afab volume 3 issue 4

Volume 3, Issue 42013

ISSN: 2159-8967www.AFABjournal.com

262 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 4 - 2013

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 4 - 2013 263

Sooyoun Ahn University of Florida, USA

Walid Q. AlaliUniversity of Georgia, USA

Kenneth M. Bischoff NCAUR, USDA-ARS, USA

Debabrata BiswasUniversity of Maryland, USA

Claudia S. Dunkley University of Georgia, USA

Lawrence GoodridgeColorado State University, USA

Leluo GuanUniversity of Alberta, Canada

Joshua GurtlerERRC, USDA-ARS, USA

Yong D. HangCornell University, USA

Armitra Jackson-DavisAlabama A&M University, USA

Divya JaroniOklahoma State University, USA

Weihong Jiang Shanghai Institute for Biol. Sciences, P.R. China

Michael JohnsonUniversity of Arkansas, USA

Timothy KellyEast Carolina University, USA

William R. KenealyMascoma Corporation, USA

Hae-Yeong Kim Kyung Hee University, South Korea

Woo-Kyun KimUniversity of Georgia, USA

M.B. KirkhamKansas State University, USA

Todd KostmanUniversity of Wisconsin, Oshkosh, USA

Y. M. Kwon University of Arkansas, USA

Maria Luz Sanz MuriasInstituto de Quimica Organic General, Spain

Melanie R. MormileMissouri University of Science and Tech., USA

Rama NannapaneniMississippi State University, USA

Jack A. Neal, Jr.University of Houston, USA

Benedict OkekeAuburn University at Montgomery, USA

John PattersonPurdue University, USA

Toni Poole FFSRU, USDA-ARS, USA

Marcos RostagnoLBRU, USDA-ARS, USA

Roni ShapiraHebrew University of Jerusalem, Israel

Kalidas ShettyNorth Dakota State University, USA

EDITORIAL BOARD


EDITOR-IN-CHIEFSteven C. RickeUniversity of Arkansas, USA

EDITORSTodd R. CallawayFFSRU, USADA-ARS, USA

Philip G. CrandallUniversity of Arkansas, USA

Janet Donaldson Mississippi State University, USA

Ok-Kyung KooKorea Food Research Institute, South Korea

MANAGING and LAYOUT EDITOREllen J. Van LooGhent, Belgium

TECHNICAL EDITORJessica C. ShabaturaFayetteville, USA

ONLINE EDITION EDITORC.S. ShabaturaFayetteville, USA

ABOUT THIS PUBLICATION

Agriculture, Food & Analytical Bacteriology (ISSN

2159-8967) is published quarterly, beginning with

this inaugural issue.

Instructions for Authors may be obtained at the

back of this issue, or online via our website at

www.afabjournal.com

Manuscripts: All correspondence regarding pend-

ing manuscripts should be addressed Ellen Van Loo,

Managing Editor, Agriculture, Food & Analytical

Bacteriology: [email protected]

Information for Potential Editors: If you are interested

in becoming a part of our editorial board, please con-

tact Editor-in-Chief, Steven Ricke, Agriculture, Food &

Analytical Bacteriology: [email protected]

Advertising: If you are interested in advertising with

our journal, please contact us at advertising@afab-

journal.com for a media kit and current rates.

Reprint Permission: Correspondence regarding re-

prints should be addressed Ellen Van Loo, Managing

Editor, Agriculture, Food & Analytical Bacteriology

[email protected]

Ordering Print Copies: print editions of this journal

may be purchased and shipped internationally from

our website order form at www.afabjournal.com

Subscription Rates: Subscriptions are not available

at this time. To be advised when subscriptions plans

are made available, please join our newsletter at

www.afabjournal.com

Mailing Address: 2138 Revere Place . Fayetteville, AR . 72701 Website: www.AFABjournal.com

EDITORIAL STAFF


Development of Non-Forage Based Incubation System For Culturing Ruminal Lipase-Pro-ducing Bacteria In VitroH. D. Edwards, R. C. Anderson, T. M. Taylor, R. K. Miller, M. D. Hardin, N. A. Krueger, D. J. Nisbet

293

Prevalence of Foodborne Pathogens and Spoilage Microorganisms and their Drug Resis-tant Status in Different Street Foods of DhakaZ. Tabashsum, I. Khalil, Md. N. Uddin, A.K.M. M. Mollah, Y. Inatsu and Md. L. Bari

281

Effect of Citrus Pulp on the Viability of Saccharomyces boulardii in the Presence of Enteric Pathogens

J. G. Wilson, T. C. McLaurin, J. A. Carroll, S. Shields-Menard, T. B. Schmidt, T. R. Callaway, and J. R. Donaldson

303

Persistence of erythromycin resistance gene erm(B) in cattle feedlot pens over timeA. R. Mantz, D. N. Miller, M. J. Spiehs, B. L. Woodbury, and L. M. Durso

312

ARTICLES

The Role of Cellular Prion Proteins (PrPC) on Neuronal Brucella InfectionsM. Aydin, D. F. Gilmore, S. Erdogan, V. Duzguner, and S. Ahn

268

Instructions for Authors327

Introduction to Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

TABLE OF CONTENTS


Biography of new AFAB Editor: Dr. Janet Donaldson

It is the pleasure of the AFAB editorial staff to welcome Dr. Janet Donaldson as a newly appointed editor beginning in January, 2014. Dr. Janet Donaldson is currently an Associ-ate Professor in the Department of Biological Sciences at Mississippi State University. She is a microbiologist with special interests in determining mechanisms by which bacteria are able to grow and adapt to conditions within the gastrointestinal tract. Her research is primarily focused upon identifying these mechanisms in Listeria monocytogenes and Escherichia coli O157:H7. Her work has identified variations in survival as related to strain diversity, which sheds light on the mechanisms by which these dangerous pathogens sur-vive and cause disease. She has also identified a novel probiotic that provides additional energy to the host. Other research interests include probiotics mechanisms of actions and applicability. She also has research interests related to improving bioenergy sources through microbial community manipulations. She has been the recipient of the National Pork Board Innovations in Research Award in 2013 and also the Randall Lectureship award for her research. Dr. Donaldson has published 21 peer-reviewed journal articles. She has been a PI or Co-PI on several grants, with funded research totaling over $12.5 million. She and her students have given over 50 presentations since 2008 at both national and inter-national conferences and venues. She is an associate editor for five journals in her field, has been an invited reviewer for 23 journals, and is currently the president elect of the South Central Branch of the American Society for Microbiology.

Biography of new AFAB Editor: Ok-Kyung Koo

Ok-Kyung Koo, PhD, is a senior scientist in Food Safety Research Group at Korea Food Research Institute since 2012. She completed both bachelor’s and master’s degrees in the Department of Food and Animal Biotechnology from Seoul National University in South Korea. Then Koo joined PhD program in Dr. Arun K. Bhunia’s Molecular Food Microbiol-ogy lab at Purdue University in 2006. Her doctoral dissertation was on “Listeria adhesion protein and heat shock protein 60: Application in pathogenic Listeria detection and im-plication in listeriosis prevention”. After the degree, she moved to Fayetteville, Arkansas to work as a postdoctoral research associate in Center for Food Safety at the University of Arkansas in 2010. With Dr. Steven Ricke and Dr. Philip Crandall in UA, she conducted research on understanding the microbial ecology of food processing environment and application of probiotics as well as different chemical and physical methods to control the contamination of L. monocytogenes and Salmonella spp.. Her current research focuses on food safety epidemiology, pathogenesis of foodborne pathogens and their interaction with background bacteria in the food system, and natural antimicoribal agents including probiotics. She is also an assistant professor at the University of Science and Technology in South Korea.

NEW EDITORIAL STAFF


Dr. Armitra Jackson-Davis appointed to AFAB editorial Board

Dr. Armitra Jackson-Davis is an Assistant Professor of Food Microbiology at Alabama A&M University. At Alabama A&M University, she has teaching and research responsibili-ties in addition to the advisement of undergraduate and graduate students. She earned her Bachelor of Science degree in Animal Science from the University of Arkansas-Pine Bluff and her Master of Science and Doctor of Philosophy degrees from Iowa State Uni-versity in the area of Meat Science with a food microbiology emphasis. While at Iowa State University, she received the Iowa State University Teaching Excellence Award. Dr. Jackson-Davis believes that children should be educated at an early age when it comes to safe food handling practices. As a result, she authored “The Birthday to Remember Forever”, which is the first in the series “Eating safe with Ace and Mace”. The series is de-signed to teach safe food-handling practices to children in a storytelling manner. Her re-search interests include investigating the microbiological safety of food products labeled as “natural” and organic. Her work related to this research area has been published in the Journal of Food Protection and Meat Science. She was recently awarded the 2013-2014 Grant for Minority Serving Institutions to conduct research that evaluates multiple-hurdle antimicrobial technologies on the inactivation of Escherichia coli O157:H7 in beef trim. She has travelled internationally to learn more about different food systems around the world.


www.afabjournal.comCopyright © 2013

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Brucella species can settle and proliferate in microglial cells of host animals. The primary focus of this

mini-review is to discuss the biochemical and pathogenic processes that could potentially develop be-

tween the host and the agent. Brucella’s antioxidant responses to host’s oxidative reactions, which are one

of the defense systems in neuronal cells against the Brucella infection, are believed to be an important

element in its pathogenicity; however their exact mechanisms to exert pathogenicity are not fully under-

stood. In this review, the effects of cellular prion proteins (PrPC) on entrance of Brucella into host cells and

on development of oxidative defenses in the host cells will be discussed. Additionally, we will discuss the

potential of utilizing small interference RNA or short interference RNA to suppress the expression of PrPC

and determine the subsequent effect on Brucella infection on microglial cells. Finally the effects of PrPC on

oxidative events, and roles of the Brucella virulence factors during the entrance into the host cells will also

be discussed.

Keywords: Brucella, prions, microglial, infections

INTRODUCTION

Although brucellosis has been eradicated in most

developed countries, it is still an endemic disease in

many regions in the Middle East and Mediterranean

countries (Al-Sekait, 2000; Boschiroli et al., 2001; Pap-

Correspondence: Soohyoun Ahn, [email protected]: +1 352-392-1991 Ext. 310 Fax: +1 352-392-9467

pas et al., 2006; Mantur et al., 2007). Consequently

brucellosis remains an important public and animal

health problem in many countries in these regions.

This results in tremendous economic losses in these

respective geographical regions. Brucellosis is a

zoonotic disease that can easily be transmitted to

humans from raw or inadequately heated milk and

products derived from raw milk such as cream, butter,

and cheese. Brucellosis can be considered an occu-

The Role of Cellular Prion Proteins (PrPC) on Microglial Brucella Infections

M. Aydin1, D. F. Gilmore2, S. Erdogan3, V. Duzguner4, and S. Ahn5*

1Molecular Biosciences Program, Arkansas State University, Jonesboro, AR 724012Department of Biological Sciences, Arkansas State University, Jonesboro, AR 724013Department of Medical Biochemistry, Emine-Bahaeddin Nakiboglu Medical School,

Zirve University, 27260 Gaziantep, Turkey4Ardahan University, Health Services Vocational School, Ardahan, Turkey

5Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611

Agric. Food Anal. Bacteriol. 3: 268-280, 2013


pational disease as it is mostly observed in farmers,

livestock keepers, veterinarians, butchers, or employ-

ees of meat and dairy production facilities (Corbel,

1997). Direct contact with sick animals and their urine,

blood, etc. could transmit Brucella from sick animals

to humans (Megid et al., 2010). Additionally, oral

route, respiratory tract, eyes, and open wounds are

other important ways for Brucella to enter the body.

