonia levels from each pen to ascertain if dietary treatment influenced the

production of ammonia. Litter samples were taken at 20, 27, and 34 days of

age from 6 evenly distributed positions within each pen and mixed to create a

composite sample per pen. Percentage moisture content was determined by

weight loss (AOAC, 1998) after sufficient drying in a Genlab Mino Oven

(N175CF Genlab MINO/175/F) at 80°C for 48h. Litter pH was determined by

diluting samples with distilled water (1:10) as per manufacturer’s instructions

and using a pH meter (Hanna instruments, Fischer scientific, UK).

On days 14 and 35, two broilers per pen were randomly selected,

anaesthetised then killed by cervical dislocation. The abdominal cavity was

opened, and the total GI tract immediately exposed. From each euthanised

bird, the ileum, starting from the Meckel’s diverticulum to 10cm above the ileo-

caecal junction, was quickly dissected (Mohiti-Asli and Ghanaatparast-Rashti,

2017) and the ileal contents collected and immediately frozen. Ileal contents

were sufficiently dried in a Genlab Mino Oven (N175CF Genlab MINO/175/F)

at 80°C for 72h. Ileal samples were powdered through a 1mm screen using a

hammer mill (Christie and Norris, AFBI) according to manufacturer

Chapter 5

157

instructions. Ileal samples were combined on a per pen basis to give six pen

replicates of ileal per treatment.

5.2.3. Laboratory analysis

The dried samples of plants, diets and ileal digesta were analysed for various

chemical constituents according to methods outlined in AOAC (1990) unless

described otherwise. All results are reported on a DM basis. The GE content

was measured using the isoperibol bomb calorimeter (Parr Instruments Co.,

Moline, IL). Titanium dioxide content was measured according to the

procedure by Leone (1973) and modified by (Peddie et al., 1982). Amino acid

content was determined by HPLC according to methods by the International

Organisation for Standardisation namely, ISO 13903:2005 (ISO, 2005) and the

European Commission (2009). The determination of Tryptophan content in

feed was based on the method of the Swedish standards institute, (2005).

5.2.4. Lactic acid bacteria, Campylobacter spp., and E. coli

At the end of 7, 14, 21, and 35 days of age, five birds from each treatment

group were randomly selected to provide reliable results and a representative

sample of the birds (one bird from each pen). These birds were euthanised

and killed as above to determine average bacterial count from caecum

contents. Two caeca from each bird were placed into sterile 50ml tubes. The

samples were immediately chilled on ice and transported to the laboratory for

analysis within three hours from collection. The caecum samples were cut

open with a sterile scalpel and the contents were squeezed into sterile 20 ml

tubes. A 10-1 dilution was prepared by adding 1 g of the caecum contents to 9

Chapter 5

158

ml of maximum recovery diluent (MRD) (Oxoid Code CM0733B Thermo

Scientific, UK). A 10-1 to 10-6 dilution series was prepared in MRD. Lactic acid

bacteria (LAB), Escherichia coli and Campylobacter spp. were enumerated by

conventional microbiological techniques using a suitable selective agar. For

LAB, a 1 ml portion of each dilution was pour-plated, in duplicate, using De

Man, Rogosa and Sharpe (MRS ISO) agar (Oxoid Code CM1153B). Once set,

the plates were overlaid with another layer of MRS agar to restrict oxygen. To

enumerate E. coli, 100 µL of each suitable dilution was spread-plated onto

duplicate TBX agar plates (Oxoid Code CM0945B). For Campylobacter spp.,

100 µL of each suitable dilution was spread plated onto mCCDA agar plates

(Oxoid Code CM0739 supplemented with SR0155E). In addition, to help lower

the limit of detection in case pathogens are present at low levels, 1 ml of the

10-1 dilution was spread over three mCCDA plates (Khattak et al., 2018) and

the plates allowed to dry before incubation. The MRS agar plates were

incubated aerobically at 30°C for 72 hours. The TBX plates were incubated at

37°C for 24 hours. The mCCDA plates were incubated in a microaerophilic

workstation (Don Whitley Scientific, UK) at 41.5°C for 48 hours, in an

atmosphere of 5% oxygen, 10% carbon dioxide and 85% Nitrogen.

Agar plates of an appropriate dilution level with 30 to 300 bacteria colonies

were selected and colonies counted using a plate counter (Stuart Scientific,

UK).

Chapter 5

159

5.2.5. Statistical analysis and calculations

Descriptive statistics were analysed using Microsoft Excel and presented as

mean ± SD. GraphPad Prism (Version 5.0) was used for statistical analysis to

determine significant statistical differences. One-way analysis of variance

(ANOVA) was used to analyse the data followed by Bartlett’s test. A value of

P <0.05 was set to determine statistical significance between the experimental

treatments and the control groups. Pen was taken as the experimental unit.

The feed conversion ratio was calculated as the ratio of weight of feed intake

per pen to average weekly weight gain per pen (Yu et al., 2019). The FCR was

adjusted for mortality by calculating the ratio of weight of feed intake per pen

to average weekly weight gain of survivors per pen + average weekly weight

gain of mortalities per pen (Benites et al., 2008). Moisture content values were

calculated using the following equation: Percentage moisture content =[(Wet

litter weight (g) − dry litter weight (g)) / wet litter weight(g)] × 100 (Hayes et al.,

2000). Ileal digestibility was calculated through the use of titanium dioxide as

an indigestible marker (Darambazar, 2019)

The CFUs from agar plates were averaged using Excel to express 1g of CFU

per gram of caecum contents. The average CFUs from agar plates were log

transformed using GraphPad Prism (Version 5.0) and statistical significance

was determined using ANOVA.

Chapter 5

160

5.3. Results

5.3.1. Nutritional composition of plants

The crude protein content ranged from 46 g/kg (S. glabra Roxb) to 91 g/kg (A.

chinensis Bunge). Ash levels ranged from 20 g/kg (S. glabra Roxb) to 120 g/kg

(A. chinensis Bunge). Oil levels ranged from 4.2 g/kg (S. glabra Roxb) to 20.1

g/kg (A. chinensis Bunge). A. chinensis Bunge contained the highest amino

acid levels, ash, and oil levels, followed by A. pilosa Ledeb, and S. glabra Roxb

with the exception of arginine for which S. glabra Roxb contained the highest

amount. Crude fibre content was highest for A. pilosa Ledeb (349 g/kg),

followed by A. chinensis Bunge (280 g/kg), then S. glabra Roxb (258 g/kg).

Chapter 5

161

Table 5.3. Nutritional composition of dried plants as they arrived, commercially

dried

Nutritional compound Total composition g/kg

A. pilosa Ledeb

S. glabra Roxb

A. chinensis Bunge

Crude Protein (N x 6.25) (Dumas)

72 46 91

Crude Fibre 349 258 280

Ash 70 20 120

Total Oil (Oil B) 19 4.2 20.1

Alanine 3.0 0.9 3.9

Arginine 3.2 4.4 4.0

Aspartic acid 6.7 2.7 9.3

Cysteine 0.8 0.7 1.0

Glutamic acid 6.8 2.9 8.9

Glycine 3.4 1.4 4.1

Histidine 1.4 0.5 1.6

Iso-Leucine 2.9 1.0 3.8

Leucine 5.0 1.7 6.5

Lysine 3.6 1.5 4.0

Methionine 1.3 0.5 1.5

Phenylalanine 3.5 1.1 4.1

Proline 2.8 1.1 4.4

Serine 3.1 1.6 3.9

Threonine 3.0 1.4 3.8

Tyrosine 1.9 0.8 2.4

Valine 0.36 0.15 0.46

Tryptophan 0.09 0.03 0.11

Gross energy (MJ/kg) 16.32 16.04 15.50

5.3.2. Performance, and nutrient digestibility

From 0 to 14 days, birds fed with diets containing A. chinensis Bunge, S. glabra

Roxb, and amoxicillin in groups had significantly higher feed intake than the

recommended nutrient specification (positive control), (P <0.01; Table 5.4).

The birds being supplemented with amoxicillin had a significantly higher weight

gain than those offered any of the other diets apart from those offered the diet

containing A. chinensis Bunge. There was no difference in the growth of birds

Chapter 5

162

offered the recommended or reduced nutrient specification (negative control)

diet for this 0-14 day stage (Table 5.4). There was no significant difference in

FCR for the 0–14-day period but there was a tendency for birds offered the

diets containing amoxicillin, recommended nutrients, and A. pilosa Ledeb to

be more efficient (P =0.095; Table 5.4)

From 14 to 35 days birds offered the diet including A. chinensis Bunge had the

highest feed intake (P <0.05; Table 5.4). Diets containing A. chinensis Bunge,

S. glabra Roxb, and amoxicillin (positive control) significantly increased weight

gain (P <0.001) compared to the weight gain of birds given the recommended

nutrient specification, reduced nutrient specification and the diet containing A.

pilosa Ledeb. This was reflected in higher live weights at 14 days for birds

receiving diets amoxicillin and A. chinensis Bunge. Birds offered the diets

containing amoxicillin and S. glabra Roxb were significantly more efficient at

converting gain to feed (1.51 and 1.48 respectively). The diet containing the

reduced nutrient specification resulted in the least efficient conversion of 1.75

(Table 5.4).

From 0 to 35 days feed intake was highest for birds receiving diets containing

amoxicillin and A. chinensis Bunge (P <0.05; Table 5.4). Diets containing

amoxicillin (positive control), A. chinensis Bunge, and S. glabra Roxb resulted

in significantly higher weight gains and liveweights at 35 days than those

observed for birds offered the other diets. (P <0.001; Table 5.4). Feed

conversion ratios were most efficient for birds offered diets containing

amoxicillin, A. pilosa Ledeb and S. glabra Roxb and least efficient in birds

offered diets containing the reduced nutrient specification (P <0.001; Table

5.4).

Chapter 5

163

Overall, diets containing A. chinensis Bunge and S. glabra Roxb resulted in

similar growth rates and 35 days liveweights as birds offered the diets

containing Amoxicillin. All three diets significantly improved growth rate and

35d liveweight compared to birds offered the reduced nutrient specification

negative control diet. In terms of FCR, diets containing. The diet containing S.

glabra Roxb improved feed efficiency in birds above that of birds offered the

negative control and to a level equal to the that achieved by birds offered the

diet containing amoxicillin. The diets containing the recommended level of

nutrient specification and A. chinensis Bunge resulted in performance in-

between that achieved by birds offered the reduced nutrient specification diet

and the diet containing A. pilosa Ledeb a lower intake and hence growth rate

although FCR was quite efficient (Table 5.4)

There were no significant differences in the coefficient of variation in liveweight

or liveweight gain between treatment groups throughout the trial (Table 5.5)

There were no significant differences in mortality between treatment groups

(Table 5.6). Five broilers throughout the trial were euthanised as they were

unable to stand so there was no morbidity due to infections throughout the trial.

Ten of the chickens were found dead in the pen and seven were euthanised

as they displayed at least two symptoms of: inability to stand; difficulties

breathing; huddled up, subdued with eyes closed; ruffled feathers; cold and

lethargic. There were no abnormalities detected in chicken post mortem

reports throughout the trial. One chicken had ascites with secondary

septicaemia resulting in associated hepatitis, myocarditis, and broncho

interstitial pneumonia, another chicken had ascites with suspected cardiac and

Chapter 5

164

respiratory failure due to fast growth rate but not thought to be treatment

related.

At 20, 24 and 34 days there were no significant differences between treatment

groups in ammonia (NH3) output, litter moisture content, or pH of litter (Table

5.7) with one exception. Litter in pens from birds receiving diets containing

amoxicillin (positive control), the recommended nutrient specification (positive

control) and A. chinensis Bunge had significantly lower pH values than litter

from birds receiving the reduced nutrient specification (negative control), A.

pilosa Ledeb, and S. glabra Roxb (P <0.001; Table 5.7).

The ileal digestibility of dry matter was higher in the recommended nutrient

specification (positive control) compared to all other treatments (P <0.01). The

ileal digestibility of ash ranged from 0.24 for birds receiving the antibiotic to the

highest levels of 0.53 and 0.57 for birds offered the recommended nutrient

specification (positive control) and the A. chinensis Bunge supplemented

treatment group. The ileal digestibility of ash was variable. There were no

significant differences between treatment groups in the ileal digestibility of

crude protein (Table 5.8).

Chapter 5

165

Table 5.4. The effect of plant additives on feed intake (g), weight gain (g), and feed conversion ratio from 0 to 35 days of age.

Day Parameter Treatment SEM P value

T1 T2 T3 T4 T5 T6

0-14 FI (g) 581.2bc 510.0a 542.8ab 551.9ab 605.8c 571.9bc 16.72 <0.01

WG (g) 493.8b 433.0a 437.6a 465.5ab 483.0b 460.3ab 10.85 <0.01

FCR 1.18 1.18 1.24 1.19 1.25 1.24 0.024 NS (0.095)

14-35 FI (g) 2624.6a 2562.3a 2540.1a 2465.6a 2856.2b 2626.1ab 81.72 <0.05

WG (g) 1737.7b 1556.6a 1450.1a 1566.1a 1757.6b 1776.4b 39.13 <0.001

FCR 1.51a 1.65bc 1.75c 1.58ab 1.63b 1.48a 0.035 <0.001

0-35 FI (g) 3205.9ab 3072.3a 3082.9a 3017.5a 3462.0b 3197.7a 86.99 <0.05

WG (g) 2231.5b 1989.6ab 1887.7a 2031.6b 2240.6b 2236.7b 41.96 <0.001

FCR 1.44a 1.54b 1.63c 1.49ab 1.55b 1.43a 0.027 <0.001

0 Live weight (g)

44.14 43.82 43.8 43.42 44.46 43.54 0.385 NS (0.419)

14 538.0c 476.8a 481.4a 508.9abc 527.5bc 503.8ab 10.94 <0.01

35 2275.7b 2033.4ab 1931.5a 2075.0b 2285.0c 2280.2c 41.95 <0.001 Differences in superscripts (ab) indicate significant differences between treatment groups (rows). T1 = reduced nutrient specification with 40mg/kg of amoxicillin.

T2 = recommended nutrient specification. T3 = reduced nutrient specification. T4, T5, and T6 = 2% inclusion rate of A. pilosa Ledeb, A. chinensis Bunge, S.

glabra Roxb, respectively.

Chapter 5

166

Table 5.5. The effect of plant additives on Coefficient of Variation of live weight

and weight gain

Day

Parameter (%)

Treatment SEM P value

T1 T2 T3 T4 T5 T6

0 Live weight CoV

0.67 0.08 0.08 0.08 0.08 0.09 0.256 NS (0.449)

7 0.09 0.12 0.09 0.11 0.08 0.10 0.014 NS (0.361)

14 0.09 0.10 0.10 0.10 0.08 0.09 0.008 NS (0.900)

21 0.07 0.07 0.07 0.06 0.07 0.07 0.0004 NS (0.675)

28 0.06 0.06 0.06 0.06 0.07 0.05 0.007 NS (0.615)

35 0.06 0.05 0.07 0.08 0.08 0.06 0.014 NS (0.486)

0-7 Weight gain CoV

0.11 0.15 0.11 0.14 0.10 0.13 0.018 NS (0.255)

7-14 0.10 0.16 0.12 0.14 0.09 0.10 0.026 NS (0.545)

14-21 0.08 0.08 0.09 0.32 0.22 0.07 0.078 NS (0.158)

21-28 0.10 0.09 0.10 0.10 0.11 0.11 0.008 NS (0.975)

28-35 0.23 0.12 0.17 0.17 0.24 0.21 0.047 NS (0.493)

0-14 0.09 0.11 0.10 0.11 0.09 0.10 0.009 NS (0.893)

14-35 0.08 0.06 0.09 0.10 0.09 0.07 0.017 NS (0.657)

0-35 0.06 0.05 0.08 0.16 0.15 0.06 0.046 NS (0.358)

T1 = reduced nutrient specification with 40mg/kg of amoxicillin. T2 = recommended nutrient

specification. T3 = reduced nutrient specification. T4, T5, and T6 = 2% inclusion rate of A.

pilosa Ledeb, A. chinensis Bunge, S. glabra Roxb, respectively.

Table 5.6. The effect of plant additives on mortality (%) from 0 to 35 days of

age.

Day T1 (n)

T2 (n)

T3 (n)

T4 (n)

T5 (n)

T6 (n)

Total (%)

Total (n)

P value

0 0 0 0 1 0 0 0 0 NS

7 1 1 1 2 0 1 1.54 6

14 1 4 0 1 0 0 2.86 6

21 0 0 0 1 1 0 3.57 2

28 0 0 0 0 0 0 3.57 0

35 1 0 2 0 0 0 3.78 3

Total 3 5 3 5 1 1 3.78 17

T1 = reduced nutrient specification with 40mg/kg of amoxicillin. T2 = recommended nutrient



Chapter 5

167

Table 5.7. The effect of plant additives on litter ammonia, pH, and percentage

litter moisture content (LMC) from 20 to 34 days of age.

Day Parameter T1 T2 T3 T4 T5 T6 SEM P value

20 NH3 Dosi (ppm) 11.7 10.8 11.3 18.9 10.3 13.1 3.423 NS

pH 8.24 8.02 7.88 7.74 8.025 8.04 0.408 NS

LMC (%) 39.7 41.7 43.1 39.6 38.0 37.8 4.13 NS

27 NH3 Dosi (ppm) 15.5 26.0 9.5 21.0 21.0 19.0 6.24 NS

NH3 Monitor (ppm)

9.26 6.98 8.76 19.04 9.74 13.78 5.909 NS

pH 8.12 7.58 8.64 8.24 7.64 8.34 0.314 NS

LMC (%) 48.6 51.7 45.4 52.2 51.2 50.5 4.37 NS

34 NH3 Dosi (ppm) 42 53.5 47.5 64 40.5 50 14.076 NS

NH3 Monitor (ppm)

20.1 27.78 25.14 28.82 21.66 21.3 4.820 NS

pH 6.56a 7.48ab 8.34bc 8.76c 7.14a 8.50bc 0.351 <0.001

LMC (%) 55.0 62.70 56.0 62.8 61.7 61.0 3.03 NS

Differences in superscripts (ab) indicate significant differences between treatment groups

(rows). T1 = reduced nutrient specification with 40mg/kg of amoxicillin. T2 = recommended

nutrient specification. T3 = reduced nutrient specification. T4, T5, and T6 = 2% inclusion rate

of A. pilosa Ledeb, A. chinensis Bunge, S. glabra Roxb, respectively.

Table 5.8. The effect of plant additives on ileal digestibility of chickens

Digestibility T1 T2 T3 T4 T5 T6 SEM P value

Dry Matter 0.66a 0.79b 0.66a 0.70a 0.67a 0.70a 0.022 <0.01

Ash 0.24a 0.53c 0.48c 0.38b 0.57c 0.36b 0.035 <0.001

Crude Protein

0.76 0.83 0.73 0.79 0.76 0.77 0.022 NS

Differences in superscripts (ab) indicate significant differences between treatment groups

(rows). T1 = reduced nutrient specification with 40mg/kg of amoxicillin. T2 = recommended

nutrient specification. T3 = reduced nutrient specification. T4, T5, and T6 = 2% inclusion rate

of A. pilosa Ledeb, A. chinensis Bunge, S. glabra Roxb, respectively.

Chapter 5

168


Dietary treatments had no significant effect on E. coli, Campylobacter spp. or

lactic acid bacteria relative abundance within the caecal contents on days 7,

14, 21, and 35 (Figure 5.1, 5.2, and 5.3) with the exception of S. glabra Roxb

that decreased E. coli and Campylobacter spp. relative abundance on day 21

(P <0.05; Figure 5.3) and 35 (P <0.01; Figure 5.3). Amoxicillin (positive control)

also decreased Campylobacter spp. relative abundance on day 21 (P <0.05;

Figure 5.2).

The treatment groups had no significant effect on E. coli, Campylobacter spp,

or lactic acid bacteria on day 7 or 21 (Tables 5.9 and 5.11). The recommended

nutrient specification diet (positive control), the reduced nutrient specification

diet (negative control), A. pilosa Ledeb diet, A. chinensis Bunge diet, and S.

glabra Roxb diet resulted in significantly higher lactic acid bacteria relative

abundance in comparison to amoxicillin (positive control) at day 14 (P <0.001;

Table 5.10). The reduced nutrient specification diet (negative control) and S.

glabra Roxb diet also resulted in significantly higher lactic acid bacteria relative

abundance in comparison to that observed for amoxicillin diet (positive control)

at day 35 (P <0.01; Table 5.12).

Chapter 5

169

Table 5.9. The effect of plant additives on caecum E. coli, Campylobacter spp.,

and Lactic acid bacteria relative abundance at 7 days of age.

Treatment group E. coli (log10 CFU g-1)

Campylobacter spp. (log10 CFU g-1)

Lactic acid bacteria (log10 CFU g-1)

T1 7.97 2.43 8.64

T2 8.17 2.32 8.66

T3 8.22 2.48 8.72

T4 8.08 1.98 8.97

T5 8.18 2.37 9.14

T6 8.02 2.40 8.01

SEM 0.101 0.177 0.351

P value NS (0.553) NS (0.112) NS (0.333) Standard error of the mean (SEM) is indicated after each average value. T1 = reduced nutrient

specification with 40mg/kg of amoxicillin. T2 = recommended nutrient specification. T3 =

reduced nutrient specification. T4, T5, and T6 = 2% inclusion rate of A. pilosa Ledeb, A.

chinensis Bunge, S. glabra Roxb, respectively.

Table 5.10. The effect of plant additives on caecum E. coli, Campylobacter

spp., and Lactic acid bacteria relative abundance at 14 days of age.