Brucellosis causes abortions or stillbirths in female

ruminants and orchitis in male ruminants as well as

cerebral peroxidation which can lead to tremendous

economic losses (Corbel, 1990; Orozco et al., 2003;

Melek et al., 2006; Kataria et al., 2010). Humans who

have been infected by Brucella can develop chronic

symptoms such as undulant fever, loss of appetite,

extreme sweating, and arthritis (Franco et al., 2007;

Christopher et al., 2010). Due to its resistance to sim-

ple treatments and its potential as a biological weap-

on, Brucella is considered one of the most important

pathogens (Leitenberg, 2001).

Cellular events underlying the development of

brucellosis have not yet been fully revealed. There-

fore the focus of this mini-review is to discuss the

potential infection mechanism of Brucella and vari-

ous experimental approaches to gain a better under-

standing of the infection mechanism. In particular,

this review will discuss host oxidative processes that

might serve as defense mechanisms and the poten-

tial roles for cellular prion proteins (PrPC) in Brucella

infection. To the best of our knowledge there are

currently few published studies in the literature that

have examined oxidative processes as a host defen-

sive system and the roles of PrPC on the development

of the disease. In addition, the potential for a small

interference RNA-based transfection approach for

determining the role of PrPC in neurobrucellosis will

be discussed.

BRUCELLA – GENERAL CHARACTERISTICS

Brucella spp. are Gram-negative, facultative, non-

motile, and non-spore forming bacteria that can be

either intracellular or extracellular pathogens (Man-

tur and Amarnath, 2008; Christopher et al., 2010).

It has been thought that the virulence factors of

the bacteria consist of outer membrane lipopoly-

saccharides (LPS) and it has been speculated that

proteins involved in signalling, gene regulation,

and transmembrane transportation may also be

involved(Hong et al., 2000; Jimenez de Bagues et

al., 2005; Franco et al., 2007). It has also been estab-

lished that the LPS-O side chain in particular may be

an important feature of the bacteria that allows them

to protect themselves from the host defense systems

that they may encounter (Freer et al., 1996; Giam-

bartolomei et al., 2004). Brucella species, however,

contrary to what is considered typical for most other

Gram-negative bacterial pathogens, do not possess

exotoxins, invasive proteases, capsules, or virulence

plasmids. Therefore, they can easily enter the host’s

reticuloendothelial system (RES) and eventually

mononuclear phagocytes, and remain viable while

replicating themselves in the intracellular environ-

ment of the host cells (Ficht, 2003; Gross et al., 2003).

Brucella ensure their intracellular survival by

avoiding fusion of the phagosome in which they are

contained with lysosomes in macrophages (Celli and

Gorvel, 2004). Thus, these bacteria can gain entry

into the intracellular environment of the host via the

host’s phagocytic cells, and after successful entry,

may be carried to and remain latent in a variety of

organ tissues such as the spleen, brain, heart, and

bone marrow for years after the initial exposure (Leit-

enberg, 2001; Gross et al., 1998, 2003). This obvious-

ly makes them problematic over an extended period

of time and creates an ongoing risk for the host and

susceptible individuals exposed to the organism.

The next section addresses the virulence properties

of Brucella and the standard assays that can be used

to assess their virulence.

BRUCELLA – GENERAL PATHOGENESIS AND STANDARD ASSAYS

In most Brucella infection studies, Brucella meli-

tensis M16 strain has been used to infect tissue cul-

ture cells because this strain is known for its virulence

in humans. The main differences between Brucella


and other pathogens such as Yersinia, Salmonella,

and Listeria are its survival ability and reproducibility

within the immune defense system of the host cells

(i.e. microglia). These abilities are due to a variety of

virulence factors that have been identified over the

years. For example, Brucella species can activate the

virB operon and in turn initiate the synthesis of Type

IV secretion systems (T4SS; O’Callaghan et al., 1999).

By utilizing this virulence factor, Brucella species can

invade the host cell and live in it without causing any

reaction within the endocytic vacuole (phagosome)

(O’Callaghan et al., 1999). It has also been reported

that Brucella infection prevents the synthesis of in-

flammatory cytokines tumor necrosis factor alpha

(TNF-α), interferon-gamma (IFN-γ), and interleukin-1

(IL-1β) in humans and domestic animals (Gross et al.,

2003; Erdogan et al., 2007, 2008). However, some

recent studies reported that Brucella species could

cause secretion of IL-1β and TNF-α in mouse macro-

phages and microglia cells (Covert et al., 2009; Sa-

martino et al., 2010).

The classical way to assess and quantitate patho-

genesis for any microorganism is invasion assay, also

known as the gentamicin protection assay, in which

the organism of interest is incubated with the corre-

sponding type of tissue cell culture to determine the

ability of pathogenic bacteria to invade the target

eukaryotic cells. In an invasion assay with B. meliten-

sis, the number of the bacterial cells can simply be

determined after serial dilution by colony counting

or by a spectrophotometer to arrive at a specified

inoculation level. Subsequently the resulting bacte-

rial suspension is typically washed several times with

a phosphate buffered saline (PBS) solution, and an

aliquot of this suspension is introduced to the tissue

cell culture. In most studies the preferred bacteria/

tissue culture cell ratio is set at 20:1. The flasks con-

taining these bacterial/tissue culture cells are incu-

bated at 37°C, with a 5% CO2 atmosphere in a sterile

incubator for 30 minutes to allow for phagocytosis

of the bacteria. The bacteria that are not phagocy-

tized by tissue culture cells are initially washed away

with PBS containing 30 µg/mL gentamicin to ensure

elimination of all remaining bacteria on the cell sur-

faces. At this concentration that is routinely used in

any invasion assay, gentamicin has been shown not

to affect internalized bacteria (Durant et al.,1999,

2000a,b; Ficht, 2003; Howard et al., 2005).

To assess internalized cells as well as intracellular

metabolites requires preparation of cell homog-

enates. After incubation of host cells with Brucella,

trypsin is typically added to remove the tissue culture

cells from the bottom of the flasks. After incubation

with trypsin for 1 to 2 minutes with gentle shaking,

suspended cells are collected by pipette and centri-

fuged to remove them from the culture medium. At

this point, a tissue culture cell lysis buffer is added

and cytoplasmic contents are collected by centrifu-

gation. The generated cellular homogenates then

can be subjected to either direct culture to quantify

the internalized bacterial cells or biochemical analy-

ses to measure the effect of Brucella invasion on the

production of specific metabolite effects by tissue

culture cells.

Production of reactive oxygen species (ROS) such

as superoxide, nitric oxide, and hydroxyl ions are

among the most important defense mechanisms

that are developed by infected host cells (Fang,

1997; Kaymak et al., 2011). Bactericidal and apop-

tosis-stimulating properties of these molecules can

help eliminate pathogens. Nitric oxide is an antimi-

crobial molecule produced in the macrophages from

L-arginine by inducible nitric oxide synthase (iNOS)

that serves as an immune defense mechanism dur-

ing inflammations and infections (Fang, 1997). To

determine the level of nitric oxide, nitrite and nitrate

content can be measured by the Griess reaction af-

ter deproteinization (Green et al., 1982; Sun et al.,

2003). Typically when conducting the assay, copper-

coated cadmium granules and glycine buffer (pH 9.7)

are incubated for 90 minutes with test samples and

the reduction of nitrate to nitrite are measured by

spectrophotometry at 545 nm. This reduction results

in the generation of a pink color, which is formed by

diazotization of sulfanilamide and related N-naph-

thylethylene diamine (NNDA).

To counter host defense mechanisms, Brucella

can produce antioxidant enzymes such as superox-

ide dismutase and catalase. Therefore, assessing

the levels of these enzymes in infected cells can be


a critical component of evaluating Brucella patho-

genicity. Superoxide dismutase is an antioxidant

enzyme, which strongly inhibits phagocytosis-asso-

ciated nitroblue tetrazolium (NBT) reduction by re-

moving superoxide anion (O2-) radical produced by

xanthine/xanthine oxidase reaction (Johnston et al.,

1975; Choi et al., 2006). When estimating the activity

of this enzyme in homogenate samples, a mixture of

chloroform/ethanol is added to the sample, mixed by

vortexing and subsequently centrifuged. After cen-

trifugation, the upper aqueous phase can be used

for the test by adding this collected supernatant to

a mixture that contains xanthine, NBT, bovine serum

albumin and xanthine oxidase enzymes (Sun et al.,

1988). Following incubation at 25°C, CuCl2 is added

to stop the reaction and each sample can be read

on a spectrophotometer at 560 nm. Catalase activity

can be measured directly from homogenates using a

spectrophotometric assay based on the absorbance

of hydrogen peroxide at a wavelength of 240 nm. A

decrease in absorbance can be used as an indicator

of catalase production. With the production of these

enzymes, Brucella can eliminate the free radicals that

may be produced by the host cell (Kim et al., 2000).

For example, it has been suggested that B. abortus

after early infection may survive by either expressing

genes to counteract the impact of a high nitric oxide

environment or activate genes that allow it to use

nitric oxide as a potential nitrogen source (Wang et

al., 2001). Consequently, Brucella spp. may be able

to resist the host oxidative defense system and pro-

tect themselves from host’s bactericidal defensive

attacks (Orozco et al. 2003; Gross et al. 2004). The

following sections describe the interaction between

the central nervous system and Brucella.

BRUCELLA INTERACTION WITH THE CENTRAL NERVOUS SYSTEM

It has been reported that in 1.3 to 11% of brucel-

losis cases, Brucella settle and cause disease in the

central nervous system (Mousa et al., 1986; McLean

et al., 1992; Gul et al., 2008, 2009; Buzgan et al., 2010;

Erdem et al., 2012). Neurological complications are

infrequent, but are clinically important for their se-

verity and high morbidity (Ceran et al., 2011). The

most common symptom of neurobrucellosis is head-

ache with meningeal irritation (Mousa et al., 1986;

Shakir et al., 1987). It is still not clear how Brucella

gain entry into the central nervous system. Two pro-

teins that have been shown to have a role in intracel-

lular pathogenicity and invasion are BvrR and BvrS

((Brucella virulence, Sola-Landa et al., 1998; Gándara

et al., 2001). These proteins regulate production of

the Type IV secretion system in B. abortus and may

be involved in allowing Brucella spp. to enter central

nervous system cells (Nunez-Martinez et al., 2010). It

has been previously shown that the entrance of Bru-

cella to macrophages and intracellular replication is

reduced by inactivation of the BvrR-BvrS system in

Brucella mutants (Sola-Landa et al., 1998; Gándara et

al., 2001; Guzmán-Verri et al., 2002). In a later study,

Guzmán-Verri et al. (2002) determined that the BvrR-

BvrS system regulates the expression of outer mem-

brane proteins (Omp), especially Omp3a (Omp25)

and Omp3b, in B. abortus. Another study reported

that Brucella were able to enter and invade the host

cells by a sialic acid-mediated lectin recognition re-

ceptor (Del Carmen Rocha-Gracia et al., 2002).

BRUCELLA INFECTION AND PRION PRO-TEINS

Cellular prion proteins (PrPC) and scrapie-type pri-

on proteins (PrPSC) are also called sialoglycoproteins

because they contain sialic acid in their structures

(Prusiner, 1991, 1995). PrPSC is morphologically differ-

ent than PrPC. Although the exact three dimensional

structure of PrPSC is unknown, it has a higher propor-

tion of β-sheet structure in place of the normal α-helix

structure (Pan et al., 1993). PrPSC is an infectious pro-

tein that is responsible for several prion diseases

including bovine spongiform encephalopathy (mad

cow disease), Creutzfeldt-Jakob disease (CJD), and

scrapie (Schreuder et al., 1994; Prusiner et al., 1995;

Foster et al., 2000). PrPC is a glycoprotein that con-

tains a disulfide bond structure, two N-glycosylation

sites, and a glycosyl-phosphatidyl anchor (Biasini et


al., 2012). These proteins are localized on clathrin-

coated membrane rafts on the cell surface (Vey et

al., 1996). Neurotransmitter metabolism of the PrPC

has been associated with several important roles in

biological functions including such functions as cell

adhesion, signal transmission, copper metabolism

(Cashman et al., 1990; Mazzoni et al., 2005), and pro-

grammed cell death (apoptosis) (Kim et al., 2004).

Watarai et al. (2003) determined that these prion

proteins also play an important role in the entrance

of B. abortus to macrophages particularly with the

presence of heat shock protein (Hsp60). However,

in a later study Fontes et al. (2005) disagreed with

Watarai et al. (2003) and reported that PrPC did

not seem to play a noticeably effective role during

the entrance and uptake of the Brucella into mac-

rophages. Nevertheless, in B. melitensis infected

hosts, the role of the PrPC during the invasion of the

host cells and the following oxidative-antioxidative

metabolic reactions still remains largely unknown.

The involvement of the host cells and B. melitensis

needs to be further characterized before distinctive

roles of the PrPC can be identified.

During Brucella infection, host defense mecha-

nisms are initiated and as part of these mechanisms,

nitric oxide reacts with superoxide anion and gen-

erates hydroxyl (OH-) ion and peroxynitrite (ONOO-)

radical to kill the invading bacteria. However, in this

process, Brucella weakens the corresponding de-

fense of the host cell by increasing their superoxide

dismutase enzyme activity and in doing so scaveng-

ing the free radicals which would have been lethal

to the bacterial cells (Kim et al., 2000). As discussed

previously, Brucella possesses both superoxide

dismutase and catalase activities that are also ca-

pable of dissipating these free radicals produced

by the host cells. These antioxidative enzyme activi-

ties can in turn lead to ineffectiveness in oxidative

capacity in the host cells (Kim et al., 2000). In addi-

tion, an increase in the intracellular concentration

of Cu++ directly up-regulates the expression of the

PrPC proteins and as a result increases the activity

of superoxide dismutase (Brown et al., 1999). Oct-

arepeats that are on the N-terminal of PrPC activate

superoxide dismutase on the endoplasmic reticulum

by binding Cu++ to themselves and transmitting it to

the superoxide dismutase enzyme (McMahon et al.,

2001). In brucellosis, the roles of antioxidative super-

oxide dismutase and its activator (PrPC) have not yet

been elucidated. In the current review, it is specu-

lated that the PrPC protein can be silenced with the

use of small interference RNA, and this in turn would

impact the central nervous system cell viability as

well as the number of bacteria that enter the host

cells. The following sections discuss central nervous

system tissue culture approaches and small interfer-

ence siRNA (small interference RNA or short interfer-

ence RNA; from here on through the remainder of

the text will be referred to as small interference RNA)

characteristics and methodology that could be used

to elucidate these mechanisms.

TRANSIENT TRANSFECTION OF SMALL INTERFERENCE RNA

The small interference RNA molecules are a class

of double-stranded RNA molecules that usually con-

sist of 19 to 25 base pairs in length (Elbashir et al.,

2001). In a series of steps, small interference RNA are

incorporated into RNA-induced silencing complexes

(RISC) which are capable of binding specifically to

mRNA molecules, leading to their destruction, thus

blocking expression of that gene. Although first

discovered as a natural phenomenon, small inter-

ference RNA are mostly seen as effective tools for

knocking out expression of specific genes. Small in-

terference RNA can be introduced into a cell as small

interference RNA DNA in a vector. Transcription of

this DNA eventually leads to stably-produced ac-

tive small interference RNA. More commonly, cells

are transiently transfected by bringing small interfer-

ence RNA directly into the cell using electroporation

or by mixing the small interference RNA with a re-

agent such as Lipofectamine® 2000 (Life Technolo-

gies Corp., Grand Island, NY), a cationic liposome

which binds nucleic acid and delivers it to the cell

cytoplasm. Based on the mechanism of small inter-

ference RNA, it is hypothesized that these molecules

can be used to suppress the expression of the cel-


lular prion proteins. This becomes particularly useful

for characterizing Brucella infection as a means to

sort out the possible effects of PrPC transition from

the host and corresponding oxidative responses (Er-

dogan et al., 2013). Using small interference RNA

to selectively degrade the mRNA would in turn al-

low for the PrPC levels to be reduced or completely

silenced in a controlled fashion. The following sec-

tions describe some of the tissue culture cellular as-

say approaches that can be used to differentiate the

mechanisms associated with Brucella interactions

with the central nervous system.

CENTRAL NERVOUS SYSTEM-ORIENTED TISSUE CULTURE APPROACH

Astrocytes, oligodendrocytes, and microglia are

found in the central nervous system (Guillemin and

Brew, 2004; Hanisch and Kettenmann, 2007; Ber-

trand and Venero, 2013). Microglial cells belong to

the macrophage defense system and are able to

phagocytize pathogens in the central nervous system

(Bertrand and Venero, 2013; Norden, and Godbout,

2013). For central nervous system-oriented tissue

culture studies, microglial cell lines are widely used

as a model for functional studies with a number of in

vitro systems having been developed and evolved

over the years (Guillemin and Brew, 2004; Ponomarev

et al., 2005; Flode and Combs, 2007; Moussaud and

Draheim, 2010; Bertrand and Venero, 2013; Erdogan

et al., 2013). These will not be discussed in detail but

rather a brief overview of generalized methods will

be described as follows. Generally, microglia cells

have the ability to adhere to and grow on the bot-

toms of flasks or plates. Microglial cells are typically

incubated at 37°C with 5% CO2 in Dulbecco’s Modi-

fied Eagle’s Medium (DMEM) liquid medium in the

presence of variations of the following components:

inactivated fetal bovine serum (FBS), sodium pyru-

vate, sodium bicarbonate, HEPES, glutamine, glu-

cose, and along with the antibiotics penicillin and

streptomycin. Microglia cells are typically passaged

every 2 to 3 days and during this time the number of

microglia cells can be determined with trypan blue

staining using a hemocytometer or other means to

enumerate cells.

It is critical in any tissue culture assay to retain con-

sistent levels of viable cells for the duration of the

study or series of experiments. For viability assess-

ment of tissue culture cells, an MTT (Thiazolyl Blue

Tetrazolium Bromide) assay is one of several colo-

rimetric systems that have been used (Mosmann,

1983; Watts et al., 1989; Nikš and Otto. 1990; Ciapetti

et al., 1993; Vega-Avila and Pugsley, 2011). With the

MTT assay the ratio of live cells in a cell community

(containing both live and dead cells) can quantita-

tively be determined with a spectrophotometer. This

approach is based on being able to detect cleavage

of the MTT’s tetrazolium ring by mitochondrial re-

ductases resulting in change of dye color from yel-

low to the blue-purple of the formazan product. This

cleavage reaction is dependent on the activity of mi-

tochondrial succinate dehydrogenase and therefore

can only occur in healthy viable cells.

BRUCELLA INFECTION OF HUMAN MICROGLIA

Apoptosis is an important host defense system

against intracellular infections; however, apoptosis is

not stimulated in Brucella infections. Samartino et al.

(2010) infected astrocytes and microglia with B. abor-

tus. Their study suggested that in neurobrucellosis,

although proinflammatory mediators were induced

in both astrocytes and microglia, apoptosis was in-

duced only in astrocytes but not in microglia. Other

studies have shown that PrPC prevents apoptosis by

inhibiting apoptotic caspase 3 and 9 (Sakudo et al.,

2003; Kim et al., 2004; Erdogan et al., 2013). Silencing

the prion protein gene prnp results in stimulation of

apoptosis. It is still unknown why apoptosis is sup-

pressed in brucellosis, and whether PrPC has a role

in this process (Sakudo et al., 2003). PrPC also acts in

clearing superoxide from the environment. Sakudo

et al. (2003) have speculated that PrPC takes an in-

termediary place in transfer of Cu++ to superoxide

dismutase.

One approach to dissecting out the role of PrPC


would be to degrade and thus silence the PrPC

mRNA by administering siRNA prior to Brucella in-

fection occurring in the host cells. Once PrPC mRNA

is silenced, the resulting impacts on Brucella infec-

tion can be assessed based on analysis of cellular

metabolism and the corresponding metabolites (i.e.

antioxidative metabolism, proinflammatory media-

tors’ levels, cytokine levels etc.) that are produced.

The role of PrPC could be further determined by

directly enumerating invasive bacterial cells during

and after the infection process.

To answer these questions, the human microglia

C13-NJ cell line can be infected with B. melitensis

for various times (for example, 0, 15 minutes, 3 hours

and 24 hours). After this infection period of the cell

line, the cellular viability can be monitored by the en-

zymatic MTT test discussed in the previous section

as well as measuring nitric oxide levels in cellular su-

pernatants, and superoxide dismutase, catalase and

glutathione peroxidase activity. For example, gluta-

thione peroxidase activity is typically determined by

using a commercial kit. In the presence of hydrogen

peroxide, glutathione peroxidase produces oxidized

glutathione and the oxidized glutathione is in turn

reduced by glutathione reductase to NADPH and

reduced glutathione. The determination of the glu-

tathione peroxidase activity is usually calculated as

the decline in the absorbance measured on a spec-

trophotometer at a wavelength of 340 nm during the

oxidation of NADPH to NADP+.

Transcriptional analyses of PrPC, and induced and

neuronal nitric oxide synthase (iNOS and nNOS)

mRNA can be easily performed using a reverse tran-

scriptase polymerase chain reaction (RT-PCR). Re-

covering mRNA for RT-PCR analysis typically involves

the addition of reagents for RNA isolation to a set

quantity of cells, using protocols pre-described by

the commercial manufacturers (Bustin, 2002; Hanna

et al., 2005; Maciorowski et al. 2005; Sirsat et al. 2010;

Erdogan et al., 2013) and yet it is important to assess

the amount and purity of the RNA. This is usually

done by using a spectrophotometer at two different

wavelengths (260/280 nm) where RNA/DNA ratios

that are greater than 1.7 are considered acceptable

for RT-PCR application.