T1 8.52 3.05 3.68a

T2 7.80 2.56 9.16b

T3 8.01 2.94 10.05b

T4 8.16 2.66 9.69b

T5 7.81 2.08 9.54b

T6 8.21 3.33 9.40b

SEM 0.234 0.286 0.339

P value NS (0.274) NS (0.125) <0.001 Differences in superscripts (ab) indicate significant differences between treatment groups

(columns). Standard error of the mean (SEM) is indicated after each average value. T1 =

reduced nutrient specification with 40mg/kg of amoxicillin. T2 = recommended nutrient

Chapter 5

170






T1 8.27 1.80 7.35

T2 7.90 2.68 9.67

T3 7.93 2.29 8.74

T4 7.62 2.32 8.81

T5 7.94 2.07 8.56

T6 7.42 2.23 9.20

SEM 0.237 0.247 0.779

P value NS (0.208) NS (0.582) NS (0.592) Standard error of the mean (SEM) is indicated after each average value. T1 = reduced nutrient

specification with 40mg/kg of amoxicillin. T2 = recommended nutrient specification. T3 =

reduced nutrient specification. T4, T5, and T6 = 2% inclusion rate of A. pilosa Ledeb, A.

chinensis Bunge, S. glabra Roxb, respectively.






T1 8.39 2.70 7.80a

T2 7.78 1.94 8.65ab

T3 7.98 2.36 9.30c

T4 8.11 2.09 8.61ab

T5 8.26 2.05 8.93b

T6 7.97 2.11 9.64c

SEM 0.223 0.189 0.308

P value NS (0.503) NS (0.095) <0.01 Differences in superscripts (ab) indicate significant differences between treatment groups

(columns). Standard error of the mean (SEM) is indicated after each average value. T1 =

reduced nutrient specification with 40mg/kg of amoxicillin. T2 = recommended nutrient

Chapter 5

171

Figure 5.1. The effect of plant additives on relative abundance of lactic acid

bacteria in the caecum from 7 to 35 days of age

7 14 21 35

8

10

12T1

T2

T3

T4

T5

T6

Day

To

tal

via

ble

co

un

t(l

og

10 C

FU

/g)

Standard error of the mean (SEM) is shown. P value = NS. T1 = reduced nutrient specification

with 40mg/kg of amoxicillin. T2 = recommended nutrient specification. T3 = reduced nutrient

specification. T4, T5, and T6 = 2% inclusion rate of A. pilosa Ledeb, A. chinensis Bunge, S.


Chapter 5

172

Figure 5.2. The effect of plant additives on relative abundance of

Campylobacter spp. in the caecum from 7 to 35 days of age

7 14 21 35

1

2

3

4T1*

T2

T3

T4

T5

T6**

ab

b

a

ab

ab

b

a

a

Day

To

tal

via

ble

co

un

t(l

og

10 C

FU

/g)

Differences in superscripts (ab) indicate significant differences between days. P value of *

indicates <0.05, P value of ** indicates <0.01. Standard error of the mean (SEM) is shown. T1

= reduced nutrient specification with 40mg/kg of amoxicillin. T2 = recommended nutrient

Chapter 5

173

Figure 5.3. The effect of plant additives on relative abundance of E. coli in the

caecum from 7 to 35 days of age

7 14 21 35

7

8

9T1

T2

T3

T4

T5

T6*

abb

a

ab

Day

To

tal

via

ble

co

un

t (l

og

10 C

FU

/g)

Differences in superscripts (ab) indicate significant differences between days. Standard error

of the mean (SEM) is shown. P value of * indicates <0.05. T1 = reduced nutrient specification

with 40mg/kg of amoxicillin. T2 = recommended nutrient specification. T3 = reduced nutrient

specification. T4, T5, and T6 = 2% inclusion rate of A. pilosa Ledeb, A. chinensis Bunge, S.


5.4. Discussion


Previous in vivo broiler trials of plant additives and antibiotics have shown

effects of plant additives in poultry that make them suitable as alternatives to

antibiotics in poultry farming, such as modulation of GI tract microbiota,

improved health, performance, and digestibility (Shan et al., 2007; Hashemi

and Davoodi, 2010; Han et al., 2012; Gadde et al., 2017; Lillehoj et al., 2018).

Results from chapter 4 have identified antibacterial activity of a variety of plant

extracts including A. pilosa Ledeb, A. chinensis Bunge, and S. glabra Roxb in

vitro and in an in vitro poultry caecum model (McMurray et al., 2020). There

Chapter 5

174

appears to be no references to in vivo broiler trials using the plants in this study

in the research literature.

Previous studies have shown that birds fed a diet supplemented with plants or

antibiotics mixed in feed can improve their performance by modifying the GI

microbiota (Maki et al., 2019). The bird caecum functions in nutrient absorption

which effects overall performance and health (Gong et al., 2002). Firmicutes

such as lactic acid bacteria primarily colonise the caecum and catalyse the

breakdown of indigestible starch cellulose promoting nutrient utilisation

(Clavijo and Florez, 2017). Any increase in pathogens such as Campylobacter

spp. or E. coli could have detrimental impacts on nutrient absorption, feed

conversion ratios, and performance. Campylobacter spp., E. coli, and lactic

acid bacteria were enumerated from the broiler caecum to determine the mode

of action of plants and antibiotics for increasing performance. If the plants

increased lactic acid bacteria and inhibited the pathogens Campylobacter spp.

or E. coli, then that would modulate the functions of the GI tract microbiota and

have positive impacts on feed conversion ratios and performance.

The nutritional chemical composition of A. pilosa Ledeb, A. chinensis Bunge,

and S. glabra Roxb was analysed. A. chinensis Bunge had the highest amino

acid content followed by A. pilosa Ledeb, and S. glabra Roxb. High amino acid

contents have been shown to increase poultry weight gain and improve

performance (Toghyani et al., 2017). There appears to be no previous

research which demonstrates the nutritional chemical composition of these

plants, but the analysis enabled more accurate formulation of the diets as

inclusion rates of other ingredients could be modified accordingly to balance

diets for total amino acids and other essential nutrients. Due to a lack of

Chapter 5

175

published information, it is difficult to draw meaningful comparisons on amino

acid content based on existing literature. The results showed that S. glabra

Roxb had the lowest crude protein (4.6%) and amino acid content compared

to the other plants in this study. The findings indicated substantial differences

in plant nutritional value. They also highlighted the need for analysis prior to

dietary inclusion.

5.4.2. A. chinensis Bunge, S. glabra Roxb and amoxicillin improved

performance

The findings form this study showed that birds on poultry diets which contained

A. chinensis Bunge and S. glabra Roxb had similar growth rates and 35d

liveweights to those on diets which included amoxicillin. The results for each

of the three poultry diets significantly improved growth rate and 35d liveweight

compared to birds offered the reduced nutrient specification negative control

diet. In terms of FCR, the poultry diet which contained S. glabra Roxb was

shown to improve the feed efficiency of the birds on that diet compared to the

birds in the negative control group. The FCR was similar to that for birds on

the poultry diet which contained amoxicillin. The poultry diets which contained

the recommended level of nutrient specification and A. chinensis Bunge

resulted in performance in- between that achieved by birds offered the reduced

nutrient specification diet (Table 5.4).

The addition of amoxicillin in the poultry diet (positive control) led to increased

weight gain and feed conversion ratios of birds, as expected. Other studies

have also found the addition of antibiotics such as, salinomycin and 500g zinc

Chapter 5

176

bacitracin (Nkukwana et al., 2015; Yu et al., 2019), flavophospholipol antibiotic

(Ghazanfari, 2015) and penicillin (Singh et al., 2013) in poultry diets have led

to increased weight gain.

The addition of A. pilosa Ledeb, A. chinensis Bunge, and S. glabra Roxb in

poultry diets all resulted in improved bird growth rates. S. glabra Roxb was

also shown to improve the FCR of the birds on that diet. This is a new finding

as there seems to be no research to show the effects of A. pilosa Ledeb, A.

chinensis Bunge, and S. glabra Roxb in birds.


Overall, broilers receiving diets containing amoxicillin had a significant

reduction in lactic acid bacteria at day 14, lactic acid bacteria increased again

by day 21 but was lowest compared to the birds on the other diets by day 35

(Table 5.10 to Table 5.12). Broilers receiving diets containing S. glabra Roxb

had the highest amount of lactic acid bacteria at day 35 compared to birds

receiving amoxicillin (Table 5.12). With the exception of S. glabra Roxb, the

dietary treatments did not significantly reduce populations of E. coli, and

Campylobacter spp. S. glabra Roxb decreased E. coli and Campylobacter spp.

populations from day 14 to day 21 (Figure 5.2 and 5.3). The reduction of these

bacteria was maintained until day 35. Poultry diets containing amoxicillin also

decreased populations of Campylobacter spp. from day 14 day 21 (Figure 5.2)

with the Campylobacter spp. populations gradually increasing again by day 35.

The microbiota results showed that the addition of S. glabra Roxb in the poultry

diet increased the lactic acid bacteria in birds on this diet by comparison with

Chapter 5

177

the birds on the diet which contained amoxicillin. The increased lactic acid may

also be associated with the improved performance of birds on this diet. This is

supported by other poultry trial studies which included poultry diet supplements

of oregano, Moringa oleifera leaf, and citrus (Nkukwana et al., 2015; Mohiti-

Asli and Ghanaatparast-Rashti, 2017; Yu et al., 2019).

Birds receiving the poultry diet containing amoxicillin demonstrated

fluctuations in lactic acid bacteria, Campylobacter spp., and E. coli. Birds on

diets which contained amoxicillin initially showed reduced levels of lactic acid

bacteria after starting the diet and then gradually increased until day 35. The

gradual increase in lactic acid bacteria in birds on the diet which contained

amoxicillin was lower than the levels of lactic acid found in birds on the other

poultry diets. In this study the findings suggest amoxicillin did not increase

performance through modulation of the microbiota but rather through the

modification of the GI tract microbiota by decreasing pathogens.

These changes in levels of lactic acid bacteria have been reported in other

poultry trials which have included antibiotics in poultry diets; avilamycin (Costa

et al., 2017), monensin (Danzeisen et al., 2011), zinc bacitracin (Yu et al.,

2019), chlorotetracycline, virginiamycin and amoxicillin (Banerjee et al., 2018).

The poultry diet which contained S. glabra Roxb was more effective than the

diet containing amoxicillin indicated by higher levels of lactic acid bacteria in

the caecum.by day 35. There appears to be no literature on in vivo broiler trials

which include S. glabra Roxb. However other poultry trial studies have shown

one of the beneficial effects of including plants in the poultry diet is increased

Chapter 5

178

abundance of lactic acid bacteria in the caecum. For example, rosemary

(Petričević et al., 2018), and citrus extract (Yu et al., 2019).

The findings of this study suggest that poultry diets which contain S. glabra

Roxb can modulate poultry caecum microbiota by decreasing E. coli and

Campylobacter spp., and increasing lactic acid. Other research also shows

that adding plants such as oregano oil, rosemary, eugenol and carvacrol can

reduce E coli and C. jejuni in the broiler caecum (Johny et al., 2010; Norouzi

et al., 2015; Roofchaee et al.,2011; Yang et al., 2019; Yu et al., 2019) and

increase the abundance of Lactobacillus (Yu et al., 2019).

The study found there was a low relative abundance of Campylobacter spp.

across all control and treatment groups. Previous in vivo broiler trials have also

found that the Campylobacter spp. counts have been too low to enumerate

(Viveros et al., 2009). Research evidence suggests that poultry can acquire

Campylobacter spp. from the environment which can colonise the broiler GI

tract (Callicott et al., 2006). Disinfecting the poultry environment can reduce

this (Corry et al., 2017). Therefore, the low levels of Campylobacter spp. in this

study may be due to AFBI management practices which require high levels of

sanitation. The poultry pens were regularly disinfected prior to the trial.

5.4.4. Morbidity and mortality of birds

In this study the mortality of broilers was recorded. Reductions in mortality and

morbidity caused by infections are important to the poultry industry as these

can lead to improved welfare, production, sustainability, and less antibiotic use

for treatments (Meseret, 2016; Jong et al., 2020). There were no significant

Chapter 5

179

differences in mortality or morbidity across treatment groups in this study. This

suggests that mortality was not a result of adverse effects of dietary

treatments. In this context of this study A. chinensis Bunge, and S. glabra Roxb

could be considered safe as they did not cause any mortality in the broiler

chicken or suppress intake. There was a total of 3.78% mortality, ten chickens

died, and seven chickens were euthanised as they were unable to stand or

had breathing difficulties and associated with common causes of mortality.

Lung congestion, endocarditis, ascites, and leg problems are common

conditions in commercial breed broilers (Scientific Committee on Animal

Health and Animal Welfare, 2000; Kittelsen et al., 2015). None of the broilers

in this trial were considered to have died from infections. This level of mortality

was expected due to the nature of commercial breed broiler production

(RSPCA, 2020) and comparable to mortality rates reported for commercial

broiler production with mortality rates ranging from production ranged from 1.4-

14.7% with average mortality being 4.1%. Dawkins, et al., (2004). More

recently mortality rates comparable to those in this study (3.78%) were

reported for broilers kept under perceived higher welfare systems – 4% for Red

Tractor and 3% for Better Chicken Commitment (ADAS, 2019).

5.4.5. Nutrient digestibility

Modulation of the GI tract microbiota is commonly associated with improved

digestibility which can lead to improved growth and performance (Rubio et al.,

2015). In this study the ileal digestibility of dry matter was highest in birds

offered the recommended nutrient specification diet (positive control)

compared to that of birds offered other treatments (Table 5.8). This could be

Chapter 5

180

explained due to the absence of rapeseed meal and distiller's dried grains with

solubles in the recommended nutrient specification (positive control).

Rapeseed meal and distiller's dried grains with solubles (DDGS) contain more

fibrous components which leads to decreased digestibility (Everts et al., 1986).

There were no significant differences between treatment groups in the

digestibility of crude protein, despite significant differences in performance

(Table 5.8). The ileal digestibility of ash was variable between treatments. The

variability in digestibility results indicates that the mechanism of action for

improved performance in this study is due to the modulation of the microbiota

more than nutrient utilisation.

5.4.6. Litter quality and ammonia output

In this study litter moisture content, pH, and ammonia output were measured

from 20 to 34 days because they could have an impact on the GI tract

microbiota, health, and performance of birds.

Plants mixed in feed have been demonstrated to decrease ammonia output.

Park and Kim (2019) found that excreta gas ammonia concentration

decreased in birds as levels of Achyranthes japonica solvent extract were

increased in poultry feed. Modulation of the caecal microbiota can lead to less

nitrogen in the uric acid in poultry droppings and in turn, less conversion of

urea to ammonia. High ammonia levels above 25ppm can cause weight loss

in birds and above 60ppm can lead to respiratory problems (de Toledo et al.,

2020). This has a negative impact on performance. Ammonia levels were

Chapter 5

181

therefore measured to determine if the plants in the poultry feed changed

ammonia levels and in turn performance.

Litter moisture content and pH are also associated with bird health and

performance, so these were measured to determine if these factors had an

impact on poultry performance or health. A litter pH <8.0 and decreased litter

moisture content does not favour bacterial pathogen growth in the poultry litter

and also reduces ammonia emissions as the decomposition of uric acid from

poultry droppings is favoured under alkaline pH conditions, not lower pH

conditions. This reduces the exposure of birds to ammonia and reduces the

number of bacterial pathogens in the environment that could colonise the GI

tract. This is beneficial to poultry health and weight gain (Carr et al., 1990; Pra

et al., 2009).

There were no significant differences between treatment groups in NH3 output,

litter moisture content, or pH of litter, with the exception of the pH of litter at

day 34 in which the treatment groups with amoxicillin as an additive, the

recommended nutrient specification (positive control), and A. chinensis Bunge

had significantly lower pH values than the reduced nutrient specification, A.

pilosa Ledeb, and S. glabra Roxb (Table 5.7).

The results of this study suggest that litter moisture, pH, and ammonia output

were not affected by treatments and the significant differences in performance

was due to the addition of S. glabra Roxb. In the case of S. glabra Roxb this

was most likely due to the changes in the Gl tract microbiota.

Chapter 5

182

5.5. Conclusion

These results demonstrate the selective effects of S. glabra Roxb mixed in

broiler feed towards improving Lactic acid bacteria and reducing E. coli and

Campylobacter spp. S. glabra Roxb mixed in broiler feed also improved the

feed conversion ratio and weight gain to levels comparable to the antibiotic

supplemented group and therefore the performance of broilers from 0 to 35

days whilst modulating the microbiota. The additive of A. chinensis Bunge also

increased weight gain but was less effective at improving feed efficiency. The

solid dried plants were mixed in feed as opposed to a plant extract solution in

drinking water as this was considered to be a more effective method. This was

because it would allow more time for the compounds in the plant extracts to

be digested and have an effect on the GI microbiota. This method also

increased the bioavailability of the nutrients as they would be retained in the

digestive system for longer, whereas the plant extracts in solution in the

drinking water would be flushed through the urinary system of the birds

relatively quickly resulting in less time for the absorption of chemical

compounds in the plants. This study highlights the benefits of using S. glabra

Roxb and A. chinensis Bunge as additives in broiler feed and justifies further

experiments into their effects on broiler performance, health, and GI tract

microbiota. Further experiments that determine the effect of different

concentrations of S. glabra Roxb on a on the poultry GI tract microbiota, health,

and performance would be useful in establishing the optimal concentration to

mix in poultry feed.

Future research

183

6. General discussion

This concluding discussion provided an opportunity for self-reflection and to

highlight key research findings and to capture the main points of what was an

interesting, rewarding, and challenging, but valuable learning journey.

This main reasons for undertaking this study emerged and were supported by

evidence from a comprehensive review of the literature around the antibiotic

crisis, the threat this poses to human and animal health, and the need to find

practical alternatives to using antibiotics. The literature review represented a

growing body of consensual evidence that the global use and misuse of

antibiotics in animal farming, and in particular poultry farming was one of the

main causes of increasing levels of antibiotic resistance worldwide. A common

theme was the serious threat this posed to human and animal health. It was

also clear from the literature that poultry farming was a growing global industry

and that its dependence on using antibiotics as poultry feed supplements will

result in increased antibiotic consumption, and consequently to increased

levels of global antibiotic resistance. This was reflected in reports by all

countries globally of increasing levels of antibiotic resistance and multidrug

resistant strains of bacterial pathogens including those in this study, E. coli, C.

jejuni, L. monocytogenes, and S. enteritidis, and with corresponding increasing

rates of pathogen related diseases. The wealth of literature on the

mechanisms of antibiotics and antibiotic resistance provided the essential

understanding that both mechanisms are genetically encoded in bacteria. This

also helped to explain their duality and the inevitability of bacteria developing

resistance to antibiotics, even when exposed to low doses.

Future research

184

A common theme in literature around the use of antibiotics in poultry farming

was the wide range of research evidence which highlighted the potential of

plant extracts and in particular those used in traditional Chinese medicine

(TCM) as potential alternatives to antibiotics in poultry farming. Many of the

plants used in TCM were shown to demonstrate antibacterial activity against

MDR pathogens including those commonly found in the poutly GI tract such

as, E. coli, C. jejuni, L. monocytogenes, and S. enteritidis. A significant volume

of research emphasised the benefits of using TCM plant extracts in poultry

feed. Their benefits included, reduced susceptibility of bacteria to developing

antibiotic resistance, proven efficacy against MDR pathogens and overall

improvements in the health and performance of the birds. This was related to

the ability of TCM plant extracts to modulate the microbiota in the poultry GI

tract by reducing intestinal pathogens whilst enhancing beneficial bacteria,

such as Lactobacillus. The literature review also revealed a gap in the research

as there was a vast number of plants (over 300,000) most of which had not

been researched. Hence, the aim of this PhD was to develop natural botanical

supplements (phytogenics) that can be used in animal feeds as sustainable

alternatives to conventional antibiotics to promote animal health and

performance.

This study was planned and undertaken as four main investigations each

defined by aims and objectives and designed to build on the research findings

of the previous investigation culminating with a poultry trial. Each investigation

was informed by a range of relevant literature which highlighted appropriate

methods and standard practices. At each stage of the research consideration

Future research

185

was given to selecting appropriate methods which were fit for purpose and to

conducting quality research which could be repeated and or advanced by

others.

In the first investigation the literature review was used to identify a wide

selection of plants that were used in TCM with known antibacterial properties

against a range of pathogens including C. jejuni, L. monocytogenes, E. coli,

and S. enteritidis. From this, 40 plant extracts were selected for in vitro

screening using the broth microdilution method. It was hoped and anticipated

that a reasonable number of these would demonstrate antibacterial activity, as

this was essential for the further investigations which were planned. The

results provided a sound basis for the other investigations, with 29 of the 40

plant extracts inhibiting growth of at least one pathogen. Importantly, four of

the plant extracts, A. pilosa Ledeb, S. glabra Roxb, A. chinensis Bunge, and I.

domestica (L.) Goldblatt and Mabb demonstrated broad spectrum antibacterial

activity against C. jejuni, L. monocytogenes, E. coli and S. enteritidis. Notably,

A. pilosa Ledeb demonstrated a comparable antibacterial activity of ampicillin

against E. coli.

The second investigation moved from considering the antibacterial activities of

individual plant extracts to exploring the synergistic effects of combinations of

A. pilosa Ledeb, A. chinensis Bunge, S. glabra Roxb, and each respectively in

combination with ampicillin and erythromycin against E. coli, S. enteritidis, C.

jejuni, and L. monocytogenes. The rationale for this was also based on

previous synergy studies which demonstrated that such combinations could

Future research

186

lead to enhanced antibacterial activity. This was worth consideration. If this

was the case, then the amount of antibiotics used in poultry feed could be

reduced as this may be compensated for by combined synergistic effects. Most

of the results for the antibacterial activity of combinations of plant extracts were

reported as indifferent, with two exceptions. A. chinensis Bunge and A. pilosa

Ledeb demonstrated potential synergistic effects against C. jejuni and E. coli.

Antagonistic effects against E. coli were reported for combinations of A. pilosa

Ledeb with S. glabra Roxb, and A. pilosa Ledeb with A. chinensis Bunge. Each

of the plant extracts when combined with ampicillin and erythromycin against

C. jejuni, L. monocytogenes, E. coli, S. enteritidis all indicated additive effects.

Perhaps not surprisingly, given the vast number of plants in nature, there was

a lack of previously published research on most of the plant extracts in the

screening assay and in synergistic combinations. This and the fact the

literature highlighted that there were no standardised approaches to both of

these types of study made it challenging to make meaningful comparisons. On

the other hand, this lack of available literature enhanced the value of this PhD

as it provided new and additional information about the antibacterial activity of

the number of individual plants used in TCM, and their synergistic effects.