For the actual PCR reactions, a proportion of RNA

can be removed from the corresponding samples

after synthesis of the complementary DNA (cDNA)

by reverse transcriptase (RT). After an aliquot of

cDNA is taken and combined with the commercial

PCR reaction mix, the previously designed and con-

structed primer set (specific for each reaction) for the

amplification of iNOS, nNOS and PrPC genes can

be added. PCR products are then analyzed by gel

electrophoresis. Normalizations can be done using

a constitutive gene such as β-actin gene as a con-

trol. Although semi-quantitative evaluations can be

achieved by determining the intensity of the bands,

direct quantitative PCR approaches are considered

more precise and have been used in a multitude of

studies (Bustin, 2002; Hanna et al., 2005; Maciorows-

ki et al., 2005; Saengkerdsub etal, 2007a,b; Jarquin

et al., 2009; Sirsat et al., 2010; Dunkley et al., 2007,

2008, 2012; Park et al., 2009, 2011a,b, 2013).

Cellular prion proteins levels can also be assessed

directly with the use of Western Blotting approaches

involving specific monoclonal antibodies that have

been generated to the protein(s) of interest. The de-

tails of a variety of methodologies and approaches

for producing specific monoclonal antibodies to

PrPC and perspectives on their specificity are de-

scribed elsewhere (Barry and Prusiner, 1986; Bode-

mer, 1999; Furuoka et al., 2007; Liu et al., 2010) and

will only be discussed in general terms here. Briefly,

in order to analyze PrPC, an aliquot of the respective

protein containing sample is typically removed from

the cell homogenates and subsequently denatured

in Laemmli Buffer, which contains sodium dodecyl

sulfate (SDS) as the denaturing agent. After the pro-

teins have been sufficiently denatured, the resulting

preparations are applied to SDS-polyacrylamide gel

electrophoresis (SDS-PAGE) to separate out the re-

spective proteins and allow for further characteriza-

tion. Once the SDS-PAGE has been completed, the

separated proteins are typically transferred from the

gel to a nitrocellulose membrane via blotting. An-

tibodies (usually monoclonal generated antibodies)

that are specific to PrPC, are followed by the corre-

sponding secondary (typically polyclonal generated

antibodies) antibodies, are subsequently applied to


the nitrocellulose membrane. Once the antibodies

have been added and allowed to bind to their cor-

responding proteins, the presence of PrPC and their

densities can be determined by staining the mem-

brane with a chromogenic substance.

SUMMARY AND CONCLUSIONS

Brucellosis is a zoonotic disease caused by Bru-

cella spp. and is characterized by acute and chronic

symptoms. The pathogen prevents phagosome-

lysosome fusion and oxidative bursts, and does not

induce sufficient activity of the immune defense sys-

tem. Therefore, it is able to survive within the cellular

compartments of the host without any disturbance

for a long period of time. The relapse rate is ob-

served in 5 to 10% of the patients and the nervous

system involvement is approximately 1 to 11% in

humans. The invasion of the Brucella species in the

central nervous system and the response of the host

such as oxidative events, and other defense mecha-

nisms against the pathogen are not well known yet.

The overall aim of this review was to describe the

oxidative events that occur against Brucella infection

and the antioxidative responses that follow this ini-

tial event and potential approaches for studying this

pathogenesis mechanism. In particular, Brucella’s

antioxidant responses that ensure their survival from

host defense system are certainly factors that must

be considered in the pathogenic mechanism. With

the advent of more sophisticated genetic tools the

potential for human neuronal microglia cells to serve

as a neurobrucellosis model offer opportunities to

further explore this relationship at the molecular

level. It has been established that the cellular prion

protein has beneficial roles such as protecting nerve

cells from oxidative stress, but this may also serve to

help bacteria for entry into the cell cytoplasm. How-

ever, in neurobrucellosis, the interaction between

the host and the corresponding Brucella virulence

factors that engage the oxidative host defense sys-

tem and the concomitant effect of PrPC on these

events still remain virtually unknown. Despite these

unknowns there is believed to be a potential role for

PrPC, which possess antioxidative properties and is

thought to be associated in some fashion with the

engulfment of the pathogen through phagocytosis.

The potential of using PrPC-specific small inter-

ference RNA molecules to silence PrPC mRNA be-

fore Brucella infection in microglial cells was also

discussed as means to more precisely delineate the

sequence of events that occur during the expression

of the pathogenesis phenotype. Pathogenesis can

be assessed by quantitating the number of invasive

bacterial cells, determining host viability, and mea-

suring oxidative events that occur during the infec-

tion and invasion process. Further characterization of

Brucella infection in microglial cells using biochemi-

cal and molecular biology techniques should reveal

the interaction between the host neuronal cells and

the pathogen. In addition, the possible involvement

of PrPC in neurobrucellosis would potentially be re-

vealed if small interference RNA molecules can be

applied to block particular steps of the infection.

This ability to target certain steps will allow for an

assessment of how each of the singular infection

events that occur contribute to overall pathogenesis

of the microorganism and where effective control

measures might be most optimally targeted.

REFERENCES

Al-Sekait, M.A. 2000. Epidemiology of brucellosis in Al

Medina region. J. Family Community Med. 7:47–53.

Barry R.A., and S.B. Prusiner. 1986. Monoclonal anti-

bodies to the cellular and scrapie prion proteins. J.

Infect. Dis. 154:518-521.

Bertrand, J. and J. L.Venero, (Eds.). 2013. Microg-

lia - Methods and Protocols - Series: Methods in

Molecular Biology, Vol. 1041, Springer Protocols,

Humana Press, New York, NY, 350 pp.

Biasini, E., J.A. Turnbaugh, U. Unterberger, and D.A.

Harris. 2012. Prion protein at the crossroads of phys-

iology and disease. Trends in Neurosci. 35:92-103.

Bodemer, W. 1999. The use of monoclonal antibod-

ies in human prion disease. Naturwissenschaften

86: 212-220.

Boschiroli, M.L., V. Foulongne, and D. O’Callaghan.


2001. Brucellosis: a worldwide zoonosis. Curr.

Opin. Microbiol. 4:58-64.

Brown, D.R., B.S. Wong, F. Hafiz, C. Clive, S.J. Has-

well, and I.M. Jones. 1999. Normal prion protein

has an activity like that of superoxide dismutase.

Biochem. J. 344:1–5.

Bustin, S.A. 2002. Quantification of mRNA using re-

al-time reverse transcription PCR (RT-PCR): Trends

and problems. J. Mol. Endocrinol. 29: 23-39.

Buzgan, T., Karahocagil, M.K., Irmak, H., Baran, A.I.,

Karsen, H., Evirgen, O., and Akdeniz, H. 2010. Clini-

cal manifestations and complications in 1028 cases

of brucellosis: a retrospective evaluation and re-

view of the literature. Int. J. Infect. Dis. 14:469-478.

Cashman, N.R., R. Loertscher, J. Nalbantoglu, I.

Shaw, R.J. Kascsak, D.C. Bolton, and P.E. Bend-

heim. 1990. Cellular isoform of the scrapie agent

protein participates in lymphocyte activation. Cell

61:185–192.

Celli, J., and J.-P. Gorvel. 2004. Organelle robbery:

Brucella interactions with the endoplasmic reticu-

lum. Curr. Opinion in Microbiol. 7:93–97.

Ceran, N., R. Turkoglu, I. Erdem, A. Inan, D. Engin,

H.Tireli, and P. Goktas. 2011. Neurobrucellosis:

clinical, diagnostic, therapeutic features and out-

come. Unusual clinical presentations in an endem-

ic region. Braz. J. Infect. Dis. 15:52-59.

Choi, H.S., J.W. Kim, Y.-N. Cha, and C. Kim. 2006. A

quantitative nitroblue tetrazolium assay for deter-

mining intracellular superoxide anion production

in phagocytic cells. J. Immunoassay Immunochem.

27:31-44.

Christopher, S., B.L. Umapathy, K.L. Ravikumar. 2010.

Brucellosis: Review on the recent trends in patho-

genicity and laboratory diagnosis. J. Lab. Physi-

cians 2:55-60.

Ciapetti , G., E. Cenni , L. Pratelli, and A. Pizzofer-

rato. 1993. In vitro evaluation of cell/biomaterial in-

teraction by MTT assay. Biomaterials. 14:359–364.

Corbel, M.J. 1990. Brucella. In: T. Parker and L.H.

Collier. (Eds.) Principles of Bacteriology, Virology

and Immunity. Edward Arnold, London, 343–353.

Corbel, M.J. 1997. Brucellosis: an overview. Emerg.

Infect. Dis. 3:213-221.

Covert, J., A.J. Mathison, I. Eskra, M. Banai, and G.

Splitter. 2009. Brucella melitensis, B. neotomae

and B. ovis elicit common and distinctive macro-

phage defense transcriptional responses. Exp.

Biol. Med. 234:1450-1467.

Del Carmen Rocha-Gracia, R., E.I. Castaneda-Roldan,

S. Giono-Cerezo, and J.A. Giron. 2002. Brucella sp.

bind to sialic acid residues on human and animal

red blood cells. FEMS Microbiol. Lett. 213:219-224.

Dunkley, K.D., T.R. Callaway, C. O’Bryan, M.M.

Kundinger, C.S. Dunkley, R.C. Anderson, D.J.

Nisbet, P.G. Crandall, and S.C. Ricke. 2012. Com-

parison of real time polymerase chain reaction

quantification of changes in hilA and rpoS gene

expression of a Salmonella Typhimurium poultry

isolate grown at fast versus slow dilution rates in

an anaerobic continuous culture system. Food Bio-

technol. 26:239-251.

Dunkley, K.D., T.R.Callaway, V.I. Chalova, R.C. Ander-

son, M.M. Kundinger, C.S. Dunkley, D.J. Nisbet,

and S.C. Ricke. 2008. Glucose yields and genetic

responses in a poultry isolate of Salmonella Ty-

phimurium in an anaerobic continuous culture dur-

ing shifts in pH. Anaerobe 14:35-42.

Dunkley, K.D., J.L. McReynolds, M.E. Hume, C.S.

Dunkley, T.R. Callaway, L.F. Kubena, D.J. Nisbet,

and S.C. Ricke. 2007. Molting in Salmonella Enter-

itidis challenged laying hens fed alfalfa crumbles

I. Salmonella Enteritidis colonization and virulence

gene hilA response. Poultry Sci. 86:1633-1639.

Durant, J.A., V.K. Lowry, D.J. Nisbet, L.H. Stanker,

D.E. Corrier, and S.C. Ricke. 2000a. Short-chain fat-

ty acids alter HEp-2 cell association and invasion

by stationary growth phase Salmonella Typhimuri-

um. J. Food Sci. 65:1206-1209.


D.E. Corrier, and S.C. Ricke. 2000b. Late logarith-

mic Salmonella typhimurium HEp-2 cell-associa-

tion and invasion response to short chain volatile

fatty acid addition. J. Food Safety 20:1-11.


D.E. Corrier, and S.C. Ricke. 1999. Short-chain vol-

atile fatty acids affect the adherence and invasion

of HEp-2 cells by Salmonella typhimurium. J. Envi-

ron. Sci. Health B34:1083-1099.

Elbashir, S.M., W. Lendeckel, and T. Tuschi. 2001.


RNA interference is mediated by 21 and 22-nucle-

otide RNAs. Genes. Dev. 15:188-200.

Erdem, H., A. Ulu-Kilic, S. Kilic, M. Karahocagil, G.

Shehata, N. Eren-Tulek, F. Yetkin, M.K. Celen, N.

Ceran, H.C. Gul, G. Mert, S. Tekin-Koruk, M. Diz-

bay, A.S. Inal, S. Nayman-Alpat, M. Bosilkovski, D.

Inan, N. Saltoglu, L. Abdel-Baky, M.T. Adeva-Bar-

tolome, B. Ceylan, S. Sacar, V. Turhan, E. Yılmaz, N.

Elaldi, Z. Kocak-Tufan, K. Ugurlu, B. Dokuzoguz, H.

Yılmaz, S. Gundes, R. Guner, N. Ozgunes, A. Ulcay,

S. Unal, S.Dayan, L. Gorenek, A. Karakas, Y. Tasova,

G. Usluer, Y. Bayindir, B. Kurtaran, B. O.R. Sipahi,

and H. Leblebicioglu. 2012. Efficacy and tolerabil-

ity of antibiotic combinations in neurobrucellosis:

results of the Istanbul study. Antimicrob. Agents

Chemother. 56:1523-1528.

Erdogan S., S. Celik, O. Aslantas, T. Kontas, and S.

Ocak. 2007. Elevated cAMP levels reverse Bru-

cella melitensis-induced lipid peroxidation and

stimulate IL-10 transcription in rats. Res. Vet. Sci.

82:181–186.

Erdogan, S., O. Aslantas, S. Celik, and E. Atik. 2008.

The effects of increased cAMP content on inflam-

mation, oxidative stress and PDE4 transcripts

during Brucella melitensis infection. Res. Vet. Sci.

84:18-25.

Erdogan, S., V. Duzguner, A. Kucukgul, and O. Aslan-

tas. 2013. Silencing PrPC (prion protein) expression

does not affect Brucella melitensis infection in hu-

man derived microglia cells. Res. Vet Sci. 95:368-373.

Fang, F.C. 1997. Perspectives series: host/pathogen

interactions. Mechanisms of nitric oxide-related

antimicrobial activity. J Clin Investig. 99:2818–2825.

Ficht T.A. 2003. Intracellular survival of Brucella:

defining the link with persistence. Vet. Microb.

92:213–223.

Flode, A.M. and C.K. Combs. 2007. Microglia re-

petitively isolated from in vitro mixed glial cultures

retain their initial phenotype. J. Neurosci. Meth.

164:218–224.

Fontes, P., M.T. Alvarez-Martinez, A. Gross, C. Carn-

aud, S. Köhler, and J.P. Liautard. 2005. Absence of

evidence for the participation of the macrophage

cellular prion protein in infection with Brucella suis.

Infect. Immun. 73:6229-6236.

Foster, P.R. 2000. Prions and blood products. Annu-

als Medicine 32:501–513.

Freer, E., E. Moreno, I. Moriyon, J. Pizarro-Cerda, A.

Weintraub, and J.P. Gorvel. 1996. Brucella-Salmonel-

la lipopolysaccharide chimeras are less permeable to

hydrophobic probes and more sensitive to cationic

peptides and EDTA than are their native Brucella

spp. counterparts. J. Bacteriol. 178:5867–5876.

Franco, M.P., M. Mulder, R.H. Gilman, and H.L Smits.

2007. Human brucellosis. Lancet Infect. Dis. 7:775-

786.

Furuoka, H., A. Yabuzoe, M. Horiuchi, Y. Tagawa ,

T. Yokoyama, Y. Yamakawa, M. and T. Sata. 2007.

Species-specificity of a panel of prion protein an-

tibodies for the immunohistochemical study of

animal and human prion diseases. J Comp Pathol.

136:9-17.

Gándara, B., A.L. Merino, M.A. Rogel and E. Mar-

tínez-Romero. 2001. Limited genetic diversity of

Brucella spp. J. Clin. Microbiol. 39:235–240.

Giambartolomei, G.H., A. Zwerdling, J. Cassataro,

L. Bruno, C.A. Fossati, and M.T. Philipp. 2004. Li-

poproteins, not lipopolysaccharide, are the key

mediators of the proinflammatory response elic-

ited by heat-killed Brucella abortus. J. Immunol.

173:4635–4642.

Green, L.C., D.A. Wagner, J. Glogowski, P.L. Skipper,

J.S. Wishnok, and S.R. Tannenbaum. 1982. Analy-

sis of nitrate, nitrite, and [15N] nitrate in biological

samples. Anal. Biochem. 126:131-138.

Gross, A., S. Spiesser, A. Terraza, B. Rouot, E. Caron,

and J. Dornand. 1998. Expression and bacteri-

cidal activity of nitric oxide synthase in Brucella

suis-infected murine macrophages. Infect Immun.

66:1309–1316.

Gross, A., M. Bouaboula, P. Casellas, J.P. Liautard,

and J. Dornand. 2003. Subversion and utilization of

the host cell cyclic adenosine 5’-monophosphate/

protein kinase A pathway by Brucella during mac-

rophage infection. J. Immunol. 170:5607–5614.

Gross, A., S. Bertholet, J. Mauel, and J, Dornand.

2004. Impairment of Brucella growth in human

macrophagic cells that produce nitric oxide. Mi-

crobial Pathogenesis 36:75-82.

Guillemin, G.J. and B.J. Brew. 2004. Microglia, mac-


rophages, perivascular macrophages, and peri-

cytes: a review of function and identification J.

Leukocyte Biol. 75:388-397.

Guzmán-Verri., C., L. Manterola., A. Sola-Landa, A.

Para., A. Cloeckaert, J. Garin, J.P. Gorvel, I. Mori-

yon., E. Moreno, and I. Lopez-Goni. 2002. The two-

component system BvrR/BvrS essential for Bru-

cella abortus virulence regulates the expression

of outer membrane proteins with counterparts in

members of the Rhizobiaceae. Proc. Natl. Acad.

Sci. 99:12375–12380.

Gul, H.C., H. Erdem, L. Gorenek, M.F. Ozdag, Y. Kal-

pakci, Y. Avci, B.A. Besirbellioglu, and C.P. Eyigun.

2008. Management of neurobrucellosis: an assess-

ment of 11 cases. Intern. Med. 47:995-1001.

Gul, H.C., H. Erdem, and S. Bek. 2009. Overview of

neurobrucellosis: a pooled analysis of 187 cases.

Int. J. Infect. Dis. 13:339–343.

Hanisch, U.K. and H. Kettenmann. 2007. Microglia: Ac-

tive sensor and versatile effector cells in the normal

and pathologic brain. Nat. Neurosci. 10:1387-1394.

Hanna, S.E., C.J. Conner, and H.H. Wang. 2005.

Real-time polymerase chain reaction for the food

microbiologist: Technologies, applications, and

limitations. J. Food Sci. 70:R49-R53.

Hong, P.C., R.M. Tsolis, and G. Adams. 2000. Identifica-

tion of genes required for chronic pesistence of Bru-

cella abortus in mice. Infect. Immun. 68:4102-4107.

Howard, Z.R., R.W.Moore, I.B. Zabala-Diaz, K.L.

Landers, J.A. Byrd, L.F. Kubena, D.J. Nisbet, S.G.

Birkhold, and S.C. Ricke. 2005. Ovarian laying hen

follicular maturation and in vitro Salmonella inter-

nalization. Vet. Microbiol. 108:95-100.

Jarquin, R., I. Hanning, S. Ahn, and S.C. Ricke. 2009.

Development of rapid detection and genetic

characterization of Salmonella in poultry breeder

feeds. Sensors 9:5308-5323.

Jimenez de Bagues, M.P., A. Gross, A. Terraza, and J.

Dornand. 2005. Regulation of the mitogen-activated

protein kinases by Brucella spp. expressing a smooth

and rough phenotype: relationship to pathogen in-

vasiveness. Infect. Immun. 73:3178-3183.

Johnston, Jr, R.B., B.B. Keele, Jr, H.P. Misra, J.E. Leh-

meyer, L.S. Webb, R.L. Baehner, and K.V. RaJago-

palan. 1975. The role of superoxide anion genera-

tion in phagocytic bactericidal activity. Studies with

normal and chronic granulomatous disease leuko-

cytes. J. Clin. Invest. 55:1357–1372.

Kataria , N., A.K Kataria , R. Maan, and A.K.Gahlot.

2010. Evaluation of oxidative stress in Brucella in-

fected cows. J. Stress Physiol. Biochem. 6:19-25.

Kaymak, C., H. Basar, and S. Sardas. 2011. Reactive

oxygen species (Ros) generation in sepsis. FABAD

J. Pharm. Sci. 36:41-47.

Kim B.H., H.G. Lee, J.K. Choi, J.I. Kim, E.K. Choi, R.I.

Carp, and Y.S. Kim. 2004. The cellular prion protein

(PrPC) prevents apoptotic neuronal cell death and

mitochondrial dysfunction induced by serum de-

privation. Mol. Brain Res. 124:40–50.

Kim, J.A., Z. Sha, and J.E. Mayfield. 2000. Regula-

tion of Brucella abortus catalase. Infect. Immun.

68:3861–3866.

Lehmann S., 2002. Metal ions and prion diseases.

Curr. Opin. Chem. Biol. 6:187–192.

Leitenberg, M. 2001. Biological weapons in the

twentieth century: a review and analysis. Crit. Rev.

Microbiol. 27:267-320.

Liu, Y.S., Y.Z Ding, J. Zhang, H.T. Chen, X.L. Zhu, X.P.

Cai, X.T. Liu, and Q.G. Xie. 2010. Simple method

of monoclonal antibody production against mam-

malian cellular prion protein. Hybridoma 29:37-43.

Maciorowski, K.G., S.D. Pillai, F.T. Jones, and S.C.

Ricke. 2005. Polymerase chain reaction detection

of foodborne Salmonella spp. in animal feeds. Crit.

Rev. Microbiol. 31:45-53.

Mantur, B.G. and S.K. Amarnath. 2008. Brucellosis in

India – a review. J. Biosci. 33:539–547.

Mantur, B.G., S.K. Amarnath, and R.S. Shinde. 2007.

Review of clinical and laboratory features of human

brucellosis. Indian J. Med. Microbiol. 25:188-202.

Mazzoni, I.E., H.C. Ledebur, Jr, E. Paramithiotis, and

N. Cashman. 2005. Lymphoid signal transduction

mechanisms linked to cellular prion protein. Bio-

chem. Cell Biol. 83:644–653.