Overall, the first two investigations provided significant evidence to support the

potential use of TCM plants in poultry feed.

The third investigation provided an opportunity to explore the use of a poultry

caecum model in an in vitro assay using the time kill method. Previous

research suggested that using poultry caecum content as the growth medium

would more closely resemble the environmental conditions of endogenous

bacteria such as, E. coli, S. enteritidis, C. jejuni, and L. monocytogenes, and

Future research

187

beneficial microbiota, such as lactic acid bacteria in the GI tract of the living

bird. Hence, the caecum model was used to provide new information about

how the bactericidal activity of A. pilosa Ledeb, S. glabra Roxb, A. chinensis

Bunge, and I. domestica (L.) Goldblatt and Mabb changed over a 24 hour

period, and with different concentrations against the above pathogens. The

findings from this investigation confirmed the antibacterial activity of all four

plant extracts. Moreover, the plant extracts demonstrated significant

bactericidal activity against S. enteritidis, E. coli, and L. monocytogenes over

a 24 hour period. The findings also showed that using higher concentrations

of plant extracts resulted in rapid bactericidal activity against these pathogens.

This was not surprising as the literature suggests that the antibacterial activity

of plants was due to their secondary metabolites. So, it could reasonably be

expected that increasing the concentration of plant extracts would result in a

corresponding increase in the levels of active compounds and hence to higher

levels of bactericidal activity. Notably, some of the plant extracts were potent

with comparable results to ampicillin and were capable of decreasing

pathogens in 0.5 hours as in the cases of A. pilosa Ledeb against E. coli, and

A. chinensis Bunge and A. pilosa Ledeb against L. monocytogenes.

The fourth and final investigation was a poultry trial which used selected

extracts of A. pilosa Ledeb, S. glabra Roxb, A. chinensis Bunge, and I.

domestica (L.) Goldblatt and Mabb against E. coli, S. enteritidis, C. jejuni, and

L. monocytogenes as a feed material to the diet of 420, day-old male Ross 308

broiler chicks. The poultry trial was an essential and the most challenging part

of this PhD. It was essential because it provided an opportunity to investigate

Future research

188

the effects of these plant extracts on performance, nutrient digestibility, and GI

tract microbiota on living birds and hence to make a more meaningful

judgement about their potential use in poultry feed. The scale, scope, and

nature of the poultry trial made it challenging to effectively manage, for

example these challenges included ensuring bird welfare, applying

standardised environmental conditions to each group, consistency in

administering treatments, including feeding intervals/times, and ensuring

consistent and accurate plant extract to feed ratios. These were mitigated by

careful planning, poultry trial design, and regularly monitoring progress.

Given the positive results from the in vitro investigations it was anticipated that

the findings of poultry trial would also be promising. Indeed, this turned out to

be the case. The main findings showed that S. glabra Roxb significantly

decreased the relative abundance of bacterial pathogens, E. coli, and

Campylobacter spp. on days 21 and 35 while increasing the relative

abundance of lactic acid bacteria when compared to the antibiotic group on

days 14 and 35. S. glabra Roxb and A. chinensis Bunge significantly increased

weight gain of birds compared to the negative control and the positive control

birds receiving the recommended nutrient specification diet. Most importantly,

the above results were comparable with the birds receiving the positive

amoxicillin control treatment. This suggested that the above plant samples

were as effective as amoxicillin in improving weight gain and maintaining

performance. This study appears to be the first poultry trial to report data on

A. pilosa Ledeb, S. glabra Roxb, and A. chinensis Bunge mixed in poultry feed

on performance, nutrient digestibility, and GI tract microbiota. Overall, the

Future research

189

findings from this PhD, and in particular those from the poultry trial provide

strong evidence of the potential of using TCM plants mixed in poultry feed to

support poultry health and performance.

However, there are also important areas of further research which would add

to our knowledge and understanding around the growing area of interest of

plants as poultry feed supplements. Perhaps the most obvious is that there are

numerous TCM plants still to be explored. Another research focus might be to

consider how to address the lack of a standardised approach and lack of

inconsistency of results in antibacterial screening assays and synergy studies.

This might include a comprehensive comparison of the different research

methods used in these studies and recommending a standardised approach.

The literature also commonly highlighted the lack of understanding around the

antibacterial mechanisms of action of plant extracts. This could be a further

area of research which would include biochemical analysis of plant secondary

metabolites, their antibacterial properties, and their impact on poultry digestion

and microbiota.

The research in this PhD could be further developed by investigating different

concentrations of plant extracts in poultry feed and optimal benefits in terms of

digestion, inhibition of intestinal pathogens, enhancement of GI tract beneficial

microbiota, and performance. More specific research could consider how

different plant extracts might impact the population and distribution of the

poultry microbiota in different parts of the bird GI tract. This could lead to using

different plants for treating different diseases

References

190

7. References

Abdeltawab, A., Ammar, A., Hamouda, A., Sebaey, W. A. EL, Elhawary, S. A.

M. and El-Deen, S. S. (2018) ‘Interaction of some plant extracts with some

antibiotics against E. coli from chickens’, Benha Veterinary Medical Journal,

35(2), pp. 107–119. doi: 10.21608/bvmj.2018.95989.

Abiala, M., Olayiwola, J., Babatunde, O., Aiyelaagbe, O. and Akinyemi, S.

(2016) ‘Evaluation of therapeutic potentials of plant extracts against poultry

bacteria threatening public health’, BMC Complementary and Alternative

Medicine, 16(1), pp. 1–8. doi: 10.1186/s12906-016-1399-z.

Aboelsoud, N. (2010) ‘Herbal medicine in ancient Egypt’, J Medicinal Plants

Research, 4(2), pp. 82–86. doi: 10.5897/JMPR09.013.

Abreu, A. C., McBain, A. J. and Simões, M. (2012) ‘Plants as sources of new

antimicrobials and resistance-modifying agents’, Natural Product Reports,

29(9), pp. 1007–1021. doi: 10.1039/c2np20035j.

Abubakar, A. R. and Haque, M. (2020) ‘Preparation of medicinal plants: Basic

extraction and fractionation procedures for experimental purposes’, Journal of

pharmacy & bioallied sciences. 2020/01/29, 12(1), pp. 1–10. doi:

10.4103/jpbs.JPBS_175_19.

Acamovic, T. and Brooker, J. D. (2005) ‘Biochemistry of plant secondary

metabolites and their effects in animals’, in Proceedings of the Nutrition

Society. Cambridge University Press, pp. 403–412. doi:

10.1079/PNS2005449.

References

191

ADAS (2019) Impacts of the better chicken commitment on the UK broiler

sector.

Adeola, O. and Cowieson, A. J. (2011) ‘Board-invited review: Opportunities

and challenges in using exogenous enzymes to improve nonruminant animal

production’, Journal of Animal Science, 89(10), pp. 3189–3218. doi:

10.2527/jas.2010-3715.

Adhikari, P., Lee, C. H., Cosby, D. E., Cox, N. A. and Kim, W. K. (2019) ‘Effect

of probiotics on fecal excretion, colonization in internal organs and immune

gene expression in the ileum of laying hens challenged with Salmonella

Enteritidis’, Poultry Science, 98(3), pp. 1235–1242. doi: 10.3382/ps/pey443.

Adil, S., Banday, T., Bhat, G. A., Mir, M. S. and Rehman, M. (2010) ‘Effect of

dietary supplementation of organic acids on performance, intestinal

histomorphology, and serum biochemistry of broiler chicken’, Veterinary

Medicine International, 2010, pp. 1–7. doi: 10.4061/2010/479485.

Ahmed, S., Islam, M., Mun, H.-S., Sim, H.-J., Kim, Y.-J. and Yang, C.-J. (2014)

‘Effects of Bacillus amyloliquefaciens as a probiotic strain on growth

performance, cecal microflora, and fecal noxious gas emissions of broiler

chickens’, Poultry science, 93, pp. 1963–1971. doi: 10.3382/ps.2013-03718.

Akinyele, T. A., Igbinosa, E. O., Akinpelu, D. A. and Okoh, A. I. (2017) ‘In vitro

assessment of the synergism between extracts of Cocos nucifera husk and

some standard antibiotics’, Asian Pacific Journal of Tropical Biomedicine, 7(4),

pp. 306–313. doi: 10.1016/j.apjtb.2016.12.022.

Akyildiz, S. and Denli, M. (2016) ‘Application of plant extracts as feed additives

in poultry nutrition’, Animal Science, 59, pp. 71–74. Available at:

References

192

https://www.researchgate.net/publication/308919201.

Al-Bayati, F. A. (2008) ‘Synergistic antibacterial activity between Thymus

vulgaris and Pimpinella anisum essential oils and methanol extracts’, Journal

of Ethnopharmacology, 116(3), pp. 403–406. doi: 10.1016/j.jep.2007.12.003.

Alagawany, M., Elnesr, S. S. and Farag, M. R. (2018) ‘The role of exogenous

enzymes in promoting growth and improving nutrient digestibility in poultry’,

Iranian Journal of Veterinary Research, 19(3), pp. 157–164.

Aldred, K. J., Kerns, R. J. and Osheroff, N. (2014) ‘Mechanism of quinolone

action and resistance’, Biochemistry, 53(10), pp. 1565–1574. doi:

10.1021/bi5000564.

Alexander, T. W., Yanke, L. J., Topp, E., Olson, M. E., Read, R. R., Morck, D.

W. and McAllister, T. A. (2008) ‘Effect of subtherapeutic administration of

antibiotics on the prevalence of antibiotic-resistant Escherichia coli bacteria in

feedlot cattle’, Applied and Environmental Microbiology, 74(14), pp. 4405–

4416. doi: 10.1128/AEM.00489-08.

Alexandratos, N. and Bruinsma, J. (2012) ‘World agriculture towards

2030/2050’, Food and Agriculture Organisation of the United Nations, (12), pp.

1–147.

Allen, H. K. and Stanton, T. B. (2014a) ‘Altered egos: Antibiotic effects on food

animal microbiomes’, Annual Review of Microbiology, 68(1), pp. 297–315. doi:

10.1146/annurev-micro-091213-113052.

Allen, H. K. and Stanton, T. B. (2014b) ‘Altered egos: Antibiotic effects on food

animal microbiomes’, Annual Review of Microbiology, 68(1), pp. 297–315. doi:

References

193

10.1146/annurev-micro-091213-113052.

Allen, H. K., Trachsel, J., Looft, T. and Casey, T. A. (2014) ‘Finding alternatives

to antibiotics’, Annals of the New York Academy of Sciences, 1323(1), pp. 91–

100. doi: 10.1111/nyas.12468.

Alloui, M. N., Szczurek, W. and Światkiewicz, S. (2013) ‘The usefulness of

prebiotics and probiotics in modern poultry nutrition: A review’, Annals of

Animal Science, 13(1), pp. 17–32. doi: 10.2478/v10220-012-0055-x.

Alonso-Hernando, A., Prieto, M., García-Fernández, C., Alonso-Calleja, C.

and Capita, R. (2012) ‘Increase over time in the prevalence of multiple

antibiotic resistance among isolates of Listeria monocytogenes from poultry in

Spain’, Food Control, 23(1), pp. 37–41. doi:

https://doi.org/10.1016/j.foodcont.2011.06.006.

AOAC (1990) Official methods of analysis, Association of official analytical

chemists. Washington, DC.

AOAC (1998) Official method 934.01 loss on drying (moisture) at 95–100°C

for feeds dry matter on oven drying at 95–100°C for feeds first, Journal of

AOAC.

Arsi, K., Donoghue, A. M., Venkitanarayanan, K., Kollanoor-Johny, A.,

Fanatico, A. C., Blore, P. J. and Donoghue, D. J. (2014) ‘The efficacy of the

natural plant extracts, Thymol and Carvacrol against Campylobacter

colonization in broiler chickens’, Journal of Food Safety, 34(4), pp. 321–325.

doi: 10.1111/jfs.12129.

Ashayerizadeh, A., Dabiri, N., Ashayerizadeh, O., Mirzadeh, K. H.,

References

194

Roshanfekr, H. and Mamooee, M. (2009) ‘Effect of dietary antibiotic, probiotic

and prebiotic as growth promoters, on growth performance, carcass

characteristics and hematological indices of broiler chickens’, Pakistan Journal

of Biological Sciences, 12(1), pp. 52–57. doi: 10.3923/pjbs.2009.52.57.

Aviagen (2018) Broiler management handbook 2018. Available at:

http://online.anyflip.com/kmgi/zqpr/index.html#p=4.

Aviagen (2019) Ross 308 Broiler: Nutrition Specifications, Aviagen.

Awad, W. A., Ghareeb, K., Abdel-Raheem, S. and Böhm, J. (2009) ‘Effects of

dietary inclusion of probiotic and synbiotic on growth performance, organ

weights, and intestinal histomorphology of broiler chickens’, Poultry Science,

88(1), pp. 49–55. doi: 10.3382/ps.2008-00244.

Azwanida, N. (2015) ‘A review on the extraction methods use in medicinal

plants, principle, strength and limitation’, Medicinal and Aromatic Plants, 4(3),

pp. 3–8. doi: 10.4172/2167-0412.1000196.

Bae, J. (2004) ‘Antimicrobial effect of Lithospermum erythrorhizon extracts on

the food-borne pathogens’, Korean Journal of Food Science and Technology,

36(5), pp. 0–4.

Bai, S. P., Wu, A. M., Ding, X. M., Lei, Y., Bai, J., Zhang, K. Y. and Chio, J. S.

(2013) ‘Effects of probiotic-supplemented diets on growth performance and

intestinal immune characteristics of broiler chickens’, Poultry Science, 92(3),

pp. 663–670. doi: 10.3382/ps.2012-02813.

Balouiri, M., Sadiki, M. and Ibnsouda, S. K. (2016) ‘Methods for in vitro

evaluating antimicrobial activity: A review’, Journal of Pharmaceutical Analysis,

References

195

6(2), pp. 71–79. doi: 10.1016/j.jpha.2015.11.005.

Banday, M. T., Adil, S., Khan, A. A. and Untoo, M. (2015) ‘A study on efficacy

of fumaric acid supplementation in diet of broiler chicken’, International Journal

of Poultry Science, 14(11), pp. 589–594. doi: 10.3923/ijps.2015.589.594.

Banerjee, S., Sar, A., Misra, A., Pal, S., Chakraborty, A. and Dam, B. (2018)

‘Increased productivity in poultry birds by sub-lethal dose of antibiotics is

arbitrated by selective enrichment of gut microbiota, particularly short-chain

fatty acid producers’, Microbiology (United Kingdom), 164(2), pp. 142–153.

doi: 10.1099/mic.0.000597.

Baquero, F., Lanza, V. F., Duval, M. and Coque, T. M. (2020) ‘Ecogenetics of

antibiotic resistance in Listeria monocytogenes’, Molecular

Microbiology, 113, pp. 570–579. doi: 10.1111/mmi.14454.

Başer, K. H. C. and Demirci, F. (2007) ‘Chemistry of essential oils’, in Berger,

R. G. (ed.) Flavours and Fragrances. 1st edn. Berlin: Springer, Berlin,

Heidelberg, pp. 43–44.

Bedford, M. R. and Cowieson, A. J. (2012) ‘Exogenous enzymes and their

effects on intestinal microbiology’, Animal Feed Science and Technology,

173(1), pp. 76–85. doi: https://doi.org/10.1016/j.anifeedsci.2011.12.018.

Bell, A. (2005) ‘Antimalarial drug synergism and antagonism: Mechanistic and

clinical significance’, 253, pp. 171–184. doi: 10.1016/j.femsle.2005.09.035.

Benites, V., Gilharry, R., Gernat, A. G. and Murillo, J. G. (2008) ‘Effect of

dietary mannan oligosaccharide from bio-mos or SAF-mannan on live

performance of broiler chickens’, Journal of Applied Poultry Research, 17(4),

References

196

pp. 471–475. doi: 10.3382/japr.2008-00023.

Bennett, P. M. (2008) ‘Plasmid encoded antibiotic resistance :acquisition and

transfer of antibiotic resistance genes in bacteria’, British Journal of

Pharmacology, 153, pp. 347–357. doi: 10.1038/sj.bjp.0707607.

Bhardwaj, M., Singh, B. R., Sinha, D. K., Kumar, V., Vadhana, P., Singh, V.,

Nirupama, K., Pruthvishree and Archana, S. B. (2016) ‘Potential of herbal drug

and antibiotic combination therapy: A new approach to treat multidrug resistant

bacteria’, Pharmaceutica Analytica Acta, 7(11), pp. 1–14. doi: 10.4172/2153-

2435.1000523.

Bioline (2019) Bioline, MyTaqTM Red Mix. Available at:

https://www.bioline.com/uk/downloads/dl/file/id/2697/mytaq_red_mix_product

_manual.pdf (Accessed: 16 April 2020).

Biswas, A., Mohan, N., Raza, M., Mir, N. A. and Mandal, A. (2019) ‘Production

performance, immune response and blood biochemical parameters in broiler

chickens fed diet incorporated with prebiotics’, Journal of Animal Physiology

and Animal Nutrition, 103(2), pp. 493–500. doi: 10.1111/jpn.13042.

Van Boeckel, T. P., Brower, C., Gilbert, M., Grenfell, B. T., Levin, S. A.,

Robinson, T. P., Teillant, A. and Laxminarayan, R. (2015) ‘Global trends in

antimicrobial use in food animals’, Proceedings of the National Academy of

Sciences of the United States of America, 112(18), pp. 5649–5654. doi:

10.1073/pnas.1503141112.

Bogaard, A. E. van den, Willems, R., London, N., Top, J. and Stobberingh, E.

E. (2002) ‘Antibiotic resistance of fecal Enterococci in poultry, poultry farmers,

and poultry slaughterers’, Journal of Antimicrobial Chemotherapy, 49(3), pp.

References

197

497–505. doi: 10.1017/s0195941700082552.

Bogucka, J., Ribeiro, D. M., Bogusławska-Tryk, M., Dankowiakowska, A., da

Costa, R. P. R. and Bednarczyk, M. (2019) ‘Microstructure of the small

intestine in broiler chickens fed a diet with probiotic or synbiotic

supplementation’, Journal of Animal Physiology and Animal Nutrition, 103(6),

pp. 1785–1791. doi: 10.1111/jpn.13182.

Bonapace, C. R., Bosso, J. A., Friedrich, L. V. and White, R. L. (2002)

‘Comparison of methods of interpretation of checkerboard synergy testing’,

Diagnostic Microbiology and Infectious Disease, 44(4), pp. 363–366. doi:

10.1016/S0732-8893(02)00473-X.

Bravo, D. and Ionescu, C. (2008) ‘Meta-analysis of the effect of a mixture of

carvacrol, cinnamaldehyde and capsicum oleoresin in broilers’, Poultry

Science, 87, p. 75.

Buckland, E. L., O'Neill, D., Summers, J., Mateus, A., Church, D.,

Redmond, L. and Brodbelt, D. (2016) ‘Characterisation of antimicrobial usage

in cats and dogs attending UK primary care companion animal veterinary

practices’, Veterinary Record, 179(19), pp. 489 LP – 489. Available at:

http://veterinaryrecord.bmj.com/content/179/19/489.abstract.

Caesar, L. K. and Cech, N. B. (2019) ‘Synergy and antagonism in natural

product extracts: When 1 + 1 does not equal 2’, Natural Product Reports,

36(6), pp. 869–888. doi: 10.1039/c9np00011a.

Callicott, K. A., Friðriksdo, V., Reiersen, J., Lowman, R., Bisaillon, J.,

Gunnarsson, E., Berndtson, E., Hiett, K. L., Needleman, D. S. and Stern, N. J.

(2006) ‘Lack of evidence for vertical transmission of Campylobacter spp. in

References

198

chickens’, Applied and Enviromental Microbiology, 72(9), pp. 5794–5798. doi:

10.1128/AEM.02991-05.

Canli, K., Yetgin, A., Akata, I. and Altuner, E. M. (2016) ‘In vitro antimicrobial

activity screening of Rheum rhabarbarum roots’, International Journal of

Pharmaceutical Science Invention, 5(2), p. 104.

Cantón, R. and Morosini, M. I. (2011) ‘Emergence and spread of antibiotic

resistance following exposure to antibiotics’, FEMS Microbiology Reviews,

35(5), pp. 977–991. doi: 10.1111/j.1574-6976.2011.00295.x.

Centeno, C., Arija, I., Viveros, A. and Brenes, A. (2007) ‘Effects of citric acid

and microbial phytase on amino acid digestibility in broiler chickens’, British

Poultry Science, 48(4), pp. 469–479. doi: 10.1080/00071660701455276.

Centers for Disease Control and Prevention (2019) Antibiotic resistance

threats in the United States, Centers for Disease Control and Prevention. doi:

CS239559-B.

Centres for Disease Control and Precention (2020) Antibiotic/Antimicrobial

Resistance (AR/AMR), Centres for Disease Control and Precention. Available

at: https://www.cdc.gov/drugresistance/index.html (Accessed: 31 July 2020).

Centres for Disease Control and Prevention (2013) Antibiotic resistance

threats in the United States.

Centres for Disease Control and Prevention (2019) Glossary of terms related

to antibiotic resistance, Centres for Disease Control and Prevention. Available

at: https://www.cdc.gov/narms/resources/glossary.html (Accessed: 30 July

2020).

References

199

Chalghoumi, R., Belgacem, A., Trabelsi, I., Bouatour, Y. and Bergaoui, R.

(2013) ‘Effect of dietary supplementation with probiotic or essential oils on

growth performance of broiler chickens’, International Journal of Poultry

Science, 12(9), pp. 538–544. doi: 10.3923/ijps.2013.538.544.

Cheesman, M. J., Llanko, A., Blonk, B. and Cock, I. E. (2017) ‘Developing new

antimicrobial therapies: Are synergistic combinations of plant

extracts/compounds with conventional antibiotics the solution?’,

Pharmacognosy Reviews, 11, pp. 57–72.

Chen, Y. S., Srionnual, S., Onda, T. and Yanagida, F. (2007) ‘Effects of

prebiotic oligosaccharides and trehalose on growth and production of

bacteriocins by lactic acid bacteria’, Letters in Applied Microbiology, 45(2), pp.

190–193. doi: 10.1111/j.1472-765X.2007.02167.x.

Cheng, G., Hao, H., Shuyu, X., Xu, W., Meihong, D., Lingli, H. and Zonghui,

Y. (2014) ‘Antibiotic alternatives : The substitution of antibiotics in animal

husbandry’, (May). doi: 10.3389/fmicb.2014.00217.

Choct, M. (2009) ‘Managing gut health through nutrition’, British Poultry

Science, 50(1), pp. 9–15. doi: 10.1080/00071660802538632.

Clavijo, V. and Florez, M. (2017) ‘The gastrointestinal microbiome and its

association with the control of pathogens in broiler chicken production: A

review’, Poultry Science, 97(3). doi: 10.3382/ps/pex359.

Clinical and Laboratory Standards Institute (1999) Methods for determining

bactericidal activity of antimicrobial agents; approved guideline.

Clinical and Laboratory Standards Institute (2009) Methods for dilution:

References

200

Antimicrobial susceptibility tests for bacteria that grow aerobically; approved

standard. 8th edn.

Co, M., Zettersten, C., Nyholm, L., Sjöberg, P. J. R. and Turner, C. (2012)

‘Analytica Chimica Acta Degradation effects in the extraction of antioxidants

from birch bark using water at elevated temperature and pressure’, Analytica

Chimica Acta, 716, pp. 40–48. doi: 10.1016/j.aca.2011.04.038.

Collett, S. R. and Dawson, K. A. (2002) ‘How effective are the alternatives to

AGPS?’, Feed Mix, 10, pp. 16–19.

Collignon, P. J. and McEwen, S. A. (2019) ‘One health-its importance in

helping to better control antimicrobial resistance’, Tropical Medicine and

Infectious Disease, 4(22), pp. 1–21. doi: 10.3390/tropicalmed4010022.

Connell, S. R., Tracz, D. M., Nierhaus, K. H. and Taylor, D. E. (2003)

‘Ribosomal protection proteins and their mechanism of tetracycline resistance’,

Antimicrobial Agents and Chemotherapy, 47(12), pp. 3675–3681. doi:

10.1128/AAC.47.12.3675-3681.2003.

Corry, J., Jørgensen, F., Purnell, G., James, C., Pinho, R. and James, S. J.

(2017) Reducing Campylobacter cross-contamination during poultry

processing.

Costa, M. C., Bessegatto, J. A., Alfieri, A. A., Weese, J. S., Filho, J. A. B. and

Oba, A. (2017) ‘Different antibiotic growth promoters induce specific changes

in the cecal microbiota membership of broiler chicken’, PLoS ONE, 12(2), pp.

1–13. doi: 10.1371/journal.pone.0171642.

Coutinho, H. D. M., Costa, J. G. M., Lima, E. O., Falcão-Silva, V. S. and

References

201

Siqueira-Júnior, J. P. (2009) ‘In vitro interference of Momordica charantia in

the resistance to aminoglycosides’, Pharmaceutical Biology, 47(11), pp. 1056–

1059. doi: 10.3109/13880200902991540.

Cowieson, A. J. and Kluenter, A. M. (2019) ‘Contribution of exogenous

enzymes to potentiate the removal of antibiotic growth promoters in poultry

production’, Animal Feed Science and Technology, 250(April 2018), pp. 81–

92. doi: 10.1016/j.anifeedsci.2018.04.026.

Dahiya, P. and Purkayastha, S. (2012) ‘Phytochemical screening and

antimicrobial activity of some medicinal plants against multi-drug resistant

bacteria from clinical isolates’, Indian journal of pharmaceutical sciences,

74(5), pp. 443–450. doi: 10.4103/0250-474X.108420.

Dal Pont, G. C., Farnell, M., Farnell, Y. and Kogut, M. H. (2020) ‘Dietary factors

as triggers of low-grade chronic intestinal inflammation in poultry’,

Microorganisms, 8(1), pp. 1–10. doi: 10.3390/microorganisms8010139.

Danzeisen, J. L., Kim, H. B., Isaacson, R. E., Tu, Z. J. and Johnson, T. J.

(2011) ‘Modulations of the chicken cecal microbiome and metagenome in

response to anticoccidial and growth promoter treatment’, PLoS ONE, 6(11),

pp. 1–14. doi: 10.1371/journal.pone.0027949.

Darambazar, E. (2019) ‘Determination of titanium dioxide as a livestock

digestibility marker. Assay protocol’, University of Saskatchewan, p. 10. doi:

10.13140/RG.2.2.14265.31842.

Das, B. K., Das, P. D., Pradhan, J., Priyadarshinee, B., Sahu, I., Roy, P. and

Mishra, B. K. (2012) ‘Evaluation of antimicrobial actiivty and phytochemical

screening of ethanolic extract of greater duckweed, Spirodela polyrrhiza’,

References

202

Internationl Journal of Pharma and Bio Sciences, 3(3), pp. 822–833.

Das, S., Akhter, R., Khandaker, S., Huque, S., Das, P., Anwar, M. R., Tanni,

K. A., Shabnaz, S. and Shahriar, M. (2017) ‘Phytochemical screening,

antibacterial and anthelmintic activities of leaf and seed extracts of Coix

lacryma-jobi L.’, Journal of Coastal Life Medicine, (January), pp. 360–364. doi:

10.12980/jclm.5.2017j7-65.

Davies, J. and Davies, D. (2010) ‘Origins and evolution of antibiotic resistance’,

Microbiology and Molecular Biology Reviews, 74(3), pp. 417–433. doi:

10.1128/MMBR.00016-10.

Davila, A.-M., Blachier, F., Gotteland, M., Andriamihaja, M., Benetti, P.-H.,

Sanz, Y. and Tomé, D. (2013) ‘Re-print of “Intestinal luminal nitrogen

metabolism: Role of the gut microbiota and consequences for the host”’,

Pharmacological Research, 69(1), pp. 114–126. doi:

https://doi.org/10.1016/j.phrs.2013.01.003.

Dawkins, M. S., Donnelly, C. A. and Jones, T. A. (2004) ‘Chicken welfare is

influenced more by housing conditions than by stocking density.’, Nature,

427(6972), pp. 342–344. doi: 10.1038/nature02226.

Diarra, M. S., Hassan, Y. I., Block, G. S., Drover, J. C. G., Delaquis, P. and

Oomah, B. D. (2020) ‘Antibacterial activities of a polyphenolic-rich extract

prepared from American cranberry (Vaccinium macrocarpon) fruit pomace

against Listeria spp.’, LWT - Food Science and Technology, 123. doi:

10.1016/j.lwt.2020.109056.

Dibner, J. J. and Buttin, P. (2002) ‘Use of organic acids as a model to study

the impact of gut microflora on nutrition and metabolism’, Journal of Applied

References

203

Poultry Research, 11(4), pp. 453–463. doi: 10.1093/japr/11.4.453.

Dibner, J. J. and Richards, J. D. (2005) ‘Antibiotic growth promoters in

agriculture: History and mode of action’, Poultry Science, 84(4), pp. 634–643.

doi: 10.1093/ps/84.4.634.

Dittoe, D. K., Ricke, S. C. and Kiess, A. S. (2018) ‘Organic acids and potential

for modifying the avian gastrointestinal tract and reducing pathogens and

disease’, Frontiers in Veterinary Science, 5(216), pp. 1–12. doi:

10.3389/fvets.2018.00216.

Doern, C. D. (2014) ‘When does 2 plus 2 equal 5? A review of antimicrobial

synergy testing’, Journal of Clinical Microbiology, 52(12), pp. 4124–4128. doi:

10.1128/JCM.01121-14.

Dumonceaux, T. J., Hill, J. E., Hemmingsen, S. M. and Van Kessel, A. G.

(2006) ‘Characterization of intestinal microbiota and response to dietary

virginiamycin supplementation in the broiler chicken’, Applied and

Environmental Microbiology, 72(4), pp. 2815–2823. doi:

10.1128/AEM.72.4.2815-2823.2006.

Dunislawska, A., Slawinska, A., Stadnicka, K., Bednarczyk, M., Gulewicz, P.,

Jozefiak, D. and Siwek, M. (2017) ‘Synbiotics for broiler chickens - In vitro

design and evaluation of the influence on host and selected microbiota

populations following in ovo delivery’, PLoS ONE, 12(1), pp. 1–20. doi:

10.1371/journal.pone.0168587.

E. Carr, L., W. Wheaton, F. and W. Douglass, L. (1990) ‘Empirical models to

determine ammonia concentrations from broiler chicken litter’, Transactions of

the ASAE, 33(4), pp. 1337–1342. doi: https://doi.org/10.13031/2013.31478.

References

204

Ebrahim, N., Nasser, K. R. M., Awadh, A. A., Ahmeda, T. A. and Ramzy, M.

(2013) ‘Antioxidant activity and anthocyanin content in flower of Mirabilis jalab

L. collected from Yemen’, Banat’s Journal of Biotechnology, 4(8), pp. 85–92.

doi: 10.7904/2068.

EC (2013) ‘Report on the implementation of national residue monitoring plans

in the Member States in 2013 (Council Directive 96/23/EC).’, 2013(178), p.

239. Available at: http://ec.europa.eu/food/safety/docs/cs_vet-med-

residues_workdoc_2013_en.pdf.

EFSA (2012) ‘Scientific opinion on the substantiation of health claims related

to a combination of Lactobacillus rhamnosus CNCM I-1720, Lactobacillus

helveticus CNCM I-1722, Bifidobacterium longum subsp. longum CNCM I-

3470 and Saccharomy’, EFSA Journal, 10(8), p. 2853. doi:

10.2903/j.efsa.2012.2853.Available.

Eiamphungporn, W., Schaduangrat, N. and Malik, A. A. (2018) ‘Tackling the

antibiotic resistance caused by class A β-lactamases through the use of β-

lactamase inhibitory protein’, International Journal of Molecular Sciences,

19(2222), pp. 1–24. doi: 10.3390/ijms19082222.

Elhadidy, M., Miller, W. G., Arguello, H. and Álvarez-ordóñez, A. (2018)

‘Genetic basis and clonal population structure of antibiotic resistance in

Campylobacter jejuni isolated from broiler carcasses in Belgium

bacterial strains and growth conditions’, Frontiers in Microbiology, 9(May), pp.

1–9. doi: 10.3389/fmicb.2018.01014.

Eloff, J. N. (2019) ‘Avoiding pitfalls in determining antimicrobial activity of plant

extracts and publishing the results’, BMC Complementary and Alternative

References

205

Medicine, 19(1), pp. 1–8. doi: 10.1186/s12906-019-2519-3.

Elston, D. M. (2009) ‘Topical antibiotics in dermatology: Emerging patterns of

resistance’, Dermatologic Clinics, 27, pp. 25–31. doi:

10.1016/j.det.2008.07.004.

Esimone, C. O., Iroha, I. R., Ibezim, E. C., Okeh, C. O. and Okpana, E. M.

(2006) ‘In vitro evaluation of the interaction between tea extracts and penicillin

G against Staphylococcus aureus’, African Journal of Biotechnology, 5(11),

pp. 1082–1086. doi: 10.5897/AJB06.043.

European Centres for Disease Prevention and Control, Cassini, A., Högberg,

L. D., Plachouras, D., Quattrocchi, A., Hoxha, A., Simonsen, G. S., Colomb-

Cotinat, M., Kretzschmar, M. E., Devleesschauwer, B., Cecchini, M., Ouakrim,

D. A., Oliveira, T. C., Struelens, M. J., Suetens, C., Monnet, D. L., Strauss, R.,

Mertens, K., Struyf, T., Catry, B., Latour, K., Ivanov, I. N., Dobreva, E. G.,

Tambic Andraševic, A., Soprek, S., Budimir, A., Paphitou, N., Žemlicková, H.,

Schytte Olsen, S., Wolff Sönksen, U., Märtin, P., Ivanova, M., Lyytikäinen, O.,

Jalava, J., Coignard, B., Eckmanns, T., Abu Sin, M., Haller, S., Daikos, G. L.,

Gikas, A., Tsiodras, S., Kontopidou, F., Tóth, Á., Hajdu, Á., Guólaugsson, Ó.,

Kristinsson, K. G., Murchan, S., Burns, K., Pezzotti, P., Gagliotti, C., Dumpis,

U., Liuimiene, A., Perrin, M., Borg, M. A., de Greeff, S. C., Monen, J. C., Koek,

M. B., Elstrøm, P., Zabicka, D., Deptula, A., Hryniewicz, W., Caniça, M.,

Nogueira, P. J., Fernandes, P. A., Manageiro, V., Popescu, G. A., Serban, R.

I., Schréterová, E., Litvová, S., Štefkovicová, M., Kolman, J., Klavs, I., Korošec,

A., Aracil, B., Asensio, A., Pérez-Vázquez, M., Billström, H., Larsson, S., Reilly,

J. S., Johnson, A. and Hopkins, S. (2019) ‘Attributable deaths and disability-

References

206

adjusted life-years caused by infections with antibiotic-resistant bacteria in the

EU and the European economic area in 2015: a population-level modelling

analysis’, The Lancet Infectious Diseases, 19(1), pp. 56–66. doi:

10.1016/S1473-3099(18)30605-4.

European Commission (2009) ‘Commission regulation (EC) No 152/2009 of

27 January 2009 laying down the methods of sampling and analysis for the

official control of feed’, Official Journal of the European Union, (L54), pp. 1–

130.

European Committee for Antimicrobial Susceptibility Testing (2017)

‘Breakpoint tables for interpretation of MICs and zone diameters’, European

Committee for Antimicrobial Susceptibility Testing, pp. 0–77. Available at:

http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_t

ables/v_5.0_Breakpoint_Table_01.pdf.

European Committee on Antimicrobial Susceptibility Testing (2018)

‘Breakpoint tables for interpretation of MICs and zone diameters’, The

European Committee on Antimicrobial Susceptibility Testing., 8, pp. 0–77.

Available at:

http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_t

ables/v_8.0_Breakpoint_Tables.pdf.

European Food and Safety Authority (2018) EU Register on nutrition and

health claims EU Register on nutrition and health claims.

European Food and Safety Authority (2019) ‘One health zoonoses report’,

EFSA Journal, 17(12), pp. 1–276. doi: 10.2903/j.efsa.2019.5926.

European Food Safety Authority (2010) Annual report.

References

207

Everts, H., Smits, B. and Jongbloed, A. W. (1986) ‘Effect of crude fibre, feeding

level and body weight on apparent digestibility of compound feeds by swine.’,

Netherlands Journal of Agricultural Science, 34(4), pp. 501–503. doi:

10.18174/njas.v34i4.16775.

Faden, A. A. (2018) ‘Evaluation of antibacterial activities of aqueous and

methanolic extracts of Areca catechu against some opportunistic oral bacteria’,

Biosciences Biotechnology Research Asia, 15(3), pp. 655–659.

Fischer, G., Maier, J. C., Rutz, F. and Bermudez, V. L. (2002) ‘Performance of

broilers fed corn soybean meal based diets with or without inclusion of

enzymes’, Revista Brasileira de Zootecnia, 31(1), pp. 402–410.

Fong, S. C., Mulyana, Y. and Girawan, D. (2016) ‘Antibacterial effect of

Pulsatilla chinensis towards Staphylococcus aureus, Shigella dysenteriae, and

Salmonella typhi’, Althea Medical Journal, 3(2), pp. 292–295. doi:

10.15850/amj.v3n2.467.

Food and Agriculture Organisation of the United Nations (2016) Probiotics in

animal nutrition, Physiological reviews. doi: 10.1152/physrev.1954.34.1.25.

Food and Agriculture Organisation and World Health Organization (2006)

Probiotics in food, Chemical and functional properties of food components. doi:

10.1201/9781420009613.ch16.

Food Safety Authority of Ireland (2020) Food Safety Authority of Ireland,

Probiotic health Claims.

Franz, C., Baser, K. H. C. and Windisch, W. (2010) ‘Essential oils and aromatic

plants in animal feeding – a European perspective. A review.’, Flavour and

References

208

Fragrance Journal, 25(5), pp. 327–340. doi: 10.1002/ffj.1967.

Franzblau, S. G. and Cross, C. (1986) ‘Comparative in vitro antimicrobial

activity of Chinese medicinal herbs’, Journal of Ethnopharmacology, 15(1), pp.

279–288.

Friedman, M., Henika, P. R. and Mandrell, R. E. (2002) ‘Bactericidal activities

of plant essential oils and aome of their isolated constituents against

Campylobacter jejuni, Escherichia coli ,Listeria

monocytogenes , and Salmonella enterica’, Journal of Food

Protection, 65(10), pp. 1545–1560.

Fung, S., Rempel, H., Forgetta, V. and Dewar, E. T. K. (2013) ‘Ceca

microbiome of mature broiler chickens fed with or without Salinomycin’, in In

the Gut Microbiome: The Effector/Regulatory Immune Network Conference

Keystone Symposia on Molecular and Cellular Biology, p. B3.

Gadde, U., Kim, W. H., Oh, S. T. and Lillehoj, H. S. (2017) ‘Alternatives to

antibiotics for maximizing growth performance and feed efficiency in poultry: A

review’, Animal Health Research Reviews, 18(1), pp. 26–45. doi:

10.1017/S1466252316000207.

Gaggìa, F., Mattarelli, P. and Biavati, B. (2010) ‘Probiotics and prebiotics in

animal feeding for safe food production’, International journal of food

microbiology, 141 Suppl, pp. S15-28. doi: 10.1016/j.ijfoodmicro.2010.02.031.

Ganguly, S. (2015) ‘A comprehensive review on physiological and nutritional

properties of prebiotics as poultry feed supplement’, Octa Journal of

Biosciences, 3(1), pp. 5–6.

References

209

Ghazanfari, M. and A. M. (2015) ‘Effects of coriander essential oil on the

performance, blood characteristics, intestinal microbiota and histological of

broilers’, Brazilian Journal of Poultry Science, 17(4), pp. 419–426.

Gibbons, S., Oluwatuyi, M., Veitch, N. C. and Gray, A. I. (2003) ‘Bacterial

resistance modifying agents from Lycopus europaeus’, Phytochemistry, 62,

pp. 83–87.

Gibson, G. R., Hutkins, R., Sanders, M. E., Prescott, S. L., Reimer, R. A.,

Salminen, S. J., Scott, K., Stanton, C., Swanson, K. S., Cani, P. D., Verbeke,

K. and Reid, G. (2017) ‘Expert consensus document: The International

Scientific Association for Probiotics and Prebiotics (ISAPP) consensus

statement on the definition and scope of prebiotics’, Nature Reviews

Gastroenterology and Hepatology, 14(8), pp. 491–502. doi:

10.1038/nrgastro.2017.75.

Gibson, G. R. and Roberfroid, M. B. (1995) ‘Dietary modulation of the human

colonic microbiota: Introducing the concept of prebiotics’, The Journal of

Nutrition, 125(6), pp. 1401–1412. doi: 10.1093/jn/125.6.1401.

Gomes, S. M. F., Xavier, J. da C., Santos, J. F. S. dos, Matos, Y. M. L. S. de,

Tintino, S. R., Freitas, T. S. de and Coutinho, H. D. M. (2017) ‘Evaluation of

antibacterial and modifying action of catechin antibiotics in resistant strains’,

Microbial Pathogenesis, (2018). doi: 10.1016/j.micpath.2017.12.058.

Gong, J., Forster, R. J., Yu, H., Chambers, J. R., Wheatcroft, R., Sabour, P.

M. and Chen, S. (2002) ‘Molecular analysis of bacterial populations in the ileum

of broiler chickens and comparison with bacteria in the cecum’, FEMS

Microbiology Ecology, 41(3), pp. 171–179. doi: 10.1016/S0168-

References

210

6496(02)00291-X.

Górniak, I., Bartoszewski, R. and Króliczewski, J. (2019) Comprehensive

review of antimicrobial activities of plant flavonoids, Phytochemistry Reviews.

doi: 10.1007/s11101-018-9591-z.

Gullberg, E. (2014) Selection of antibiotic resistance at very low antibiotic

concentrations., Upsala journal of medical sciences. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/24694026.

Hall, M. J., Middleton, R. F. and Westmacott, D. (1983) ‘The fractional

inhibitory concentration (FIC) index as a measure of synergy’, Journal of

Antimicrobial Chemotherapy, 11(5), pp. 427–433. doi: 10.1093/jac/11.5.427.

Han, X., Piao, X. S., Zhang, H. Y., Li, P. F., Yi, J. Q., Zhang, Q. and Li, P.

(2012) ‘Forsythia suspensa extract has the potential to substitute antibiotic in

broiler chicken’, Asian-Australasian Journal of Animal Sciences, 25(4), pp.

569–576.

Harris, P. N. A. (2015) ‘Clinical management of infections caused by

Enterobacteriaceae that express extended- spectrum β -lactamase and

AmpC enzymes’, Seminars in Respiratory and Critical Care Medicine, 36(1),

pp. 56–73.

Hashemi, S. R. and Davoodi, H. (2010) ‘Phytogenics as new class of feed

additive in poultry industry’, Journal of Animal and Veterinary Advances, 9(17),

pp. 2295–2304. doi: 10.3923/javaa.2010.2295.2304.

Hashemi, S. R. and Davoodi, H. (2011) ‘Herbal plants and their derivatives as

growth and health promoters in animal nutrition’, Veterinary Research

References

211

Communications, 35(3), pp. 169–180. doi: 10.1007/s11259-010-9458-2.

Hashemi, S. R., Zulkifli, I., Bejo, M. H., Farida, A. and Somchit, M. N. (2008)

‘Acute toxicity study and phytochemical screening of selected herbal aqueous

extract in broiler chickens’, International Journal of Pharmacology, 4(5), pp.

352–360. doi: 10.3923/ijp.2008.352.360.

Hashemipour, H., Kermanshahi, H., Golian, A. and Veldkamp, T. (2013) ‘Effect

of thymol and carvacrol feed supplementation on performance, antioxidant

enzyme activities, fatty acid composition, digestive enzyme activities, and

immune response in broiler chickens’, Poultry Science, 92(8), pp. 2059–2069.

doi: 10.3382/ps.2012-02685.