McLean, D.R., N. Russell, and M.Y. Khan. 1992. Neu-

robrucellosis: clinical and therapeutic features.

Clin. Infect. Dis. 15:582–590.

McMahon, H.E., A. Mange, N. Nishida, C. Creminon,

D. Casanova, and S. Lehmann. 2001. Cleavage of

the amino terminus of the prion protein by reactive


oxygen species. J. Biol. Chem. 276:2286–2291.

Megid, J., L.A. Mathias, and C.A. Robles. 2010. Clini-

cal manifestations of brucellosis in domestic ani-

mals and humans. Open Vet. Sci. J. 4:119-126.

Melek, I.M., S. Erdogan, S. Celik, O. Aslantas, and

T. Duman. 2006. Evaluation of oxidative stress and

inflammation in long term Brucella melitensis in-

fection. Mol. Cell. Biochem. 293:203-209.

Mosmann, T. 1983. Rapid colorimetric assay for cellu-

lar growth and survival: Application to proliferation

and cytotoxicity assays. J. Immunol. Meth. 65: 55-63.

Mousa, A.R., T.S. Koshy, G.F. Araj, A.A. Marafie, S.A.

Muhtaseb, D.S. Al-Mudallal, and M.S. Busharetulla

1986. Brucella meningitis: presentation, diagnosis

and treatment—a prospective study of ten cases.

Q. J. Med. 60:873–885.

Moussaud, S. and H.J. Draheim. 2010. A new meth-

od to isolate microglia from adult mice and culture

them for an extended period of time. J. Neurosci.

Meth. 187:243–253.

Nikš, M. and M. Otto. 1990. Towards an optimized

MTT assay. J. Immunol. Meth. 130:149-151.

Norden, D.M. and J. P. Godbout. 2013. Review: Mi-

croglia of the aged brain: primed to be activated

and resistant to regulation. Neuropathol. Appl.

Neurobiol. 39:19–34.

Nunez-Martinez, C., P. Altamirano-Silva, F. Alvara-

do-Guillen, E. Moreno, C. Guzman-Verri, and E.

Chaves-Olarte.2010. The two-component system

BvrR/BvrS regulates the expression of the type IV

secretion system VirB in Brucella abortus. J. Bacte-

riol. 192:5603-5608.

O’Callaghan, D., C. Cazevieille, A. Allardet-Servent,

M.L. Boschiroli, G. Bourg, V. Foulongne, P. Frutos,

Y. Kulakov, and M. Ramuz. 1999. A homologue

of the Agrobacterium tumefaciens VirB and Bor-

detella pertussis Ptl type IV secretion systems is

essential for intracellular survival of Brucella suis.

Mol. Microbiol. 33:1210-1220.

Orozco, G., E. Sánchez, M.A. Lopez-Nevot, A. Ca-

ballero, M.J. Bravo, P. Morata, J.D. Colmenero, A.

Alonso, and J. Martin. 2003. Inducible nitric oxide

synthase promoter polymorphism in human bru-

cellosis. Microb. Infect. 5:1165-1169.

Pan, K.M., M. Baldwin, J. Nguyen, M. Gosset, A.

Serban, D. Groth, I. Mehlhorn, Z. Huang, R.J. Flet-

tericj, and F.E. Cohen. 1993. Conversion of alpha-

helices into beta-sheets features in the formation

of the scrapie prion proteins. Proc. Natl. Acad. Sci.

USA. 90:10962–10966.

Pappas, G., P. Papadimitriou, N. Akritidis, L. Chris-

tou, and E.V. Tsianos. 2006. The new global map of

human brucellosis. Lancet Infect Dis. 6: 91-99.

Park, S.H., I. Hanning, A. Perrota, B. J. Bench, E. Alm,

and S. C. Ricke. 2013. Modifying the gastrointesti-

nal ecology in alternatively raised poultry and the

potential for molecular and metabolomic assess-

ment. Poultry Sci. 92:546–561.

Park, S.H., I. Hanning, R. Jarquin, P. Moore Jr., D.J.

Donoghue, A.M. Donoghue, and S.C. Ricke.

2011a. Multiplex PCR assay for the detection and

quantification of Campylobacter spp., Escherichia

coli O157:H7 and Salmonella serotypes in water

samples. FEMS Microbiol. Lett. 316:7-15.

Park, S.H., R. Jarquin, I. Hanning, G. Almeida, and

S.C. Ricke. 2011b. Detection of Salmonella spp.

survival and virulence in poultry feed by targeting

the hilA gene. J. Appl. Microbiol. 111:426-432.

Park, S. H., H.J. Kim, W.H. Cho, J.H. Kim, M.H. Oh,

S.H. Kim, B.K. Lee, S.C. Ricke, and H.Y. Kim. 2009.

Identification of Salmonella enterica subspecies I,

Salmonella enterica serovars Typhimurium, Enter-

itidis and Typhi using multiplex PCR. FEMS Micro-

biol. Letts. 301:137-146.

Ponomarev, E.D., M. Novikova, K. Maresz, L.P. Shriv-

er, and B.N. Dittel. 2005. Development of a culture

system that supports adult microglial cell prolifera-

tion and maintenance in the resting state. J. Im-

munol. Meth. 300:32–46.

Prusiner, S.B. 1995. The prion diseases. Scientific

American. 272: 48-51, 54-57.

Prusiner, S.B. 1991. Molecular biology of prion dis-

eases. Science. 252:1515-1522.

Saengkerdsub, S., R.C. Anderson, H.H. Wilkinson,

W.-K. Kim, D.J. Nisbet, and S.C. Ricke. 2007a.

Identification and quantification of methanogenic

archaea in adult chicken ceca. Appl. Environ. Mi-

crobiol. 73:353-356.

Saengkerdsub, S., P. Herrera, C.L. Woodward, R.C.

Anderson, D.J. Nisbet, and S.C. Ricke. 2007b. De-


tection of methane and quantification of methano-

genic archaea in faeces from young broiler chick-

ens using real-time PCR. Letts. Appl. Microbiol.

45:629-634.

Sakudo, A., D.C. Lee, K. Saeki, Y. Nakamura, K. In-

oue, Y. Matsumoto, S. Itohara, and T. Onodera.

2003. Impairment of superoxide dismutase activa-

tion by N-terminally truncated prion protein (PrP)

in PrP deficient neuronal cell line. Biochem. Bio-

phys. Res. Commun. 308:660–667.

Samartino, C.G., M.V. Delpino, G.P. Godoy, M.S. Di

Genaro, K.A. Pasquevich, A. Zwerdling, P. Barrion-

uevo, P. Mathieu, J. Cassataro, F. Pitossi, and G.H.

Giambartolomei. 2010. Brucella abortus induces

the secretion of proinflammatory mediators from

glial cells leading to astrocyte apoptosis. Am. J.

Pathol. 176:1323-1338.

Schreuder, B.E.C. 1994. Animal spongiform enceph-

alopathies – an update Part I. Scrapie and lesser

known animal spongiform encephalopathies. Vet.

Quarterly 16:173-181.

Shakir, R.A., A.S. Al-Din, G.F. Araj, A.R. Lulu, A.R.

Mousa, and M.A. Saadah. 1987. Clinical catego-

ries of neurobrucellosis. A report on 19 cases.

Brain 110:213–223.

Sirsat, S.A., A. Muthaiyan, S.E. Dowd, Y.M. Kwon,

and S.C. Ricke. 2010. The potential for applica-

tion of foodborne Salmonella gene expression

profiling assays in postharvest poultry process-

ing. pp. 195-222. In: Perspectives on Food Safety

Issues of Food Animal Derived Foods, S.C. Ricke

and F.T. Jones (eds.) University of Arkansas Press,

Fayetteville, AR.

Sola-Landa, A., J. Pizarro-Cerdá, M.-J. Grilló, E.

Moreno, I. Moriyón, J.-M. Blasco, J. P. Gorvel, and

I. López-Goñi. 1998. A two-component regulatory

system playing a critical role in plant pathogens

and endosymbionts is present in Brucella abortus

and controls cell invasion and virulence. Mol. Mi-

crobiol. 29:125–138.

Sun, J., X. Zhang, M. Broderick and H. Fein. 2003.

Measurement of nitric oxide production in biologi-

cal systems by using Griess reaction assay. Sensors

3:276-284.

Sun, Y., L.W. Oberley, L.W., and L.A. Ying. 1988.

Simple method for clinical assay of superoxide dis-

mutase. Clin. Chem. 34:497-500.

Vega-Avila, E. and M.K. Pugsley. 2011. An overview

of colorimetric assay methods used to assess sur-

vival or proliferation of mammalian cells. Proc.

West. Pharmacol. Soc. 54:10-14.

Vey, M., S. Pilkuhn, H. Wille, R. Nixon, S.J. DeAr-

mond, E.J. Smart, R.G. Anderson, A. Taraboulos,

and S.B. Prusiner. 1996. Subcellular colocalization

of the cellular and scrapie prion proteins in cave-

olae-like membranous domains. Proc. Natl. Acad.

Sci. 93:14945–14949.

Wang, M., N. Qureshi, N. Soeurt, and G. Splitter.

2001. High levels of nitric oxide production de-

crease early but increase late survival of Brucella

abortus in macrophages. Microbial Pathogenesis

5:221-230.

Watarai, M., S. Kim, J. Erdenebaatar, S. Makino, M.

Horiuchi, T. Shirahata, S. Sakaguchi, and S. Kat-

amine. 2003. Cellular prion protein promotes Bru-

cella infection into macrophages. J. Exp. Med.

198:5-17.

Watts , M.E., I.J. Roberts , and M.Woodcock. 1989.

A comparison of colorimetric and clonogenic as-

says for hypoxic-specific toxins with hamster and

human cells. Int. J. Oncol., Biol., Phys. 16:939–942.




ABSTRACT

The street foods play an important socio-economic role in meeting food and nutritional requirements of city

consumers at affordable prices. This study was designed to evaluate the detailed microbial status including

foodborne pathogen and spoilage bacteria and their drug sensitivity status in different street foods of Dhaka

city. For this assessment, 39 street foods samples of 13 kinds were collected from Motijheel area, the busiest

part of the Dhaka city area. These samples were analyzed for foodborne pathogens including, Salmonella

spp., Escherichia coli O157, O111, O26 and other E. coli, other coliforms, Cronobacter sakazakii, Yersinia spp.,

Listeria spp., Staphylococcus spp., and spoilage microorganisms including Enterococcus spp., Pseudomonas

spp., Bacillus spp., and lactic acid fermenting bacteria (LAB). The average natural aerobic bacterial population

varied from 3.0 ± 0.04 log CFU/g to 8.8 ± 0.02 log CFU/g and the average coliform count varied from 2.0 ±

0.01 log CFU/g to 7.5 ± 0.02 log CFU/g. In addition, Salmonella spp. and Escherichia coli (O157, O111, O26)

were identified in 2 street food samples, other E. coli were found in 5 samples, coliform bacteria was found

in 28 samples and Enterococcus spp. in 10 samples, out of 39 food sample analyzed. Moreover, Listeria spp.

were detected in 15 samples, Yersinia spp. in 10 samples, Enterobacter sakazakii in 8 samples, and Staphylo-

coccus spp. in all 39 samples. Among the spoilage organisms, Bacillus spp. were identified in 12 food samples,

Pseudomonas spp. in 15 food samples and lactic acid fermenting bacteria (LAB) in 24 samples, out of the 39

samples tested. The isolated pathogens were then checked for antibiotic sensitivity and the results revealed

that all the Salmonella spp. exhibited multi drug resistance (at least 7 antibiotics), all Escherichia coli O157,

O111, O26 and other E. coli were multi drug resistant (at least 6 antibiotics), Enterobacter sakazakii (at least 6

drugs) and the similar results were found for all the coliform (at least 5 antibiotics), Listeria spp., Pseudomonas

spp. and lactic acid fermenting bacteria (LAB). In addition, Staphylococcus spp., Bacillus spp., isolates were

resistant to most of the antibiotics and some isolates were resistant to all the antibiotics tested. Enterococcus

spp. was found to be sensitive to vancomycin. These study result demonstrated that foods sold in the street of

Dhaka City constitutes a potential microbial hazard to human health.

Correspondence: Md. Latiful Bari, [email protected]: 8801971560560 Fax: : 8802-8615583

Prevalence of Foodborne Pathogens and Spoilage Microorganisms and Their Drug Resistant Status in Different Street Foods of Dhaka city

Z. Tabashsum1,6, I. Khalil2, Md. Nazimuddin3 , A.K.M. M. Mollah4 , Y. Inatsu5 and Md. L. Bari1

1Center for advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh2Bangladesh Standards and Testing Institution, Tejgaon Industrial Area, Dhaka-1205, Bangladesh

3Bangladesh Agricultural Research Institute, Gazipur 1701, Bangladesh4Faculty of Life Sciences, Asian University for Women, Chittagong 4000, Bangladesh

5National Food Research Institute, 2-1-12 Kannondai, Tsukuba-shi, Japan6Faculty of Life Science, Independent University Bangladesh, Dhaka-1229, Bangladesh



INTRODUCTION

Foodborne diseases are now becoming a great

concern involving a wide range of illnesses caused

by bacterial, viral, parasitic or chemical contamina-

tion of food. In addition, resistance of these microor-

ganisms to multi-drugs made this situation more of

a concern to public health. Approximately, 30 million

people in Bangladesh suffer from food borne illness-

es each year (FAO, 2012). Diarrheal diseases are the

most common food poisoning cases in Bangladesh

and in some cases, these can cause death. The dis-

eases are caused by either the toxin produced by

the microorganism, or by the human body’s reac-

tions to the microorganism.

Street foods are described as a wide range of

ready-to-eat foods and beverages, or prepared at

home and consumed on the streets without further

preparation (Dardano, 2003). The food items are

sold by vendors and hawkers especially in the streets

and other similar public places. While street-vend-

ed foods are appreciated for their unique flavors as

well as their convenience, they are also important in

contributing to the nutritional status of the people.

Street food vending assures food security for low-

income urban populations and provides a livelihood

for a large number of workers who would otherwise

be unable to establish a business for want of capital.

In contrast to these potential benefits, it is also rec-

ognized that street-food vendors are often poor, un-

educated and lack knowledge in safe food handling

practices, environment, sanitation and hygiene,

mode of food display, food service and hand wash-

ing, sources of raw materials, and use of portable

water. Consequently, street foods are perceived to

be a major public health risk.

A study of the socio-economic conditions and de-

termination of the hygienic and sanitary practices of

street food vendors in Dhaka City Corporation was

carried out by FAO 2010. The study result demon-

strated that 25% street food vendors are illiterate

and cannot write their names and have no formal

education. As street food business requires low in-

vestment, most of the vendors (88%) were found to

own their business. They reportedly work for 13 to

18 hours a day without having toilet facilities. Most

of the vending shops (68%) were located on the

footpath irrespective of areas surveyed and 30%

vending carts were placed near municipal drains

and 18% near sewage. A microbiological study on

different foods items, drinking water and hand swab

samples revealed the prevalence of overwhelmingly

high numbers of aerobic bacteria and coliform bac-

teria. The study also indicated that a significant por-

tion of drug resistant bacteria are spreading in the

community through the street foods. This study also

suggested the need to conduct a detailed microbial

study and profile their drug resistance characteristics

to assess the potential public health hazards. There-

fore, this study was designed to evaluate the detailed

microbial status including foodborne pathogen and

spoilage bacterial content and their drug sensitivity

status from different street foods of Dhaka city.

MATERIALS AND METHODS

Sample collection

Street food samples (36) of thirteen categories

were purchased from the vendors at Motijheel area

of Dhaka between October 15 and November 15,

2012. All the samples were transported to the Food

Analysis and Research Laboratory, Center for Ad-

vanced Research in Sciences (CARS) of University

of Dhaka at the earliest convenience for processing

and further assessment. All the analysis was carried

out according to the standard methods described in

the U.S. Food and Drug Administration (FDA) Bacte-

riological Analytical Manual and the schematic dia-

gram is presented in Figure 1.

Total aerobic count and total coliform count

Twenty five (25) g of each sample were homog-

enized in 225 milliliters of saline water (0.85% NaCl).

Decimal dilutions were prepared upto 10-6 and ap-

propriate dilutions were spread plated on Tryptic

soy agar (Oxoid Ltd., Hampshire, England) and incu-


Figure 1. Flow diagram for the identification of foodborne pathogens and food spoilage bacteria using conventional, immunological, and molecular methods.


bated at 35ºC for 24 hours for total aerobic bacterial

count and on MacConkey agar (Oxoid Ltd., Hamp-

shire, England) and incubated at 35ºC and 42ºC

for 24 hours for total coliform counts. Total aerobic

counts were used to indicate the quality and shelf

life of the products and total coliform counts to indi-

cate the unhygienic condition of the food prepara-

tion surfaces.

Escherichia coli O157, O111, O26

Twenty five (25) g of each samples were homog-

enized in 225 milliliters mEC medium (Nissui Co.,

Ltd., Tokyo, Japan) and incubated at 42ºC for 20

hours. The enriched cultures were streaked onto

Sorbitol MacConkey agar (Oxoid Ltd., Hampshire,

England) complemented with Cefixime and potas-

sium tellurite supplement (Fluka, Sigma-Aldrich,

Bangalore, India) and characteristic colonies were

subjected to biochemical tests (IMViC). Biochemi-

cally confirmed isolates were screened through

Rainbow agar (Biolog, France) and CHROM agar

(Kanto Co. Ltd., Kyoto, Japan). The colonies which

gave characteristic color were subsequently sero-

typed by O157, O111 and O26 specific antisera. The

isolates were subsequently tested for stx1 and stx2

by NH-Immunochromato VT1/2 and by PCR using

primer 5’-CAGTTAATGTGGTGGCGAAGG-3’ and

5’-CACCAGACAAATGTAACCGCTC-3’ for stx1 and

5’-ATCCTATTCCCGGGAGTTTACG-3’ and 5’-GC-

GTCATCGTATACACAGGAGC-3’ for stx2 (Vidal et

al., 2004).

Escherichia coli, Coliform bacteria, En-terobacter sakazakii


enized in 225 milliliters Enterobacteria enrichment

broth-Mossel pre-enrichment medium (Oxoid Ltd.,

Hampshire, England) and incubated at 35ºC for 20

hours. One milliliter aliquots of pre-enriched cultures

were mixed with nine milliliters of 2x EC medium (Nis-

sui Co., Ltd., Tokyo, Japan) and incubated at 35ºC for

20 hours. To confirm the existence of fecal coliforms,

one loopful of the culture was inoculated into 10

milliliters 1x EC medium with Durham fermentation

tubes and incubated at 42ºC for 20 hours. Gas pro-

duction in the tubes were used to indicate the pres-

ence of fecal coliforms. To isolate E. coli, one loopfull

of gas produced 1x EC culture broth was streaked

on EMB agar plates (Nissui Co., Ltd., Tokyo, Japan)

and developed typical colonies were then confirmed

using biochemical characterization (IMViC) and API

20E kit (bioMérieux, Durham, NC, USA). The same

pre-enrichment culture was also used for isolation

and characterization of coliform bacteria on Sorbitol

MacConkey agar (Nissui Co., Ltd., Tokyo, Japan) and

isolated strains were subjected to further character-

ization using an API 20E kit. Pre-enriched culture was

streaked onto Chromocult Enterobacter sakazakii

(Merck, Darmstadt, Germany) agar plates to isolate

Cronobacter sakazakii. The typical colonies were fur-

ther characterized using an API 20E kit (BioMérieux,

Durham, NC, USA). Presence of Escherichia coli or

fecal coliform bacteria was used to indicate that the

food had become contaminated with fecal material

in some fashion.

Salmonella spp.


enized in 225 milliliters of buffered peptone water

(Merck, Darmstadt, Germany) and incubated at 35ºC

for 20 hours. One milliliter pre-enrichment cultures

were mixed with nine milliliters of Hanja Tetrathion-

ate Broth (Eiken Chemical Co. Ltd., Tokyo, Japan)

and incubated at 35ºC for 20 hours and nine milli-

liters of Rappaport-Vassiliadis Broth (Eiken Chemi-

cal Co. Ltd., Tokyo, Japan) and incubated at 42ºC

for 20 hours. The culture broths were subsequently

streaked onto DHL and MLCB and characteristics

of isolates from candidate colonies was determined

through biochemical tests (TSI and LIM). Biochemi-

cally confirmed isolates were re-confirmed using Sal-

monella LA latex agglutination test and API 20E kits.

Yersinia spp.



enized in 225 milliliters of 0.85% NaCl and incubated

at 10ºC for 7 days. These enriched cultures were

streaked on Yersinia selective agar (Fluka, Sigma-

Aldrich, Bangalore, India) and incubated at 30ºC for

20 hours. The candidate colonies were subsequently

confirmed by API 20E (BioMérieux, Durham, NC,

USA).

Bacillus spp., Staphylococcus spp. and Pseudomonas spp.

Twenty five (25) g each samples were homog-



for 20 hours. The respective pre-enrichment cul-

tures were streaked on NaCl Glycine Kim Goepfert

(NGKG) agar (Nissui Co., Ltd., Tokyo, Japan) with

20% egg yolk, mannitol salt agar (Nissui Co., Ltd.,

Tokyo, Japan) and NAC agar (Nissui Co., Ltd., To-

kyo, Japan) to isolate Bacillus spp., Staphylococcus

spp. and Pseudomonas spp. Typical colonies were

subjected to biochemical characterization tests us-

ing suitable API kits. API 50CH with API CHB and API

Staph (BioMérieux, Durham, NC, USA) were used

for the identification of Bacillus spp. and Staphy-

lococcus spp., respectively. The isolates of Bacillus

spp. were subsequently checked for CRS gene by

polymerase chain reaction (PCR) using the sense

strand primer 5’-GGTGAATTGTGTCTGGGAGG-3’

and antisense strand primer 5’-ATTTTTATTAAGAG-

GCAATG-3’. Typical colonies of Pseudomonas spp.

were further checked by Oxidase and Catalase tests

and API 20NE (BioMérieux, Durham, NC, USA) diag-

nostic kits.