Hayes, J. R., Carr, L. E., Mallinson, E. T., Douglass, L. W., Joseph, S. W. and

Al, H. E. T. (2000) ‘Characterization of the contribution of water activity and

moisture content to the population distribution of Salmonella spp. in

commercial poultry houses’, Poultry Science, 79(11), pp. 1557–1561. doi:

10.1093/ps/79.11.1557.

Hemaiswarya, S., Kruthiventi, A. K. and Doble, M. (2008) ‘Synergism between

natural products and antibiotics against infectious diseases’, Phytomedicine,

15(8), pp. 639–652. doi: 10.1016/j.phymed.2008.06.008.

Hernandez-Patlan, D., Solis-Cruz, B., Hargis, B. M. and Tellez, G. (2016) ‘The

use of probiotics in poultry production for the control of bacterial infections and

aflatoxins’, Prebiotics and Probiotics - Potential Benefits in Human Nutrition

and Health, p. 13. doi: http://dx.doi.org/10.5772/57353.

Hernández, F., Madrid, J., García, V., Orengo, J. and Megías, M. D. (2004)

‘Influence of two plant extracts on broilers performance, digestibility, and

References

212

digestive organ size’, Poultry Science, 83(2), pp. 169–174. doi:

10.1093/ps/83.2.169.

Higgins, D., Mukherjee, N., Pal, C. and Sulaiman, I. M. (2020) ‘Association of

virulence and antibiotic resistance in Salmonella — statistical and

computational insights into a selected set of clinical isolates’, Microorganisms,

8(1465), pp. 1–17.

HM Government (2019) Tackling antimicrobial resistance 2019–2024 – The

UK’s five-year national action plan, HM Government. doi:

10.1016/j.jhin.2019.02.019.

Hong, Y., Cheng, Y., Li, Y., Li, X., Zhou, Z., Shi, D., Li, Z. and Xiao, Y. (2019)

‘Preliminary study on the effect of Bacillus amyloliquefaciens TL on cecal

bacterial community structure of broiler chickens’, BioMed Research

International, 2019. doi: 10.1155/2019/5431354.

Hooge, D. M. and Connolly, A. (2011) ‘Meta-analysis summary of broiler

chicken trials with dietary Actigen (2009-2011)’, International Journal of Poultry

Science, 10(10), pp. 819–824.

Hossain, A., Park, H., Lee, K., Park, Sung-won, Park, Seung-chun and Kang,

J. (2020) ‘In vitro synergistic potentials of novel antibacterial combination

therapies against Salmonella enterica serovar Typhimurium’, BMC

Microbiology, 20(118), pp. 1–14.

Hua, S., Zhang, Y., Liu, J., Dong, L., Huang, J., Lin, D. and Fu, X. (2018)

‘Ethnomedicine, phytochemistry and pharmacology of Smilax glabra: An

important Traditional Chinese Medicine’, The American Journal of Chinese

Medicine, 46(2), pp. 261–297. doi: 10.1142/S0192415X18500143.

References

213

Huovinen, P. (2001) ‘Resistance to trimethoprim-sulfamethoxazole’, Clinical

Infectious Diseases, 2001(32), pp. 1608–1614. doi: 10.1086/320532.

Hwang, H., Miller, E. A., Johnson, A., Valeris-Chacin, R., Nault, A. J., Singer,

R. S. and Johnson, T. J. (2020) Efficacy of prebiotics and probiotics on growth

performance in poultry: A protocol for a systematic review.

Interagency Coordination Group on Antimicrobial Resistance (2019) No time

to wait: Securing the future from drug resistant infections, Interagency

Coordination Group on Antimicrobial Resistance.

International organisation for standardisation (2005) ISO 13903:2005 Animal

feeding stuffs — Determination of amino acids content.

Ionescu, D., Mihele, D., Predan, G., Rizea, G. D., Dune, A. and Ivopol, G.

(2013) ‘Antimicrobial activity of some hydroalcoholic extracts of artichoke

(Cynara scolymus), burdock (Arctium lappa) and dandelion (Taraxacum

officinale)’, Agricultural Food Engineering, 6(55).

Iovine, N. M. (2013) ‘Resistance mechanisms in Campylobacter jejuni’,

Virulence, 4, pp. 230–240.

Irshad, S., Butt, M. and Younus, H. (2011) ‘In vitro antibacterial activity of Aloe

Barbadensis Miller (Aloe Vera)’, International Research Journal of

Pharmaceuticals, 01(02), pp. 59–64.

Jammoul, A. and Darra, N. El (2019) ‘Evaluation of antibiotics residues in

chicken meat samples in Lebanon’, Antibiotics, 8(69), pp. 1–11.

Jamwal, K., Bhattacharya, S. and Puri, S. (2018) ‘Plant growth regulator

mediated consequences of secondary metabolites in medicinal plants’, Journal

References

214

of Applied Research on Medicinal and Aromatic Plants, 9, pp. 26–38. doi:

https://doi.org/10.1016/j.jarmap.2017.12.003.

Jang, I. S., Ko, Y. H., Kang, S. Y. and Lee, C. Y. (2007) ‘Effect of a commercial

essential oil on growth performance, digestive enzyme activity and intestinal

microflora population in broiler chickens’, Animal Feed Science and

Technology, 134(3), pp. 304–315. doi:

https://doi.org/10.1016/j.anifeedsci.2006.06.009.

Jeong, J. and Kim, I.-S. (2014) ‘Effect of Bacillus subtilis C-3102 spores as a

probiotic feed supplement on growth performance, noxious gas emission, and

intestinal microflora in broilers’, Poultry science, 93(12), pp. 3097–3103. doi:

10.3382/ps.2014-04086.

Ji-Hyun, B. and Mee-Aae, S. (2005) ‘Effect of Agrimonia pilosa Ledeb extract

on the growth of food-borne pathogens’, The Korean Nutrition Society, 38(2),

pp. 112–116.

Johny, A. K., Baskaran, S. A., Charles, A. S., Amalaradjou, M. A. R., Darre, M.

J., Khan, M. I., Hoagland, T. A., Schreiber, D. T., Donoghue, A. M., Donoghue,

D. J. and Venkitanarayanan, K. (2009) ‘Prophylactic supplementation of

caprylic acid in feed reduces Salmonella enteritidis colonization in commercial

broiler chicks’, Journal of food protection, 72(4), pp. 722–727.

Johny, A. K., Darre, M. J., Donoghue, A. M., Donoghue, D. J. and

Venkitanarayanan, K. (2010) ‘Antibacterial effect of trans-cinnamaldehyde,

eugenol, carvacrol, and thymol on Salmonella enteritidis and Campylobacter

jejuni in chicken cecal contents in vitro’, Journal of Applied Poultry Research,

19(3), pp. 237–244. doi: 10.3382/japr.2010-00181.

References

215

de Jong, I. C., van Hattum, T., van Riel, J. W., De Baere, K., Kempen, I.,

Cardinaels, S. and Gunnink, H. (2020) ‘Effects of on-farm and traditional

hatching on welfare, health, and performance of broiler chickens’, Poultry

Science, 99(10), pp. 4662–4671. doi: 10.1016/j.psj.2020.06.052.

Joung, D. K., Mun, S. H., Lee, K. S., Kang, O. H., Choi, J. G., Kim, S. B., Gong,

R., Chong, M. S., Kim, Y. C., Lee, D. S., Shin, D. W. and Kwon, D. Y. (2014)

‘The antibacterial assay of tectorigenin with detergents or ATPase inhibitors

against methicillin-resistant Staphylococcus aureus’, Evidence-based

Complementary and Alternative Medicine, 2014(1), pp. 1–7. doi:

10.1155/2014/716509.

Juanpere, J., Pérez-Vendrell, a M., Angulo, E. and Brufau, J. (2005)

‘Assessment of potential interactions between phytase and glycosidase

enzyme supplementation on nutrient digestibility in broilers.’, Poultry science,

84(4), pp. 571–580.

Jung, S. J., Houde, R., Baurhoo, B., Zhao, X. and Lee, B. H. (2008) ‘Effects of

galacto-oligosaccharides and a Bifidobacteria lactis-based probiotic strain on

the growth performance and fecal microflora of broiler chickens’, Poultry

Science, 87(9), pp. 1694–1699. doi: 10.3382/ps.2007-00489.

Kaakoush, N. O., Castaño-Rodríguez, N., Mitchell, H. M. and Man, S. M.

(2015) ‘Global epidemiology of Campylobacter infection’, Clinical Microbiology

Reviews, 28(3), pp. 687–720. doi: 10.1128/CMR.00006-15.

Kamatou, G. P. P., Viljoen, A. M., van Vuuren, S. F. and van Zyl, R. L. (2006)

‘In vitro evidence of antimicrobial synergy between Salvia chamelaeagnea and

Leonotis leonurus’, South African Journal of Botany, 72(4), pp. 634–636. doi:

References

216

10.1016/j.sajb.2006.03.011.

Kambai, J., Jekada, J. Z. and Audu, J. A. (2019) ‘Phytochemical screening and

antimicrobial effects of Aloe vera juice on some common skin pathogens’,

International Invention of Scientific Journal, 03(07), pp. 619–626.

Kasai, S., Watanabe, S., Kawabata, J., Tahara, S. and Mizuntani, J. (1992)

‘Antimicrobial catechin derivatives of Agrimonia pilosa’, Phytochemistry, 31(3),

pp. 787–789.

Khameneh, B., Iranshahy, M., Soheili, V., Sedigheh, B. and Bazzaz, F. (2019)

‘Review on plant antimicrobials : a mechanistic viewpoint’, Antimicrobial

Resistance and Infection Control, 8(118), pp. 1–28.

Khan, M. I., Ahhmed, A., Shin, J. H., Baek, J. S., Kim, M. Y. and Kim, J. D.

(2018) ‘Green tea seed isolated aaponins exerts antibacterial effects against

various strains of Gram positive and Gram negative bacteria, a comprehensive

study in vitro and in vivo’, Evidence-based Complementary and Alternative

Medicine, 2018. doi: 10.1155/2018/3486106.

Khan, S. H. and Iqbal, J. (2016) ‘Recent advances in the role of organic acids

in poultry nutrition’, Journal of Applied Animal Research, 44(1), pp. 359–369.

doi: 10.1080/09712119.2015.1079527.

Khattak, F., Paschalis, V., Green, M., Houdijk, G. M., Soultanas, P. and

Mahdavi, J. (2018) ‘TYPLEX® Chelate, a novel feed additive, inhibits

Campylobacter jejuni biofilm formation and cecal colonization in broiler

chickens’, Poultry Science, 97(4), pp. 1391–1399. doi: 10.3382/ps/pex413.

Kiarie, E., Romero, L. F. and Nyachoti, C. M. (2013) ‘The role of added feed

References

217

enzymes in promoting gut health in swine and poultry’, Nutrition Research

Reviews, 26(1), pp. 71–88. doi: 10.1017/S0954422413000048.

Kim, G., Gan, R., Zhang, D., Farha, A. K., Habimana, O., Mavumengwana, V.,

Li, H., Wang, X. and Corke, H. (2020) ‘Large-scale screening of 239 traditional

Chinese medicinal plant extracts for their antibacterial activities against

Multidrug-resistant Staphylococcus aureus and cytotoxic activities’,

Pathogens, 9(189).

Kim, J. W., Kim, J. H. and Kil, D. Y. (2015) ‘Dietary organic acids for broiler

chickens: A review’, Revista Colombiana de Ciencias Pecuarias, 28(2), pp.

109–123. doi: 10.17533/udea.rccp.v28n2a01.

Kim, K., Owens, G., Kwon, S., So, K., Lee, D. and Ok, Y. S. (2011) ‘Occurrence

and environmental fate of veterinary antibiotics in the terrestrial environment’,

Water, Air, and Soil Pollution, 214(1), pp. 163–174. doi: 10.1007/s11270-010-

0412-2.

Kim, K. S., Manocchio, M. and Anthony, B. F. (1984) ‘Paradox between the

responses of Escherichia coli K1 to ampicillin and chloramphenicol in vitro and

in vivo’, Antimicrobial Agents and Chemotherapy, 26(5), pp. 689–693. doi:

10.1128/AAC.26.5.689.

Kim, M. and Lee, H. (2015) ‘Growth-inhibiting effects and chemical

composition of essential oils extracted from Platycladus orientalis leaves and

stems toward human intestinal bacteria’, Food Science and Biotechnology,

24(2), pp. 427–431. doi: 10.1007/s10068-015-0056-5.

Kim, S., Kang, D., Kim, J., Ha, Y., Hwang, J. Y., Kim, T. and Lee, S. (2011a)

‘Antimicrobial activity of plant extracts against Salmonella typhimurium,

References

218

Escherichia coliO157 : H7, and Listeria monocytogenes on fresh lettuce’,

76(1). doi: 10.1111/j.1750-3841.2010.01926.x.

Kim, S., Kang, D., Kim, J., Ha, Y., Hwang, J. Y., Kim, T. and Lee, S. (2011b)

‘Antimicrobial activity of plant extracts against Salmonella typhimurium,

Escherichia coliO157 : H7, and Listeria monocytogenes on fresh lettuce’,

Journal of Food Science, 76(1). doi: 10.1111/j.1750-3841.2010.01926.x.

Kim, S., Lee, K., Kang, C. and An, B. (2016) ‘Growth performance, relative

meat and organ Weights, cecal microflora, and blood characteristics in broiler

chickens fed diets containing different nutrient density with or without essential

oils’, Asian-Australasian Journal of Animal Sciences, 29(4), pp. 549–554.

Kirchhelle, C. (2018) ‘Pharming animals: a global history of antibiotics in food

production (1935–2017)’, Palgrave Communications, 4(1), pp. 1–13. doi:

10.1057/s41599-018-0152-2.

Kirk, M. D., Pires, S. M., Black, R. E., Caipo, M., Crump, J. A.,

Devleesschauwer, B., Döpfer, D., Fazil, A., Fischer-Walker, C. L., Hald, T.,

Hall, A. J., Keddy, K. H., Lake, R. J., Lanata, C. F., Torgerson, P. R., Havelaar,

A. H. and Angulo, F. J. (2015) ‘World Health Organization estimates of the

global and regional disease burden of 22 foodborne bacterial, protozoal, and

viral diseases, 2010: A data synthesis’, PLoS Medicine, 12(12), pp. 1–21. doi:

10.1371/journal.pmed.1001921.

Kittelsen, K. E., Granquist, E. G., Kolbjørnsen, O., Nafstad, O. and Moe, R. O.

(2015) ‘A comparison of post-mortem findings in broilers dead-on-farm and

broilers dead-on-arrival at the abattoir’, Poultry Science, 94(11), pp. 2622–

2629. doi: 10.3382/ps/pev294.

References

219

Klančnik, A., Piskernik, S., Jeršek, B. and Možina, S. S. (2010) ‘Evaluation of

diffusion and dilution methods to determine the antibacterial activity of plant

extracts’, Journal of Microbiological Methods, 81(1), pp. 121–126. doi:

10.1016/j.mimet.2010.02.004.

Klein, E. Y., Van Boeckel, T. P., Martinez, E. M., Pant, S., Gandra, S., Levin,

S. A., Goossens, H. and Laxminarayan, R. (2018) ‘Global increase and

geographic convergence in antibiotic consumption between 2000 and 2015’,

Proceedings of the National Academy of Sciences, 115(15), p. E3463 LP-

E3470. doi: 10.1073/pnas.1717295115.

Koch, B. J., Hungate, B. A. and Price, L. B. (2017) ‘Food-animal production

and the spread of antibiotic resistance: the role of ecology’, Frontiers in

Ecology and the Environment, 15(6), pp. 309–318. doi: 10.1002/fee.1505.

Konaklieva, M. I. (2014) ‘Molecular targets of β-lactam-based antimicrobials:

Beyond the usual suspects’, Antibiotics, 3, pp. 128–142. doi:

10.3390/antibiotics3020128.

Kraemer, S. A., Ramachandran, A. and Perron, G. G. (2019) ‘Antibiotic

pollution in the environment: From microbial ecology to public policy’,

Microorganisms, 7(6), pp. 1–24. doi: 10.3390/microorganisms7060180.

Krause, K. M., Serio, A. W., Kane, T. R. and Connolly, L. E. (2016)

‘Aminoglycosides: An overview’, Cold Spring Harbor Perspectives in Medicine,

6, pp. 1–19.

Krawczyk-Balska, A. and Markiewicz, Z. (2016) ‘The intrinsic cephalosporin

resistome of Listeria monocytogenes in the context of stress response, gene

regulation, pathogenesis and therapeutics’, Journal of Applied Microbiology,

References

220

120(2), pp. 251–265. doi: 10.1111/jam.12989.

Kris-Etherton, P., Hecker, K. D., Bonanome, A., Coval, S. M., Binkoski, A. E.,

Hilpert, K. F., Griel, A. E. and Etherton, T. D. (2002) ‘Bioactive compounds in

foods: their role in the prevention of cardiovascular disease and cancer.’, The

American journal of medicine, 113 Suppl(01), pp. 71S-88S. doi:

10.1016/S0002-9343(01)00995-0.

Krishan, G. and Narang, A. (2014) ‘Use of essential oils in poultry nutrition: A

new approach’, Journal of Advanced Veterinary and Animal Research, 1(4),

pp. 156–162. doi: 10.5455/javar.2014.a36.

Kumar, M., Kumar, V., Roy, D., Kushwaha, R. and Vaswani, S. (2014)

‘Application of herbal feed additives in animal nutrition - A review’, International

Journal of Livestock Research, 4(9), p. 1. doi: 10.5455/ijlr.20141205105218.

Kumar, S., Madaan, R., Farooq, A. and Sharma, A. (2008) ‘Plant review the

genus Pulsatilla: A review’, Pharmacognosy Reviews, 2(3), pp. 116–124.

Lai, W., Cock, I. E. and Cheesman, M. J. (2018) ‘Interactive antimicrobial

profiles of Astragalus membranaceus (Fisch.) Bunge extracts and

conventional antibiotics against pathogenic and non-pathogenic

gastrointestinal bacteria’, Pharmacognosy Communications, 8(4), pp. 158–

164. doi: 10.5530/pc.2018.4.33.1.

Latorre, M., Revuelta, J., García-Junceda, E. and Bastida, A. (2016) ‘6-O-

Nucleotidyltransferase: an aminoglycoside-modifying enzyme specific for

streptomycin/streptidine’, Medicinal Chemistry Communications, 7, pp. 177–

183.

References

221

Lazarus, B., Paterson, D. L., Mollinger, J. L. and Rogers, B. A. (2015) ‘Do

human extraintestinal escherichia coli infections resistant to expanded-

spectrum cephalosporins originate from food-producing animals? A systematic

review’, Clinical Infectious Diseases, 60(3), pp. 439–452. doi:

10.1093/cid/ciu785.

Leclercq, R. (2002) ‘Mechanisms of resistance to macrolides and

lincosamides: Nature of the resistance elements and their clinical implications’,

Clinical Infectious Diseases, 34(4), pp. 482–492. doi: 10.1086/324626.

Lee, C.-F., Han, C.-K. and Tsau, J.-L. (2004) ‘In vitro inhibitory activity of

Chinese leek extract against Campylobacter species’, International Journal of

Food Microbiology, 94(2), pp. 169–174. doi:

https://doi.org/10.1016/j.ijfoodmicro.2004.01.009.

Lee, D., Das, S., Dawson, N. L., Dobrijevic, D., Ward, J. and Orengo, C. (2016)

‘Novel computational protocols for functionally classifying and characterising

serine beta-lactamases’, PLOS Computational Biology, 12(6), pp. 1–33. doi:

10.1371/journal.pcbi.1004926.

Lee, J., Ji, Y., Yu, M., Bo, M. H., Seo, H., Lee, S. and Lee, I. (2009)

‘Antimicrobial effect and resistant regulation of Glycyrrhiza uralensis on

methicillin-resistant Staphylococcus aureus’, Natural Product Research, 6419.

doi: 10.1080/14786410801886757.

Lee, K., Cha, J., Choi, S., Jang, E., Ko, E., Cha, S. and Yun, S. (2018)

‘Antibacterial activity of the ethanol extract of Paeonia lactiflora on growth of

oral bacteria’, Journal of Oral Health and Dental Science, 2(2).

Lee, K. W., Hong, Y., Lee, S.-H., Jang, S., Park, M., Bautista, D., Ritter, D.,

References

222

Jeong, W., Jeoung, H. Y., An, D.-J., Lillehoj, E. and Lillehoj, H. (2012) ‘Effects

of anticoccidial and antibiotic growth promoter programs on broiler

performance and immune status’, Research in veterinary science, 93(2), pp.

721–728. doi: 10.1016/j.rvsc.2012.01.001.

Lee, K. W., Lee, S. H., Lillehoj, H. S., Li, G. X., Jang, S. I., Babu, U. S., Park,

M. S., Kim, D. K., Lillehoj, E. P., Neumann, A. P., Rehberger, T. G. and

Siragusa, G. R. (2010) ‘Effects of direct-fed microbials on growth performance,

gut morphometry, and immune characteristics in broiler chickens.’, Poultry

science, 89(2), pp. 203–216. doi: 10.3382/ps.2009-00418.

Lee, N. K., Jung, B. S., Na, D. S., Yu, H. H., Kim, J. S. and Paik, H. D. (2016)

‘The impact of antimicrobial effect of chestnut inner shell extracts against

Campylobacter jejuni in chicken meat’, LWT - Food Science and Technology,

65, pp. 746–750. doi: 10.1016/j.lwt.2015.09.004.

Lee, S.-H. and Kim, M.-J. (2019) ‘Antimicrobial effect of natural plant extracts

against periodontopathic bacteria’, Journal of the Korea Contents Association,

19(1), pp. 242–255.

Lei, X., Li, J., Liu, B., Zhang, N. and Liu, H. (2015) ‘Separation and

identification of four new compounds with antibacterial activity from Portulaca

olercacea L.’, Molecules, 20, pp. 16375–16387. doi:

10.3390/molecules200916375.

Lemaire, S., Van Bambeke, F., Mingeot-Leclercq, M. P. and Tulkens, P. M.

(2005) ‘Activity of three β-lactams (ertapenem, meropenem and ampicillin)

against intraphagocytic Listeria monocytogenes and Staphylococcus aureus’,

Journal of Antimicrobial Chemotherapy, 55(6), pp. 897–904. doi:

References

223

10.1093/jac/dki094.

Leone, J. L. (1973) ‘Collaborative study of the quantitative determination of

titanium dioxide in cheese.’, Journal - Association of Official Analytical

Chemists, 56(3), pp. 535–537.

Leong, W. M. (2013) Screening for antibacterial activity of local plants.

Levy, S. B., Fitzgerald, G. B. and Macone, A. N. N. B. (1976) ‘Spread of

antibiotic-resistant plasmids from chicken to chicken and from chicken to man’,

Nature, 260(5546), pp. 40–42. doi: 10.1038/260040a0.

Li, N., Ho, K. W. K., Ying, G. G. and Deng, W. J. (2017) ‘Veterinary antibiotics

in food, drinking water, and the urine of preschool children in Hong Kong’,

Environment International, 108(2017), pp. 246–252. doi:

10.1016/j.envint.2017.08.014.

Li, W., Atkinson, G. C., Thakor, N. S., Lu, C., Chan, K.-Y., Tenson, T.,

Schulten, K., Wilson, K. S., Hauryliuk, V. and Jaochim, F. (2013) ‘Mechanism

of tetracycline resistance by ribosomal protection protein tet(o)’, Nature

Communications, 4(147), pp. 1–17. doi: 10.1038/ncomms2470.Mechanism.

Li, X., Wang, Z., Li, X. and Wang, Z. (2011) ‘Chemical composition ,

antimicrobial and antioxidant activities of the essential oil in leaves of Salvia

miltiorrhiza Bunge’, Journal of Essential Oil Research, 2905. doi:

10.1080/10412905.2009.9700222.

Li, Y., Xu, C., Zhang, Q., Liu, J. Y. and Tan, R. X. (2005) ‘In vitro anti

Helicobacter pylori action of 30 Chinese herbal medicines used to treat ulcer

diseases’, Journal of Ethnopharmacology, 98, pp. 329–333. doi:

References

224

10.1016/j.jep.2005.01.020.

Lillehoj, H., Liu, Y., Calsamiglia, S., Fernandez-Miyakawa, M. E., Chi, F.,

Cravens, R. L., Oh, S. and Gay, C. G. (2018) ‘Phytochemicals as antibiotic

alternatives to promote growth and enhance host health’, Veterinary Research,

49(76), pp. 1–18. doi: 10.1186/s13567-018-0562-6.

Lin, J. (2011) ‘Effect of antibiotic growth promoters on intestinal microbiota in

food animals: A novel model for studying the relationship between gut

microbiota and human obesity?’, Frontiers in Microbiology, 2(MAR), pp. 2002–

2004. doi: 10.3389/fmicb.2011.00053.

Lin, Y. T., Labbe, R. G. and Shetty, K. (2004) ‘Inhibition of Listeria

monocytogenes in fish and meat systems by use of oregano and cranberry

phytochemical synergies’, Applied and Environmental Microbiology, 70(9), pp.

5672–5678. doi: 10.1128/AEM.70.9.5672-5678.2004.

Liu, C., Cham, T., Yang, C. and Chang, H. (2007) ‘Antibacterial properties of

Chinese herbal medicines against nosocomial antibiotic resistant strains of

Pseudomonas aeruginosa in Taiwan’, 35(6), pp. 1047–1060.

Liu, I. X., Durham, D. G. and Richards, R. M. E. (2000) ‘Baicalin synergy with

β-lactam antibiotics against methicillin-resistant Staphylococcus aureus and

other β-lactam-resistant strains of S. aureus’, Journal of Pharmacy and

Pharmacology, 52(3), pp. 361–366. doi:

https://doi.org/10.1211/0022357001773922.

Liu, J., Huang, D., Hao, D. and Hu, Q. (2014) ‘Chemical composition,

antibacterial activity of the essential oil from roots of Radix aucklandiae against

selected food-borne pathogens’, Advances in Bioscience and Biotechnology,

References

225

05(13), pp. 1043–1047. doi: 10.4236/abb.2014.513119.

Liu, X., Steele, J. C. and Meng, X. Z. (2017) ‘Usage, residue, and human health

risk of antibiotics in Chinese aquaculture: A review’, Environmental Pollution,

223, pp. 161–169. doi: 10.1016/j.envpol.2017.01.003.

Lohakare, J. D., Chae, B. J. and Hahn, T. W. (2004) ‘Effects of feeding

methods (water vs. feed) of vitamin C on growth performance and carcass

characteristics in broiler chickens’, Asian-Australasian Journal of Animal

Sciences, 17(8), pp. 1112–1117. doi: 10.5713/ajas.2004.1112.

Lowy, F. (2003) ‘Antimicrobial resistance: the example of Staphylococcus

aureus’, The Journal of Clinical Investigation, 111(9), pp. 1267–1273. doi:

10.1172/JCI200318535.

Luong, T. M. N., Joon-Kwan, M., Jeong-Han, K., Takayuki, S. and Young-

Joon, A. (2012) ‘Growth-inhibiting effects of Paeonia lactiflora root steam

distillate constituents and structurally related compounds on human intestinal

bacteria’, World Journal of Microbiology and Biotechnology, 28, pp. 1575–

1583. doi: 10.1007/s11274-011-0961-6.

Ma, F., Chen, Y., Li, J., Qing, H., Wang, J., Zhang, Y., Long, B. and Bai, Y.

(2010) ‘Screening test for anti- Helicobacter pylori activity of traditional

Chinese herbal medicines’, World Journal of Gastroenterology, 16(44), pp.

5629–5634. doi: 10.3748/wjg.v16.i44.5629.

Mak, S., Xu, Y. and Nodwell, J. R. (2014) ‘The expression of antibiotic

resistance genes in antibiotic-producing bacteria’, Molecular Microbiology,

93(3), pp. 391–402. doi: 10.1111/mmi.12689.

References

226

Maki, J. J., Klima, C. L., Sylte, M. J. and Looft, T. (2019) ‘The microbial pecking

order: Utilization of intestinal microbiota for poultry health’, Microorganisms,

7(10), p. 376. doi: 10.3390/microorganisms7100376.

Manandhar, S., Luitel, S. and Dahal, R. K. (2019) ‘In vitro antimicrobial activity

of some medicinal plants against human pathogenic bacteria’, Journal of

Tropical Medicine, 2019. doi: 10.1155/2019/1895340.

Manyi-Loh, C., Mamphweli, S., Meyer, E. and Okoh, A. (2018) Antibiotic use

in agriculture and its consequential resistance in environmental sources:

Potential public health implications, Molecules. doi:

10.3390/molecules23040795.

Maron, D. F., Smith, T. J. S. and Nachman, K. E. (2013) ‘Restrictions on

antimicrobial use in food animal production: An international regulatory and

economic survey’, Globalization and Health, 9(48), pp. 1–11. doi:

10.1186/1744-8603-9-48.

Marshall, B. M. and Levy, S. B. (2011) ‘Food animals and antimicrobials:

Impacts on human health’, Clinical Microbiology Reviews, 24(4), pp. 718–733.

doi: 10.1128/CMR.00002-11.

Marteau, P. and Shanahan, F. (2003) ‘Basic aspects and pharmacology of

probiotics: An overview of pharmacokinetics, mechanisms of action and side-

effects’, Bailliere’s Best Practice and Research in Clinical Gastroenterology,

17(5), pp. 725–740. doi: 10.1016/S1521-6918(03)00055-6.

Matshediso, P. G., Cukrowska, E. and Chimuka, L. (2015) ‘Development of

pressurised hot water extraction ( PHWE ) for essential compounds from

Moringa oleifera leaf extracts’, Food Chemistry, 172, pp. 423–427. doi:

References

227

10.1016/j.foodchem.2014.09.047.

McMurray, R. L., Ball, M. E. E., Tunney, M. M., Corcionivoschi, N. and Situ, C.

(2020) ‘Antibacterial activity of four plant extracts extracted from traditional

Chinese medicinal plants against Listeria monocytogenes, Escherichia coli,

and Salmonella enterica subsp. enterica serovar Enteritidis’, Microorganisms,

8(962), pp. 1–12. doi: 10.3390/microorganisms8060962.

Meseret, S. (2016) ‘A review of poultry welfare in conventional production

system’, Livestock Research for Rural Development, 28(12), pp. 1–6.

Micciche, A., Rothrock, M. J., Yang, Y. and Ricke, S. C. (2019) ‘Essential oils

as an intervention strategy to reduce Campylobacter in poultry production: A

review’, Frontiers in Microbiology, 10(1058), pp. 1–22. doi:

10.3389/fmicb.2019.01058.

Miguel, M. G. (2010) ‘Antioxidant and anti-inflammatory activities of essential

oils: A short review’, Molecules, 15(12), pp. 9252–9287. doi:

10.3390/molecules15129252.

Miyasaki, Y., Nichols, W. S., Morgan, M. A., Kwan, J. A., Van Benschoten, M.

M., Kittell, P. E. and Hardy, W. D. (2010) ‘Screening of herbal extracts against

multi-drug resistant Acinetobacter baumannii’, Phytotherapy Research, 24(8),

pp. 1202–1206. doi: 10.1002/ptr.3113.

Mohiti-Asli, M. and Ghanaatparast-Rashti, M. (2017) ‘Comparing the effects of

a combined phytogenic feed additive with an individual essential oil of oregano

on intestinal morphology and microflora in broilers’, Journal of Applied Animal

Research, 2119, pp. 1–6. doi: 10.1080/09712119.2017.1284074.

References

228

Mookiah, S., Sieo, C. C., Ramasamy, K., Abdullah, N. and Ho, Y. W. (2014)

‘Effects of dietary prebiotics, probiotic and synbiotics on performance, caecal

bacterial populations and caecal fermentation concentrations of broiler

chickens’, Journal of the Science of Food and Agriculture, 94(2), pp. 341–348.

doi: 10.1002/jsfa.6365.

Morgan, D. J., Okeke, I. N., Laxminarayan, R., Perencevich, E. N. and

Weisenberg, S. (2011) ‘Non-prescription antimicrobial use worldwide: A

systematic review’, The Lancet Infectious Diseases, 11(9), pp. 692–701. doi:

https://doi.org/10.1016/S1473-3099(11)70054-8.

Mottet, A. and Tempio, G. (2017) ‘Global poultry production: current state and

future outlook and challenges’, World’s Poultry Science Journal. 2017/04/11,

73(2), pp. 245–256. doi: DOI: 10.1017/S0043933917000071.

Muller, C., Plesiat, P. and Jeannot, K. (2011) ‘A two-component regulatory

system interconnects resistance to polymyxins, aminoglycosides,

floroquinolones, and β-lactams in Psuedomonas aeruginosa’, Antimicrobial

Agents and Chemotherapy, 55(3), pp. 1211–1221. doi: 10.1128/AAC.01252-

10.

Mund, M. D., Khan, U. H., Tahir, U., Mustafa, B. E. and Fayyaz, A. (2017)

‘Antimicrobial drug residues in poultry products and implications on public

health: A review’, International Journal of Food Properties, 20(7), pp. 1433–

1446. doi: 10.1080/10942912.2016.1212874.

Munita, J. M. and Arias, C. A. (2016) ‘Mechanisms of antibiotic resistance’,

Microbiology Spectrum, 4(2), pp. 1–24.

Nakphaichit, M., Thanomwongwattana, S., Phraephaisarn, C., Sakamoto, N.,

References

229

Keawsompong, S., Nakayama, J. and Nitisinprasert, S. (2011) ‘The effect of

including Lactobacillus reuteri KUB-AC5 during post-hatch feeding on the

growth and ileum microbiota of broiler chickens’, Poultry Science, 90(12), pp.

2753–2765. doi: 10.3382/ps.2011-01637.

Nanasombat, S., Kuncharoen, N. and Ritcharoon, B. (2018) ‘Antibacterial

activity of Thai medicinal plant extracts against oral and gastrointestinal

pathogenic bacteria and prebiotic effect on the growth of Lactobacillus

acidophilus’, Chiang Mai Journal of Science, 45(1), pp. 33–44.

Nasir, B., Fatima, H., Ahmad, M. and Hsan-ul-Haq (2015) ‘Recent trends and

methods in antimicrobial drug discovery from plant sources’, Austin Journal of

Microbiology, 1(1), p. 1002. Available at:

http://austinpublishinggroup.com/microbiology/fulltext/ajm-v1-id1002.php.

Natchimuthu, K., Mani, J. and Subbiah, K. (2012) ‘Synergistic antibacterial

activity of four medicinal plants collected from Dharapuram Taluk of Tiruppur

District, South India’, Journal of Plant Sciences, 7(1), pp. 32–38.

Navarro, M., Stanley, R., Cusack, A. and Yasmina, S. (2015) ‘Combinations of

plant-derived compounds against Campylobacter in vitro’, Journal of Applied

Poultry Research, 24(3), pp. 352–363. doi: 10.3382/japr/pfv035.

Neumann, A. P. and Suen, G. (2015) ‘Differences in major bacterial

populations in the intestines of mature broilers after feeding virginiamycin or

bacitracin methylene disalicylate’, Journal of Applied Microbiology, 119(6), pp.

1515–1526. doi: 10.1111/jam.12960.

Neyestani, T. R., Khalaji, N. and Gharavi, A. (2007) ‘Selective microbiologic

effects of tea extract on certain antibiotics against Escherichia coli in vitro’,

References

230

Journal of Alternative and Complementary Medicine, 13(10), pp. 1119–1124.

doi: 10.1089/acm.2007.7033.

Nkukwana, T. T., Muchenje, V., Masika, P. J. and Mushonga, B. (2015)

‘Intestinal morphology, digestive organ size and digesta pH of broiler chickens

fed diets supplemented with or without Moringa oleifera leaf meal’, South

African Journal of Animal Sciences, 45(4), pp. 362–371. doi:

10.4314/sajas.v45i4.2.

Noll, M., Kleta, S. and Al Dahouk, S. (2018) ‘Antibiotic susceptibility of 259

Listeria monocytogenes strains isolated from food, food-processing plants and

human samples in Germany’, Journal of Infection and Public Health, 11(4), pp.

572–577. doi: 10.1016/j.jiph.2017.12.007.

Norouzi, B., Qotbi, A. A. A., Seidavi, A., Schiavone, A. and Martínez Marín, A.

L. (2015) ‘Effect of different dietary levels of rosemary (Rosmarinus officinalis)

and yarrow (Achillea millefolium) on the growth performance, carcass traits

and ileal microbiota of broilers’, Italian Journal of Animal Science, 14(3), pp.

448–453. doi: 10.4081/ijas.2015.3930.

O’Dea, E. E., Fasenko, G. M., Allison, G. E., Korver, D. R., Tannock, G. W.

and Guan, L. L. (2006) ‘Investigating the effects of commercial probiotics on

broiler chick quality and production efficiency’, Poultry Science, 85(10), pp.

1855–1863. doi: 10.1093/ps/85.10.1855.

O’Neill, J. (2016) Tackling drug-resistant infections globally: final report and

recommendations, the Review on Antimicrobial Resistance. doi:

10.1016/j.jpha.2015.11.005.

Obaji, M., Enweani, I. B. and Oli, A. N. (2020) ‘A unique combination of

References

231

Alchornea cordifolia and Pterocarpus santalinoides in the management of

multi-drug resistant diarrhoegenic bacterial infection.’, Asian Journal of

Biomedical and Pharmaceutical Sciences, 10(69). doi: 10.35841/2249-

622x.69.5059.

Odds, F. C. (2003) ‘Synergy, antagonism, and what the chequerboard puts

between them’, Journal of Antimicrobial Chemotherapy, 52(1), pp. 1–1. doi:

10.1093/jac/dkg301.

Oh, E. and Jeon, B. (2015) ‘Synergistic anti-Campylobacter jejuni activity of

fluoroquinolone and macrolide antibiotics with phenolic compounds’, Frontiers

in Microbiology, 6(OCT), pp. 1–9. doi: 10.3389/fmicb.2015.01129.

OIE (2018) Annual report on antimicrobial agents intended for use in animals.

OIE (2021) Annual report on antimicrobial agents intended for use in animals.

Olaimat, A. N., Al-Holy, M. A., Shahbaz, H. M., Al-Nabulsi, A. A., Abu Ghoush,

M. H., Osaili, T. M., Ayyash, M. M. and Holley, R. A. (2018) ‘Emergence of

antibiotic resistance in Listeria monocytogenes isolated from food products: A

comprehensive review’, Comprehensive Reviews in Food Science and Food

Safety, 17(5), pp. 1277–1292. doi: 10.1111/1541-4337.12387.

Omojate, G. C., Enwa, F. O., Jewo, A. O. and Eze, C. O. (2014) ‘Mechanisms

of antimicrobial actions of phytochemicals against enteric pathogens – A

review’, Journal of Pharmaceutical, Chemical and Biological Sciences, 2(2),

pp. 77–85.

Onyebuchi, C. and Kavaz, D. (2020) ‘Effect of extraction temperature and

solvent type on the bioactive potential of Ocimum gratissimum L .

References

232

extracts’, Scientific Reports, 10(21760), pp. 1–11. doi: 10.1038/s41598-020-

78847-5.

Ooi, L. S. M., Li, Y., Kam, S.-L., Wang, H., Wong, E. Y. L. and Ooi, V. E. C.

(2006) ‘Antimicrobial activities of cinnamon oil and cinnamaldehyde from the

Chinese medicinal herb Cinnamomum cassia Blume’, The American Journal

of Chinese Medicine, 34(03), pp. 511–522. doi:

10.1142/S0192415X06004041.

Organisation for Economic and Co-operation and Development (2019)

‘Antimicrobial resistance tackling the burden in the European Union’,

Organisation for Economic Co-operation and Development, pp. 1–20.

Available at: https://www.oecd.org/health/health-systems/AMR-Tackling-the-

Burden-in-the-EU-OECD-ECDC-Briefing-Note-2019.pdf.

Osuntokun, O. T. and Faniyi, I. T. (2020) ‘Curative, suppressive and

prophylactic phyto- medicinal therapy/synergistic efficacy of Alstonia boonei/

Capsicum frutescens ethanolic extracts against Plasmodium berghei (NK 65)’,

Asian Journal of Biotechnology and Genetic Engineering, 3(3), pp. 19–34.

Oyedeji, A. O., Msagati, T. A. M., Williams, A. B. and Benson, N. U. (2019)

‘Determination of antibiotic residues in frozen poultry by a solid-phase

dispersion method using liquid chromatography-triple quadrupole mass

spectrometry’, Toxicology Reports, 6(September), pp. 951–956. doi:

10.1016/j.toxrep.2019.09.005.

Padungtod, P., Kaneene, J. B., Hanson, R., Morita, Y. and Boonmar, S. (2006)

‘Antimicrobial resistance in Campylobacter isolated from food animals and

humans in northern Thailand’, FEMS Immunology and Medical Microbiology,

References

233

47(2), pp. 217–225. doi: 10.1111/j.1574-695X.2006.00085.x.

Pandey, A. and Tripathi, S. (2014) ‘Concept of standardization , extraction and

pre phytochemical screening strategies for herbal drug’, Journal of

Pharacognosy and Phytochemistry, 2(5), pp. 115–119.

Park, J. H. and Kim, I. H. (2014) ‘Supplemental effect of probiotic Bacillus

subtilis B2A on productivity, organ weight, intestinal Salmonella microflora, and

breast meat quality of growing broiler chicks’, Poultry Science, 93(8), pp.

2054–2059. doi: 10.3382/ps.2013-03818.

Park, J. H. and Kim, I. H. (2019) ‘Effects of dietary Achyranthes japonica

extract supplementation on the growth performance, total tract digestibility,

cecal microflora, excreta noxious gas emission, and meat quality of broiler

chickens’, Poultry_Science, 99(1), pp. 463–470. doi: 10.3382/ps/pez533.

Park, S. H., Kim, S. A., Lee, S. I., Rubinelli, P. M., Roto, S. M., Pavlidis, H. O.,

McIntyre, D. R. and Ricke, S. C. (2017) ‘Original XPCTM effect on Salmonella

typhimurium and cecal microbiota from three different ages of broiler chickens

when incubated in an anaerobic in vitro culture system’, Frontiers in

Microbiology, 8(1070), pp. 1–13. doi: 10.3389/fmicb.2017.01070.

Paterson, D. L. and Bonomo, R. A. (2005) ‘Extended-spectrum beta-

lactamases: a clinical update’, Clinical Microbiology Reviews, 18(4), pp. 657–

686. doi: 10.1128/CMR.18.4.657.

Patra, A. K. (2012) Dietary phytochemicals and microbes, Dietary

Phytochemicals and Microbes. doi: 10.1007/978-94-007-3926-0.

Patridge, E., Gareiss, P., Kinch, M. S. and Hoyer, D. (2016) ‘An analysis of

References

234

FDA-approved drugs: Natural products and their derivatives’, Drug Discovery

Today, 21(2), pp. 204–207. doi: 10.1016/j.drudis.2015.01.009.

Patterson, J. A. and Burkholder, K. M. (2003) ‘Application of prebiotics and

probiotics in poultry production’, Poultry Science, 82(4), pp. 627–631. doi:

10.1093/ps/82.4.627.

Pearlin, B. V., Muthuvel, S., Govidasamy, P., Villavan, M., Alagawany, M.,

Ragab Farag, M., Dhama, K. and Gopi, M. (2020) ‘Role of acidifiers in livestock

nutrition and health: A review’, Journal of Animal Physiology and Animal

Nutrition, 104(2), pp. 558–569. doi: 10.1111/jpn.13282.

Peddie, J., Dewar, A. B., Gilbert and Waddington, D. (1982) ‘The use of

titanium dioxide for determining apparent nutrient digestibility in mature

domestic fowls (Gallus domesticus)’, Journal of Agricultural Sciences, 99, pp.

233–236.

Peterson, E. and Kaur, P. (2018) ‘Antibiotic resistance mechanisms in

bacteria: Relationships between resistance determinants of antibiotic

producers, environmental bacteria, and clinical pathogens’, Frontiers in

Microbiology, 9(NOV), pp. 1–21. doi: 10.3389/fmicb.2018.02928.

Petričević, V., Lukić, M., Škrbić, Z., Rakonjac, S., Dosković, V., Petričević, M.

and Stanojković, A. (2018) ‘The effect of using rosemary (Rosmarinus

officinalis) in broiler nutrition on production parameters, slaughter

characteristics, and gut microbiological population’, Turkish Journal of

Veterinary and Animal Sciences, 42(6), pp. 658–664. doi: 10.3906/vet-1803-

53.

Petrova, M., Gorlenko, Z. and Mindlin, S. (2009) ‘Molecular structure and

References

235

translocation of a multiple antibiotic resistance region of a Psychrobacter

psychrophilus permafrost strain’, FEMS Microbiology Letters, 296(2), pp. 190–

197. doi: 10.1111/j.1574-6968.2009.01635.x.

Petrovska, B. B. (2012) ‘Historical review of medicinal plants’ usage’,

Pharmacognosy Reviews, 6(11), pp. 1–5. doi: 10.4103/0973-7847.95849.

Piddock, L. J. V (2015) ‘Teixobactin, the first of a new class of antibiotics

discovered by ichip technology?’, Journal of Antimicrobial Chemotherapy,

70(10), pp. 2679–2680. doi: 10.1093/jac/dkv175.

Podolsky, S. H. (2018) ‘The evolving response to antibiotic resistance (1945–

2018)’, Palgrave Communications, 4(124), pp. 1–8. doi: 10.1057/s41599-018-

0181-x.

Poe, M. (1976) ‘Antibacterial synergism: a proposal for chemotherapeutic

potentiation between trimethoprim and sulfamethoxazole’, Science,

194(4264), pp. 533–535. doi: 10.1126/science.788154.

Poole, K. (2005) ‘Efflux-mediated antimicrobial resistance’, Journal of

Antimicrobial Chemotherapy, 56(1), pp. 20–51. doi: 10.1093/jac/dki171.

Pourabedin, M., Guan, L. and Zhao, X. (2015) ‘Xylo-oligosaccharides and

virginiamycin differentially modulate gut microbial composition in chickens’,

Microbiome, 3(1), pp. 1–12. doi: 10.1186/s40168-015-0079-4.

Pourabedin, M. and Zhao, X. (2015) ‘Prebiotics and gut microbiota in

chickens’, FEMS Microbiology Letters, (April), pp. 1–8. doi:

10.1093/femsle/fnv122.

Pra, M. A. D., Corrêa, É. K., Roll, V. F., Xavier, E. G., Lopes, D. C. N.,

References

236

Lourenço, F. F., Zanusso, J. T. and Roll, A. P. (2009) ‘Quicklime for controlling

Salmonella spp. and Clostridium spp in litter from floor pens of broilers’,

Ciência Rural, 39(4), pp. 1189–1194. doi: 10.1590/s0103-

84782009005000028.

Pratiwi, T. E., Dewi, W. and Prijono, E. (2015) ‘Antibacterial activity testing of

ethanolic extract of aloe vera leaf and gel against methicillin-resistant

Staphylococcus aureus’, Padjadjaran Journal of Dentistry, 27(1), pp. 1–5.

Principi, N. and Esposito, S. (2016) ‘Antibiotic administration and the

development of obesity in children’, International Journal of Antimicrobial

Agents, 47(3), pp. 171–177. doi: 10.1016/j.ijantimicag.2015.12.017.

Prinsloo, A., van Straten, A. M. S. and Weldhagen, G. F. (2008) ‘Antibiotic

synergy profiles of multidrug-resistant Pseudomonas aeruginosa in a

nosocomial environment’, Southern African Journal of Epidemiology and

Infection, 23(3), pp. 7–9. doi: 10.1080/10158782.2008.11441315.

Qin, R., Xiao, K., Li, B., Jiang, W., Peng, W. and Zheng, J. (2013) ‘The

combination of catechin and epicatechin gallate from Fructus crataegi

potentiates β -lactam antibiotics against methicillin-resistant Staphylococcus

aureus (MRSA) in vitro and in vivo’, pp. 1802–1821. doi:

10.3390/ijms14011802.

Radulovic, N. S., Blagojevic, P. D., Stojanovic-Radic, Z. Z. and Stojanovic, N.

M. (2013) ‘Antimicrobial plant metabolites: Structural diversity and mechanism

of action’, Current Medicinal Chemistry, 20(7), pp. 932–952. doi:

10.2174/0929867311320070008.

Rahman, A. and Kang, S. C. (2009) ‘In vitro control of food-borne and food

References

237

spoilage bacteria by essential oil and ethanol extracts of Lonicera japonica

Thunb’, Food Chemistry, 116(3), pp. 670–675. doi:

10.1016/j.foodchem.2009.03.014.

Rahman, M. A., Rahman, A. K. M. A., Islam, M. A. and Alam, M. M. (2018)

‘Detection of Multi–Drug Resistant Salmonella From Milk and Meat in

Bangladesh’, Bangladesh Journal of Veterinary Medicine, 16(1), pp. 115–120.

doi: 10.3329/bjvm.v16i1.37388.

Rahman, M. A., Sultana, P., Islam, M. S., Mahmud, M. T. and Rashid, M. M.

O. (2014) ‘Comparative antimicrobial activity of Areca catechu nut extracts

using different extracting solvents’, Bangladesh Journal of Microbiology, 31(1),

pp. 19–23.

Ramatla, T., Ngoma, L., Adetunji, M. and Mwanza, M. (2017) ‘Evaluation of

antibiotic residues in raw meat using different analytical methods’, Antibiotics,

6(4), pp. 1–17. doi: 10.3390/antibiotics6040034.

Ramlucken, U., Ramchuran, S. O., Moonsamy, G., Lalloo, R., Thantsha, M. S.

and Jansen van Rensburg, C. (2020) ‘A novel Bacillus based multi-strain

probiotic improves growth performance and intestinal properties of Clostridium

perfringens challenged broilers’, Poultry Science, 99(1), pp. 331–341. doi:

10.3382/ps/pez496.

Ravindran, V. (2013) ‘Feed enzymes: The science, practice, and metabolic

realities’, Journal of Applied Poultry Research, 22(3), pp. 628–636. doi:

10.3382/japr.2013-00739.

Rebello, S., Balakrishnan, D., Anoopkumar, A. N., Sindhu, R., Binod, P.,

Pandey, A. and Annesh, E. M. (2019) ‘Industrial enzymes as feed

References

238

supplements—Advantages to nutrition and global environment’, in Green Bio-

processes, Energy, Environment, and Sustainability. Springer Singapore, pp.

293–304. doi: 10.1007/978-981-13-3263-0.

Reygaert, W. C. (2018a) ‘An overview of the antimicrobial resistance

mechanisms of bacteria’, AIMS Microbiology, 4(3), pp. 482–501. doi:

10.3934/microbiol.2018.3.482.

Reygaert, W. C. (2018b) ‘Green tea catechins: Their use in treating and

preventing infectious diseases’, 2018.

Rice, L. B. (2012) ‘Mechanisms of resistance and clinical relevance of

resistance to β-lactams, glycopeptides, and fluoroquinolones’, Mayo Clinic

Proceedings, 87(2), pp. 198–208. doi: 10.1016/j.mayocp.2011.12.003.

Ricke, S. C. (2018) ‘Impact of prebiotics on poultry production and food safety’,

Yale Journal of Biology and Medicine, 91(2), pp. 151–159.

Ríos, J. L. and Recio, M. C. (2005) ‘Medicinal plants and antimicrobial activity’,

Journal of Ethnopharmacology, 100(1–2), pp. 80–84. doi:

10.1016/j.jep.2005.04.025.

Rishi, P., Preet, S., Bharrhan, S. and Verma, I. (2011) ‘In vitro and in vivo

synergistic effects of cryptdin 2 and ampicillin against Salmonella’,

Antimicrobial Agents and Chemotherapy, 55(9), pp. 4176–4182. doi:

10.1128/AAC.00273-11.

Rodríguez-Martínez, J. M., Cano, M. E., Velasco, C., Martínez-Martínez, L.

and Pascual, Á. (2011) ‘Plasmid-mediated quinolone resistance: an update’,

Journal of Infection and Chemotherapy, 17(2), pp. 149–182. doi:

References

239

10.1007/s10156-010-0120-2.

Roofchaee, A., Irani, M., Ebrahimzadeh, M. A. and Akbari, M. R. (2011) ‘Effect

of dietary oregano (Origanum vulgare L.) essential oil on growth performance,

cecal microflora and serum antioxidant activity of broiler chickens’, African

Journal of Biotechnology, 10(32), pp. 6177–6183. doi: 10.5897/AJB10.2596.

Rosen, G. D. (2007) ‘Holo-analysis of the efficacy of Bio-Mos(R) in broiler

nutrition’, British poultry science, 48(1), pp. 21–26. doi:

10.1080/00071660601050755.

Rothrock, M. J., Davis, M. L., Locatelli, A., Bodie, A., McIntosh, T. G.,

Donaldson, J. R. and Ricke, S. C. (2017) ‘Listeria occurrence in poultry flocks:

Detection and potential implications’, Frontiers in Veterinary Science, 4(AUG).

doi: 10.3389/fvets.2017.00125.

Le Roy, C. I., Woodward, M. J., Ellis, R. J., La Ragione, R. M. and Claus, S.

P. (2019) ‘Antibiotic treatment triggers gut dysbiosis and modulates

metabolism in a chicken model of gastro-intestinal infection’, BMC Veterinary

Research, 15(1), pp. 1–13. doi: 10.1186/s12917-018-1761-0.

RSPCA (2020) The life of a typical meat chicken.

Rubinelli, P. M., Kim, S. A., Park, S. H., Roto, S. M., Nealon, N. J., Ryan, E. P.

and Ricke, S. C. (2017) ‘Differential effects of rice bran cultivars to limit

Salmonella typhimurium in chicken cecal in vitro incubations and impact on the

cecal microbiome and metabolome’, PLoS ONE, 12(9), pp. 1–20. doi:

10.1371/journal.pone.0185002.

Rubio, L. A., Peinado, M. J., Ruiz, R., Suárez-Pereira, E., Ortiz Mellet, C. and

References

240

García Fernández, J. M. (2015) ‘Correlations between changes in intestinal

microbiota composition and performance parameters in broiler chickens’,

Journal of Animal Physiology and Animal Nutrition, 99(3), pp. 418–423. doi:

10.1111/jpn.12256.

S Prajwal, VN Vasudevan, T Sathu, A Irshad, S. N. and and Kuleswan Pame

(2017) ‘Antibiotic residues in food animals: Causes and health effects’, The

Pharma Innovation Journal, 6(12), pp. 01–04.

Sabour, S., Tabeidian, S. A. and Sadeghi, G. (2019) ‘Dietary organic acid and

fiber sources affect performance, intestinal morphology, immune responses

and gut microflora in broilers’, Animal Nutrition, 5(2), pp. 156–162. doi:

10.1016/j.aninu.2018.07.004.

Sachi, S., Ferdous, J., Sikder, M. H. and Azizul Karim Hussani, S. M. (2019)

‘Antibiotic residues in milk: Past, present, and future’, Journal of Advanced

Veterinary and Animal Research, 6(3), pp. 315–332. doi:

10.5455/javar.2019.f350.

Salim, H. M., Kang, H. K., Akter, N., Kim, D. W., Kim, J. H., Kim, M. J., Na, J.

C., Jong, H. B., Choi, H. C., Suh, O. S. and Kim, W. K. (2013) ‘Supplementation

of direct-fed microbials as an alternative to antibiotic on growth performance,

immune response, cecal microbial population, and ileal morphology of broiler

chickens’, Poultry Science, 92(8), pp. 2084–2090. doi: 10.3382/ps.2012-

02947.

Santiago-Rodriguez, T., Fornaciari, G., Luciani, S., Toranzos, G., Marota, I.,

Giuffra, V. and Cano, R. (2017) ‘Gut microbiome and putative resistome of

Inca and Italian nobility mummies’, Genes.

References

241

Sato, Y., Suzaki, S., Nishikawa, T. and Kihara, M. (2000) ‘Phytochemical

flavones isolated from Scutellaria barbata and antibacterial activity against

methicillin-resistant Staphylococcus aureus’, Journal of Ethnopharmcology,

72, pp. 483–488.

Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A.,

Roy, S. L., Jones, J. L. and Griffin, P. M. (2011) ‘Foodborne illness acquired in

the United States-Major pathogens’, Emerging Infectious Diseases, 17(1), pp.

7–15. doi: 10.3201/eid1701.P11101.

Schmidt, B., Ribnicky, D. M., Poulev, A., Logendra, S. and William, T. (2010)

‘A natural history of botanical therapeutics’, Metabolism, 57(1), pp. 1–12. doi:

10.1016/j.metabol.2008.03.001.A.

Scientific Committee on Animal Health and Animal Welfare (2000) The Welfare

of chickens kept for meat production (broilers), European Union Commission.

Seidel, V. (2012) ‘Initial and bulk extraction of natural products isolation’, in

Natural Products Isolation, pp. 27–41. doi: 10.1007/978-1-61779-624-1.

Sethiya, N. K. (2016) ‘Review on natural growth promoters available for

improving gut health of poultry: An alternative to antibiotic growth promoters’,

Asian Journal of Poultry Science, 10(1), pp. 1–29. doi:

10.3923/ajpsaj.2016.1.29.

Setya, D., Marsal, D., Novita, R., Harahap, S., Nirmala, A., Kusdianti, S. and

Kharina, H. (2020) ‘Phytochemical screening and antibacterial activity Coix

lacryma-jobi oil’, Journal of Plant Biotechnology, 47, pp. 100–106.

Shahrbaf, F. G. and Assadi, F. (2015) ‘Drug-induced renal disorders’, Journal

References

242

of Renal Injury Prevention, 4(3), pp. 57–60. doi: 10.12861/jrip.2015.12.

Shan, B., Cai, Y.-Z., Brooks, J. D. and Corke, H. (2007) ‘Antibacterial

properties and major bioactive components of cinnamon stick (Cinnamomum

burmannii): Activity against foodborne pathogenic bacteria’, Journal of

Agricultural and Food Chemistry, 55(14), pp. 5484–5490. doi:

10.1021/jf070424d.

Shan, B., Cai, Y., Brooks, J. D. and Corke, H. (2007) ‘The in vitro antibacterial

activity of dietary spice and medicinal herb extracts’, International Journal of

Food Microbiology, 117, pp. 112–119. doi: 10.1016/j.ijfoodmicro.2007.03.003.

Shan, B., Cai, Y., Brooks, J. D. and Corke, H. (2008) ‘Antibacterial properties

of Polygonum cuspidatum roots and their major bioactive constituents’, Food

Chemistry, 109, pp. 530–537. doi: 10.1016/j.foodchem.2007.12.064.

Shan, B., Cai, Y. Z., Brooks, J. D. and Corke, H. (2007) ‘Antibacterial

properties and major bioactive components of cinnamon stick (Cinnamomum

burmannii): Activity against foodborne pathogenic bacteria’, Journal of

Agricultural and Food Chemistry, 55(14), pp. 5484–5490. doi:

10.1021/jf070424d.

Shang, Y., Regassa, A., Kim, J. H. and Kim, W. K. (2015) ‘The effect of dietary

fructooligosaccharide supplementation on growth performance, intestinal

morphology, and immune responses in broiler chickens challenged with

Salmonella Enteritidis lipopolysaccharides’, Poultry Science, 94(12), pp.

2887–2897. doi: 10.3382/ps/pev275.

Shikov, A. N., Pozharitskaya, O. N. and Makarov, V. G. (2018) ‘Challenges in

the investigation of combinatory modes of action of nutrients and

References

243

pharmaceuticals’, Synergy, 7, pp. 36–38. doi: 10.1016/j.synres.2018.10.002.

Shimizu, M., Shiota, S., Mizushima, T., Ito, H., Hatano, T., Yoshida, T. and

Tsuchiya, T. (2001) ‘Marked potentiation of activity of beta-lactams against

methicillin-resistant Staphylococcus aureus by corilagin’, Antimicrobial Agents

and Chemotherapy, 45(11), pp. 3198–3201. doi: 10.1128/AAC.45.11.3198.

Shiota, S., Shimizu, M., Mizusima, T. and Ito, H. (2000) ‘Restoration of

effectiveness of beta-lactams on methicillin-resistant Staphylococcus aureus

by tellimagrandin I from rose red’, Microbiology Letters, 185, pp. 135–138.

Sillanpää, M. (2015) ‘General Introduction’, Natural Organic Matter in Water,

(May 1995), pp. 1–15. doi: 10.1016/B978-0-12-801503-2.00001-X.

Silver, L. L. and Bostian, K. A. (1993) ‘Discovery and development of new

antibiotics: The problem of antibiotic resistance’, Antimicrobial Agents and

Chemotherapy, 37(3), pp. 377–383.

Singer, R. S. (2003) ‘Antibiotic resistance—the interplay between antibiotic use

in animals and human beings’, Lancet, 3, pp. 47–51.

Singh, B. R., Singh, P., Agrawal, S., Teotia, U., Verma, A., Sharma, S.,

Chandra, M., Babu, N. and Agarwal, R. K. (2007) ‘Prevalence of multidrug

resistant Salmonella in coriander, mint, carrot, and radish in Bareilly and

Kanpur, northern India’, Foodborne Pathogens and Disease, 4(2), pp. 233–

240. doi: 10.1089/fpd.2006.0082.

Singh, P., Karimi, A., Devendra, K., Waldroup, P. W., Cho, K. K. and Kwon, Y.

M. (2013) ‘Influence of penicillin on microbial diversity of the cecal microbiota

in broiler chickens’, Poultry Science, 92(1), pp. 272–276. doi:

References

244

10.3382/ps.2012-02603.

Skrivanova, E., Van Immerseel, F., Hovorkova, P. and Kokoska, L. (2016) ‘In

vitro selective growth-inhibitory effect of 8-hydroxyquinoline on Clostridium

perfringens versus Bifidobacteria in a medium containing chicken ileal digesta’,

PLoS ONE, 11(12), pp. 1–11. doi: 10.1371/journal.pone.0167638.

Skroza, D., Šimat, V., Smole Možina, S., Katalinić, V., Boban, N. and Generalić

Mekinić, I. (2019) ‘Interactions of resveratrol with other phenolics and activity

against food-borne pathogens’, Food Science and Nutrition, 7(7), pp. 2312–

2318. doi: 10.1002/fsn3.1073.

Smirnov, A., Perez, R., Amit-Romach, E., Sklan, D. and Uni, Z. (2005) ‘Mucin

dynamics and microbial populations in chicken small intestine are changed by

dietary probiotic and antibiotic growth promoter supplementation’, Journal of

Nutrition, 135(2), pp. 187–192. doi: 10.1093/jn/135.2.187.

Smith, E. C. J., Williamson, E. M., Wareham, N., Kaatz, G. W. and Gibbons,

S. (2007) ‘Antibacterials and modulators of bacterial resistance from the

immature cones of Chamaecyparis lawsoniana’, Phytochemistry, 68(2), pp.

210–217. doi: 10.1016/j.phytochem.2006.10.001.

Smith, H. W. and Crabb, W. E. (1957) ‘The effect of continuous administration

of diets containing low levels of tetracyclines on the incidence of drug-resistant

Bacterium coli in the faeces of pigs and chickens: the sensitivity of the Bact.

coli to other chemotherapeutic agents.’, Veterinary Record, 69, pp. 24–30.

Sohail, M. U., Hume, M. E., Byrd, J. A., Nisbet, D. J., Shabbir, M. Z., Ijaz, A.

and Rehman, H. (2015) ‘Molecular analysis of the caecal and tracheal

microbiome of heat-stressed broilers supplemented with prebiotic and

References

245

probiotic’, Avian Pathology, 44(2), pp. 67–74. doi:

10.1080/03079457.2015.1004622.

Solis De Los Santos, F., Donoghue, A. M., Venkitanarayanan, K., Dirain, M.

L., Reyes-Herrera, I., Blore, P. J. and Donoghue, D. J. (2008) ‘Caprylic acid

supplemented in feed reduces enteric Campylobacter jejuni colonization in

ten-day-old broiler chickens’, Poultry Science, 87(4), pp. 800–804. doi:

10.3382/ps.2007-00280.

Solis de los santos, F., Donoghue, A. M., Venkitanarayanan, K., Metcalf, J. H.,

Reyes-Herrera, I., Dirain, M. L., Aguiar, V. F., Blore, P. J. and Donoghue, D.

J. (2009) ‘The natural feed additive caprylic acid decreases Campylobacter

jejuni colonization in market-aged broiler chickens’, Poultry Science, 88(1), pp.

61–64. doi: 10.3382/ps.2008-00228.

Son, J.-H. and Ravindran, V. (2012) ‘Feed enzyme technology: Present status

and future developments’, Recent Patents on Food, Nutrition & Agriculturee,

3(2), pp. 102–109. doi: 10.2174/2212798411103020102.

Sopirala, M. M., Mangino, J. E., Gebreyes, W. A., Biller, B., Bannerman, T.,

Balada-Llasat, J. M. and Pancholi, P. (2010) ‘Synergy testing by etest,

microdilution checkerboard, and time-kill methods for pan-drug-resistant

Acinetobacter baumannii’, Antimicrobial Agents and Chemotherapy, 54(11),

pp. 4678–4683. doi: 10.1128/AAC.00497-10.

Srivastava, A. K. (2019) Significance of medicinal plants in human life,

Synthesis of Medicinal Agents from Plants. Elsevier Ltd. doi: 10.1016/B978-0-

08-102071-5/00001-5.

Stefanović, O. D. (2018) ‘Synergistic activity of antibiotics and bioactive plant

References

246

extracts: A study against Gram-positive and Gram-negative bacteria’, in

Bacterial Pathogenesis and Antibacterial Control, pp. 23–48.

Subramani, R., Narayanasamy, M. and Feussner, K.-D. (2017) ‘Plant-derived

antimicrobials to fight against multi-drug-resistant human pathogens’, 3

Biotech, 7(172). doi: 10.1007/s13205-017-0848-9.

Subramanian, S., Kumar, S. D., Arulselva, P. and Senthilkumar, G. P. (2006)

‘In vitro antibacterial and antifungal activities of ethanolic extract of Aloe vera

leaf gel’, Journal of Plant Sciences, 1(4), pp. 348–355.

Sun, P., Liu, X., Pan, Q., Zhang, X. and He, S. (2020) ‘Comparison of Chinese

licorice (Glycyrrhiza uralensis) granules and water extracts and investigation

of their antibacterial activities for veterinary application’, European Journal of

Integrative Medicine, 36(December 2019), p. 101115. doi:

10.1016/j.eujim.2020.101115.

Suresh, G., Das, R. K., Kaur Brar, S., Rouissi, T., Avalos Ramirez, A., Chorfi,

Y. and Godbout, S. (2018) ‘Alternatives to antibiotics in poultry feed: molecular

perspectives’, Critical Reviews in Microbiology, 44(3), pp. 318–335. doi:

10.1080/1040841X.2017.1373062.

Swedish standards institute (2005) Animal feeding stuffs – Determination of

amino acids content (ISO 13903:2005 ).

Tamma, P. D. and Mathers, A. J. (2021) ‘JAC- Antimicrobial Resistance

Navigating treatment approaches for presumed ESBL-producing infections’,

JAC - Antimicrobial Resistance, pp. 1–2. doi: 10.1093/jacamr/dlaa111.

Taneja, N., Sethi, S., Tahlan, A. K. and Kumar, Y. (2019) ‘Stepping into the

References

247

post-antibiotic era— Challenges and solutions’, in Sethi, S. (ed.) Antimicrobial

Resistance a Global Threat. Rijeka: IntechOpen. doi:

10.5772/intechopen.84486.

Tarabees, R., Gafar, K. M., EL-Sayed, M. S., Shehata, A. A. and Ahmed, M.

(2019) ‘Effects of dietary supplementation of probiotic mix and prebiotic on

growth performance, cecal microbiota composition, and protection against

Escherichia coli O78 in broiler chickens’, Probiotics and Antimicrobial Proteins,

11(3), pp. 981–989. doi: 10.1007/s12602-018-9459-y.

Tellez, G., Higgins, S. E., Donoghue, A. M. and Hargis, B. M. (2006) ‘Digestive

physiology and the role of microorganisms’, Journal of Applied Poultry

Research, 15(1), pp. 136–144. doi: 10.1093/japr/15.1.136.

Teng, P. Y. and Kim, W. K. (2018) ‘Review: Roles of prebiotics in intestinal

ecosystem of broilers’, Frontiers in Veterinary Science, 5(OCT), pp. 1–18. doi:

10.3389/fvets.2018.00245.

The Centre for Disease Dynamics Economics and Policy, Shankar, P. R.,

Piryani, R. M. and Piryani, S. (2015) The state of the world’s antibiotics 2015,

The Centre for Disease Dynamics Economics and Policy. doi:

10.3126/jcmc.v6i4.16721.

The National Antimicrobial Resistance Monitoring System (2018) 2016-2017

NARMS Integrated Summary, U.S. Food and Drug Administration. Available

at: https://www.fda.gov/animal-veterinary/national-antimicrobial-resistance-

monitoring-system/2016-2017-narms-integrated-summary (Accessed: 31 July

2020).

The PEW chartable Trusts (2016) ‘A scientific roadmap for antibiotic

References

248

discovery’, Report, May(May), pp. 1–47. doi: 10.1128/.

The Plant List (2013) The Plant List. Available at: http://www.theplantlist.org/

(Accessed: 24 January 2020).

Tiihonen, K., Kettunen, H., Bento, M. H. L., Saarinen, M., Lahtinen, S.,

Ouwehand, A. C., Schulze, H. and Rautonen, N. (2010) ‘The effect of feeding

essential oils on broiler performance and gut microbiota’, British Poultry

Science, 51(3), pp. 381–392. doi: 10.1080/00071668.2010.496446.

Toghyani, M., Girish, C. K., Wu, S. B., Iji, P. A. and Swick, R. A. (2017) ‘Effect

of elevated dietary amino acid levels in high canola meal diets on productive

traits and cecal microbiota population of broiler chickens in a pair-feeding

study’, Poultry Science, 96(5), pp. 1268–1279. doi: 10.3382/ps/pew388.

de Toledo, T. dos S., Roll, A. A. P., Rutz, F., Dallmann, H. M., Prá, M. A. D.,

Leite, F. P. L. and Roll, V. F. B. (2020) ‘An assessment of the impacts of litter

treatments on the litter quality and broiler performance: A systematic review

and metaanalysis’, PLoS ONE, 15(5), pp. 1–26. doi:

10.1371/journal.pone.0232853.

Toma, M., Academy, R., Vinatoru, M. and Mason, T. J. (1999) ‘Ultrasonically

assisted extraction of bioactive principles from plants and their constituents’,

Sonochemistry, 5(1), pp. 209–247. doi: 10.1016/S1569-2868(99)80007-2.

Truong, D., Nguyen, D. H., Thuy, N., Ta, A., Bui, A. V. and Do, T. H. (2019)

‘Evaluation of the use of different solvents for phytochemical constituents,

antioxidants, and in vitro anti-inflammatory activities of Severinia buxifolia’,

Journal of Food Quality, 2019(1), pp. 1–9.

References

249

Vaezirad, M. M., Keestra-Gounder, A. M., de Zoete, M. R., Koene, M. G.,

Wagenaar, J. A. and van Putten, J. P. M. (2017) ‘Invasive behavior of

Campylobacter jejuni in immunosuppressed chicken’, Virulence, 8(3), pp. 248–

260. doi: 10.1080/21505594.2016.1221559.

Valenzuela-Grijalva, N. V., Pinelli-Saavedra, A., Muhlia-Almazan, A.,

Domínguez-Díaz, D. and González-Ríos, H. (2017) ‘Dietary inclusion effects

of phytochemicals as growth promoters in animal production’, Journal of

Animal Science and Technology, 59(1), pp. 1–17. doi: 10.1186/s40781-017-

0133-9.

Vasudevan, P., Marek, P., Nair, M. K. M., Annamalai, T., Darre, M., Khan, M.

and Venkitanarayanan, K. (2005) ‘In vitro inactivation of Salmonella enteritidis

in autoclaved chicken cecal contents by caprylic acid’, Journal of Applied

Poultry Research, 14(1), pp. 122–125. doi: 10.1093/japr/14.1.122.

Vázquez-laslop, N. and Mankin, A. S. (2019) ‘How macrolide antibiotics work’,

Trends in Biochemical Sciences, 43(9), pp. 668–684. doi:

10.1016/j.tibs.2018.06.011.How.

Ventola, C. L. (2015) ‘The antibiotic resistance crisis: part 1: causes and

threats.’, P & T : A peer-reviewed journal for formulary management (2015),

40(4), pp. 277–83. doi: Article.

Vernikos, G., Medini, D. and Pontarotti, P. (2014) ‘Horizontal gene transfer and

the role of restriction-modification systems in bacterial population dynamics’,

in Evolutionary Biology: Genome Evolution, Speciation, Coevolution and

Origin of Life. Athens, pp. 169–190. doi: 10.1007/978-3-319-07623-2.

Viveros, A., Chamorro, S., Pizarro, M., Arija, I., Centeno, C. and Brenes, A.

References

250

(2009) ‘Effects of dietary polyphenol-rich grape products on intestinal

microflora and gut morphology in broiler chicks’, Poultry Science, 90(3), pp.

566–578. doi: 10.3382/ps.2010-00889.

Van Vuuren, S. and Viljoen, A. (2012) ‘Plant-based antimicrobial studies

methods and approaches to study the interaction between natural products’,

Planta Medica, 78(3), p. 302. doi: 10.1055/s-0031-1298216.

Vuuren, S. Van, Viljoen, A., Africa, S. and Africa, S. (2011) ‘Methods and

approaches to study the interaction between natural products’, Planta Medica,

77, pp. 1168–1182.

Wagner, H. and Ulrich-Merzenich, G. (2009) ‘Synergy research: Approaching

a new generation of phytopharmaceuticals’, Phytomedicine, 16(2–3), pp. 97–

110. doi: 10.1016/j.phymed.2008.12.018.

Waititu, S. M., Yitbarek, A., Matini, E., Echeverry, H., Kiarie, E., Rodriguez-

Lecompte, J. C. and Nyachoti, C. M. (2014) ‘Effect of supplementing direct-fed

microbials on broiler performance, nutrient digestibilities, and immune

responses’, Poultry Science, 93(3), pp. 625–635. doi: 10.3382/ps.2013-03575.

Wang-Li, L., Xu, Y., Padavagod Shivkumar, A., Williams, M. and Brake, J.

(2020) ‘Effect of dietary coarse corn inclusion on broiler live performance, litter

characteristics, and ammonia emission’, Poultry Science, 99(2), pp. 869–878.

doi: 10.1016/j.psj.2019.10.010.

Wang, L. and Weller, C. L. (2006) ‘Recent advances in extraction of nutra-

ceuticals from plants’, Trends in Food Science and Technology, 17, pp. 300–

312. doi: 10.1016/j.tifs.2005.12.004.

References

251

Wang, S., Li, X., Wang, W., Zhang, H. and Xu, S. (2019) ‘Application of

transcriptome analysis : Oxidative stress , inflammation and microtubule

activity disorder caused by ammonia exposure may be the primary factors of

intestinal microvilli deficiency in chicken’, Science of the Total Environment,

696(1), p. 134035. doi: 10.1016/j.scitotenv.2019.134035.

Wang, W., Jia, H., Zhang, H., Wang, J., Lv, H., Wu, S. and Qi, G. (2019)

‘Supplemental plant extracts from Flos lonicerae in combination with Baikal

skullcap attenuate intestinal disruption and modulate gut microbiota in laying

hens challenged by Salmonella pullorum’, Frontiers in Microbiology, 10(July),

pp. 1–15. doi: 10.3389/fmicb.2019.01681.

Wang, W., Jiang, H., Wu, Z., Qian, J., Wang, X., Xu, J., Hao, F. and Lanying,

F. (2016) ‘Study on the bacteriostatic effects of 7 kinds of Chinese herbal

medicines such as Ophiopogon japonicus and Comb’, Agricultural Science

and Technology, 17(11), pp. 2560–2563.

Wasiński, B., Rózanska, H. and Osek, J. (2013) ‘Occurrence of extended

spectrum ß-Lactamaseand AmpC-Producing Escherichia coli in meat

samples’, Bulletin of the Veterinary Institute in Pulawy, 57(4), pp. 513–517. doi:

10.2478/bvip-2013-0089.

White, R. L., Burgess, D. S., Manduru, M. and Bosso, J. A. (1996) ‘Comparison

of three different in vitro methods of detecting synergy: Time-kill,

checkerboard, and E test’, Antimicrobial Agents and Chemotherapy, 40(8), pp.

1914–1918. doi: 10.1128/aac.40.8.1914.

Wieczorek, K., Wolkowicz, T. and Osek, J. (2018) ‘Antimicrobial resistance

and virulence-associated traits of Campylobacter jejuni isolated from poultry

References

252

food chain and humans with diarrhea’, Frontiers in Microbiology, 9(1508). doi:

10.3389/fmicb.2018.01508.

Wiermann, R. and Conn, E. E. (1981) ‘Secondary plant products and tissue

differentiation’, in The Biochemistry of Plants, pp. 85–116.

Willis, W. L., Isikhuemhen, O. S. and Ibrahim, S. A. (2007) ‘Performance

assessment of broiler chickens given mushroom extract alone or in

combination with probiotics’, Poultry Science, 86(9), pp. 1856–1860. doi:

10.1093/ps/86.9.1856.

Wink, M. (2015) ‘Modes of action of herbal medicines and plant secondary

metabolites’, Medicines, 2(3), pp. 251–286. doi: 10.3390/medicines2030251.

Woodford, N. and Ellington, M. J. (2007) ‘The emergence of antibiotic

resistance by mutation’, Clinical Microbiology and Infection, 13(1), pp. 5–18.

doi: 10.1111/j.1469-0691.2006.01492.x.

World Health Organisation (2014) Antimicrobial resistance global report on

surveillance.

World Health Organisation (2015) WHO Estimates of the Global Burden of

Foodborne Diseases.

World Health Organisation (2017) WHO guidelines on use of medically

important antimicrobials in food-producing animals, World Health

Organisation. doi: 10.1017/S0001972000064846.

World Health Organisation (2019) WHO list of critically important

antimicrobials for human medicine, World Health Organisation. Available at:

http://who.int/foodsafety/publications/antimicrobials-fifth/en/.

References

253

World Health Organisation and Food and Agriculture Organisation of the

United Nations (2018) Codex Alimentarius Commission: Procedural Manual,

Codex Alimentarus.

World Health Organisation, Food and Agriculture Organisation of the United

Nations and World Organisation for Animal Health (2018) Monitoring global

progress on addressing antimicrobial resistance: analysis report of the second

round of results of AMR country self-assessment survey.

Woyengo, T. A. and Nyachoti, C. M. (2011) ‘Review: Supplementation of

phytase and carbohydrases to diets for poultry’, Canadian Journal of Animal

Science, 91(2), pp. 177–192. doi: 10.4141/cjas10081.

Wu, J., Goodrich, K. M., Eifert, J. D., Jahncke, M. L., Keefe, S. F. O., Welbaum,

G. E. and Neilson, A. P. (2018) ‘Inhibiting foodborne pathogens Vibrio

parahaemolyticus and Listeria monocytogenes using extracts from traditional

medicine: Chinese gallnut, pomegranate peel, baikal skullcap root and

forsythia fruit’, Open Agriculture, 3, pp. 163–170.

Xiang, H., RuiCheng, W., Tian, W. and Ran, W. (2009) ‘Effects of Pulsatilla

chinensis extract on production performance, blood parameters and immunity

index of broilers.’, Jiangsu Journal of Agricultural Sciences, 25(1), pp. 136–

141.

Xiong, J., Li, S., Wang, W., Hong, Y., Tang, K. and Luo, Q. (2013) ‘Screening

and identification of the antibacterial bioactive compounds from Lonicera

japonica Thunb. leaves’, Food Chemistry, 138(1), pp. 327–333. doi:

10.1016/j.foodchem.2012.10.127.

Xu, S., Shang, M., Liu, G., Xu, F., Wang, X., Shou, C. and Cai, S. (2013)

References

254

‘Chemical constituents from the rhizomes of Smilax glabra and their

antimicrobial activity’, Molecules, 18, pp. 5265–5287. doi:

10.3390/molecules18055265.

Xu, Y., Lin, Y. M., Stark, C. R., Ferket, P. R., Williams, C. M. and Brake, J.

(2017) ‘Effects of dietary coarsely ground corn and 3 bedding floor types on

broiler live performance, litter characteristics, gizzard and proventriculus

weight, and nutrient digestibility’, Poultry science, 96(7), pp. 2110–2119. doi:

10.3382/ps/pew485.

Yadav, S. and Jha, R. (2019) ‘Strategies to modulate the intestinal microbiota

and their effects on nutrient utilization, performance, and health of poultry’,

Journal of Animal Science and Biotechnology, 10(1), pp. 1–11. doi:

10.1186/s40104-018-0310-9.

Yamaki, M., Kashihara, M., Ishiguro, K. and Takagi, S. (1989) ‘Antimicrobial

principles of Xian He Cao (Agrimonia pilosa)’, Planta Medica, 55(2), pp. 169–

170. doi: 10.1055/s-2006-961915.

Yang, C.-H., Chang, H.-W., Lin, H.-Y. and Chuang, L.-Y. (2013) ‘Evaluation of

antioxidant and antimicrobial activities from 28 Chinese herbal medicines’,

Journal Journal of Pharmacognosy and Phytochemistry, 2(1). doi:

10.1016/254.

Yang, C. M., Cao, G. T., Ferket, P. R., Liu, T. T., Zhou, L., Zhang, L., Xiao, Y.

P. and Chen, A. G. (2012) ‘Effects of probiotic, Clostridium butyricum, on

growth performance, immune function, and cecal microflora in broiler

chickens’, Poultry Science, 91(9), pp. 2121–2129. doi: 10.3382/ps.2011-

02131.

References

255

Yang, Yichao, Feye, K. M., Shi, Z., Pavlidis, H. O., Kogut, M., J. Ashworth, A.

and Ricke, S. C. (2019) ‘A historical review on antibiotic resistance of

foodborne Campylobacter’, Frontiers in Microbiology, 10(1509), pp. 1–8. doi:

10.3389/fmicb.2019.01509.

Yang, Y., Iji, P. A. and Choct, M. (2007) ‘Effects of different dietary levels of

Mannanoligosaccharide on growth performance and gut development of

broiler chickens’, Asian-Australasian Journal of Animal Sciences, 20(7), pp.

1084–1091. doi: 10.5713/ajas.2007.1084.

Yang, Yun-feng, Zhao, L., Shao, Y., Liao, X., Zhang, L., Lu, L. and Luo, X.

(2019) ‘Effects of dietary graded levels of cinnamon essential oil and its

combination with bamboo leaf flavonoid on immune function, antioxidative

ability and intestinal microbiota of broilers’, Journal of Integrative Agriculture,

18(9), pp. 2123–2132. doi: 10.1016/S2095-3119(19)62566-9.

Yasir, M., Farman, M., Shah, M. W., Jiman-fatani, A. A., Othman, N. A.,

Almasaudi, S. B., Alawi, M., Shakil, S., Al-abdullah, N., Ismaeel, N. A. and

Azhar, E. I. (2020) ‘Journal of infection and public health genomic and

antimicrobial resistance genes diversity in multidrug-resistant CTX-M-positive

isolates of Escherichia coli at a health care facility in Jeddah’, Journal

of Infection and Public Health, 13(1), pp. 94–100. doi:

10.1016/j.jiph.2019.06.011.

Ye, Q., Wu, Q., Zhang, S., Zhang, J., Yang, G., Wang, J., Xue, L. and Chen,

M. (2018) ‘Characterization of extended-spectrum β-lactamase-producing

Enterobacteriaceae from retail food in China’, Frontiers in Microbiology,

9(AUG), pp. 1–12. doi: 10.3389/fmicb.2018.01709.

References

256

Yoon, Y. and Choi, K. H. (2012) ‘Antimicrobial activities of therapeutic herbal

plants against Listeria monocytogenes and the herbal plant cytotoxicity on

Caco-2 cell’, Letters in Applied Microbiology, 55(1), pp. 47–55. doi:

10.1111/j.1472-765X.2012.03262.x.

Yousaf, M. S., Ahmad, I., Ashraf, K., Rashid, M. A., Hafeez, A., Ahmad, A.,

Zaneb, H., Nasir, R., Numan, M., Zentek, J. and Rehman, H. (2017)

‘Comparative effects of different dietary concentrations of β-galacto-

oligosaccharides on serum biochemical metabolites, selected caecel

microbiota and immune response in broilers’, Journal of Animal and Plant

Sciences, 27(1), pp. 98–105.

Yousaf, M. S., Goodarzi Boroojeni, F., Vahjen, W., M??nner, K., Hafeez, A.,

Ur-Rehman, H., Keller, S., Peris, S. and Zentek, J. (2017) ‘Encapsulated

benzoic acid supplementation in broiler diets influences gut bacterial

composition and activity’, British Poultry Science, 58(2), pp. 122–131. doi:

10.1080/00071668.2016.1262000.

Yu, H., Wang, Y., Wang, X. and Sun, S. (2014) ‘Antibacterial activity of

aqueous and ethanolic extracts of Portulaca oleracea L and Taraxacum

mongolicum against pathogenic bacteria of cow mastitis’, International Journal

of Applied Research in Veterinary Medicine, 12(3), pp. 210–213.

Yu, J., Lei, J., Yu, H., Cai, X. and Zou, G. (2004) ‘Chemical composition and

antimicrobial activity of the essential oil of Scutellaria barbata’, Phytochemistry,

65, pp. 881–884. doi: 10.1016/j.phytochem.2004.02.005.

Yu, M., Li, Z., Chen, W., Wang, G., Cui, Y. and Ma, X. (2019) ‘Dietary

supplementation with citrus extract altered the intestinal microbiota and

References

257

microbial metabolite profiles and enhanced the mucosal immune homeostasis

in yellow-feathered broilers’, Frontiers in Microbiology, 10(2662), pp. 1–14. doi:

10.3389/fmicb.2019.02662.

Yuan, W., Lee, H. W. and Yuk, H. G. (2017) ‘Antimicrobial efficacy of

Cinnamomum javanicum plant extract against Listeria monocytogenes and its

application potential with smoked salmon’, International Journal of Food

Microbiology, 260, pp. 42–50. doi: 10.1016/j.ijfoodmicro.2017.08.015.

Zazharskyi, V. V., Davydenko, P. O., Kulishenko, O. M., Borovik, I. V. and

Brygadyrenko, V. V. (2019) ‘Antimicrobial activity of 50 plant extracts’,

Biosystems Diversity, 27(2010), pp. 163–169. doi: 10.15421/011922.

Zhai, H., Liu, H., Wang, S., Wu, J. and Kluenter, A. M. (2018) ‘Potential of

essential oils for poultry and pigs’, Animal Nutrition, 4(2), pp. 179–186. doi:

10.1016/j.aninu.2018.01.005.

Zhang, L., Ravipati, A. S., Koyyalamudi, S. R., Jeong, S. C., Bartlett, J., Smith,

P. T., Cruz, M. De, Maria, C., Jim, E. and Vicente, F. (2013) ‘Anti-fungal and

anti-bacterial activities of ethanol extracts of selected traditional Chinese

medicinal herbs’, Asian Pacific Journal of Tropical Medicine, 6(9), pp. 673–

681. doi: 10.1016/S1995-7645(13)60117-0.

Zhang, L., Wei, K., Xu, J., Yang, D., Zhang, C., Wang, Z. and Li, M. (2016)

‘Belamcanda chinensis (L.) DC-An ethnopharmacological, phytochemical and

pharmacological review’, Journal of Ethnopharmacology, 186, pp. 1–13. doi:

10.1016/j.jep.2016.03.046.

Zhao, W., Hu, Z., Okubo, S. and Hara, Y. (2001) ‘Mechanism of synergy

between epigallocatechin gallate and b-lactams against methicillin-resistant

References

258

Staphylococcus aureus’, Antimicrobial Agents and Chemotherapy, 45(6), pp.

1737–1742. doi: 10.1128/AAC.45.6.1737.

doctor of philosophy effects of phytogenics on intestinal

Documents