Enterococcus spp.




for 20 hours. One milliliter aliquots of these pre-en-

richment cultures were added to 9 milliliters of 2x AC

medium (HI media, Mumbai, India) and incubated

at 35ºC for 20 hours. After incubation, one loopfull

aliquots of these cultures were inoculated into nine

milliliters of 1x AC medium and incubated at 42ºC

for 20 hours, and then, a loopfull of each respective

culture was streaked onto EF agar (Nissui Co., Ltd.,

Tokyo, Japan.). Vancomycin containing EF (VR-EF)

antibiotic agar plates (Nissui Co., Ltd., Tokyo, Ja-

pan) were also used for the isolation of vancomycin-

resistant Enterococci spp. (VRE). Developed typical

colonies on the EF agar plates were confirmed by

biochemical tests by using an API Strep (BioMérieux,

Durham, NC, USA) kit.

Listeria spp.

Twenty five (25) g of each samples was homog-

enized in 225 milliliters of DifcoTM Listeria enrich-

ment broth (Difco, Detroit, Michigan, USA) and in-

cubated at 30ºC for 40 hours. The enriched cultures

were streaked on Listeria selective agar base (Oxoid

Ltd., Hampshire, England) with selective supplement

SR0206E (Oxoid Ltd., Hampshire, England) and in-

cubated at 30ºC for 20 hours or for extended incu-

bation times if needed. Characteristic colonies were

confirmed by NH-immunochromato Listeria and API

Listeria kits.

Lactic acid bacteria

Twenty five (25) g of each sample was homog-

enized in 225 milliliters of de Man, Rogosa and

Sharpe (MRS) broth (Difco, Detroit, Michigan, USA)

and incubated at 30ºC for 20 hours under anaero-

bic conditions. A loopfull of culture was streaked on

MRS agar and incubated at 30ºC for 20 to 40 hours

under anaerobic condition. Typical catalase negative

colonies were subjected to biochemical tests by us-

ing API 50CH kit with API CHL (BioMérieux, Durham,

NC, USA).

Antibiotic Susceptibility Test

All isolated strains were then tested for antibiotic

susceptibilities. The ranges of antibiotic susceptibil-

ity of the isolates were measured using commercially

purchased discs (Oxoid Ltd., Hampshire, England)

by the disc diffusion method on Mueller-Hinton Agar


(Oxoid Ltd., Hampshire England). Antibiotics used

in this experiment included gentamicin 10 µg (CN),

vancomycin 30 µg (VA), amoxicillin 10 µg (AML),

erythromycin 15 µg (E), streptomycin 10 µg (S), no-

vobiocin 30 µg (NV), kanamycin 30 µg (K), ampicillin

10 µg (AMP), tetracycline 30 µg (TE), cephalexin 30

µg (CL), azithromycin 15 µg (AZM), ciprofloxacin 5 µg

(CIP), cefixime 5 µg (CFM), chloramphenicol 30 µg

(C), rifampicin 5 µg (RD), nalidixic acid 30 µg (NA).

Statistical Analysis

Each category of street foods was taken three

times from the same vendor. Reported plate count

data represent the mean values obtained from three

individual trials, with each of these values being ob-

tained from duplicated samples. Data were subject-

ed to analysis of variance using the Microsoft Excel

program (Redmond, Washington DC, USA). Signifi-

cant differences in plate count data were established

by the least-significant difference at the 5% level of

significance.

RESULTS

In this study, thirteen different kinds of street food

including, singara, jhal-muri, chatpati, chetoi pitha,

sola, jilapi, drinking water, pickles, amra, tehari, veg-

etables roll, sugarcane juice and cucumber were

assessed for total aerobic bacterial populations,

total coliform and some specific food spoilage and

pathogenic bacteria.

Singara (also called samosa) is a deep fried pastry

with a savory filling, such as spiced potatoes, onions,

peas, lentils, ground lamb, ground beef or ground

chicken. The size, shape and consistency may vary,

but typically, they are distinctly triangular. Chatpati

is a type of snack. The main ingredients are boiled

yellow chickpeas and potatoes. It is spicy, savory

and tangy, combining the ingredients onion slices,

chili slices, egg slices, coriander leaves, tomato

slices, cucumber slices and tamarind sauce. In this

study, total aerobic bacterial populations and total

coliform populations were found to be greater than

the ICMSF standard limit in most of the street food

samples. The total aerobic bacterial and total coli-

form populations were the highest in the shingara

and chatpoti samples, and jar water and vegetable

rolls yielded the lowest levels of these bacteria. The

total aerobic bacterial populations and total coliform

populations are presented in Table 1.

Almost every sample contained different sero-

types of coliforms and after confirmation using API

20E diagnostic kits these strains were identified as

Enterobacter cloacae, E. aerogenes, Klebsiella oxyt-

oca, K. pneumoniae, Kluyvera spp., Citrobacter spp.,

Erwinia spp., Aeromonas spp., Pantoea spp., Serra-

tia odorifera, Raoultella ornithinolytica, R. terrigena,

Serratia odorifera.

Thirteen presumptive Salmonella spp. were iso-

lated from selective plates (Table 2), and after bio-

chemical, serological and API 20E confirmation tests,

only two isolates were confirmed as Salmonella

Choleraesuis (from chatpati and jilapi). Sola is boiled

bengal gram/chickpeas mixed with spice, onion

slices, green chili slices and boiled potato slices and

served on used newspaper or on a plate with salad.

From three street food samples (sola, vegetable roll

and cucumber), four presumptive pathogenic E. coli

serotype O157, O111 or O26 strains were detected

(Table-2) and after further investigation using bio-

chemical, serological and API 20E tests, these three

isolates were confirmed as either E. coli serotypes

O157, O111 or O26. Six E. coli other than O157,

O111, O26 serotype were isolated from five street

food samples including chetoi pitha, jilapi, tehari,

amra and cucumber (Table 2).

Fourteen presumptive Enterobacter skazakii

strains were isolated from singara, sola, chitoi pitha,

vegetable roll, sugarcane juice, and water samples

and after confirmation using API 20E kits, eight iso-

lates were confirmed as Enterobacter skazakii. Jhal

Muri is a savoury snack. It is made of puffed rice and

mixed with potatoes, onions, chili, chat masala. Oth-

er commonly used ingredients include slices of to-

matoes, onions and green chilies added to the base.

Chetoi pitha is baked ground rice or rice flour mixed

with salt and served with green chili paste or dried

fish paste. From ten street food samples (singara,


jhal-muri, chatpati, chetoi pitha, sola, drinking water,

amra, vegetables rolls, sugarcane juice and cucum-

ber), twenty presumptive Yersinia spp strains were

isolated and further confirmed by API 20E diagnostic

kits (Table 2). None of the isolated strains were con-

firmed as Yersinia enterocolitica. Jilapi is a type of

sweet and made by deep-frying a wheat-flour batter

in pretzel or circular shapes, which are subsequently

soaked in sugar syrup. The sweets are served warm

or cold. From 11 street food samples, 21 Enterococ-

cus spp were isolated, however, after confirmation

using API step. kits, only ten isolates were identified

as Enterococcus spp. that had originally been isolat-

ed from chitoi pitha, sola, jilapi, drinking water, sug-

arcane juice, and cucumber samples. These species

were identified as E. avium, E. solitaries, E. faecium

and E. faecalis. Except for achar and tehari, Listeria

spp. was identified from all other street food sample

tested. Fifteen Listeria spp were isolated and con-

firmed using API Listeria kits. Further identification

revealed that the isolated Listeria spp. were L. ivano-

vii, L. grayi, L. welshimeri, L. seeligeri and L. monocy-

togenes. From sola sample, the isolated strains were

determined to be L. monocytogenes. Twenty four

Staphylococcus spp. isolates were isolated from se-

lective plates of 39 street food samples (table-2) and

only ten isolates were confirmed as Staphylococcus

spp by API Staph kits. (singara, chatpati, muri, boiled

motor, jilapi, cucumber). The species of Staphylo-

coccus were found as S. lentus, S. xylosus, S. sciuri

and S. aureus. Staphylococcus aureus was found in

jilapi food samples only.

Table 1. Total aerobic bacterial and total coliform populations in street food samples*

Street food SamplesTotal Aerobic Bacterial population (log CFU/g)

Total Coliform Popula-tion (log CFU/g)

Singara 8.8 ± 0.02 7.5 ± 0.02

Muri 7.5 ± 0.05 5.0 ± 0.08

Chatpati 8.0 ± 0.06 5.8 ± 0.05

Chetoi pitha 7.2 ± 0.05 4.0 ± 0.01

Sola 7.5 ± 0.04 4.8 ± 0.09

Jilapi 4.9 ± 0.03 3.3 ±0.06

Jar water 3.0 ± 0.04 2.5 ± 0.08

Achar 6.5 ± 0.03 2.0 ± 0.01

Amra 6.3 ± 0.04 2.7± 0.04

Tehari 6.8 ± 0.05 2.0 ± 0.01

Vegetable Roll 5.7 ± 0.06 2.6 ± 0.05

Sugarcane juice 6.0 ± 0.04 5.1 ± 0.05

Slice Cucumber 6.2 ± 0.01 2.7 ± 0.01

* Results are expressed in average of three replicate samples ± SD, which were calculated from

duplicate plates.


Twenty three Bacillus spp. were isolated from 33

street food samples (Table 2) and twelve isolates

were identified as Bacillus spp. by API 50 CHB. These

isolates were identified as Bacillus atrophaeus, B.

globigii, B. licheniformis. No B. cereus strains were

detected throughout the study.

Fifteen Pseudomonas spp. were isolated from 39

street food samples, all Pseudomonas spp. were

confirmed by biochemical tests and API 20NE di-

agnostic kits. From 39 food samples, 24 Lactic acid

fermenting bacteria (LAB) were isolated. Different

species of LAB identified using API 50 CHL included

Lactobacillus brevis, L. pentosus, L. plantarum, L.

collinoides, L. salivarius, Lactococcus lactis, L. raf-

finolactis, Weissella confusa, Pediococcus pentosa-

ceus, Leuconostoc mesenteroiodes.

Table 2. Presence of pathogenic and spoilage bacteria on street food samples as detected on selective microbiological medium.

Number of bacterial isolates

Stre

et f

oo

d S

amp

les

Salm

one

lla s

pp

.

E.c

oli

O15

7,O

111,

O

26

E.c

oli

Co

lifo

rm

Cro

nob

acte

r sa

ka-

zaki

i

Yers

inia

sp

p.

Bac

illus

sp

p.

Stap

hylo

cocc

us s

pp

.

Pse

udo

mo

nas

spp

.

Ent

ero

cocc

us s

pp

.

List

eria

sp

p.

LAB

Singara (3)* 0 0 0 3 0 1 3 3 1 2 1 3

Muri (3) 1 0 0 3 2 1 1 3 1 1 1 3

Chatpati (3) 2 0 0 3 3 3 3 3 3 1 1 3

Pitha (3) 1 0 1 3 3 2 2 3 1 2 3 2

Boiled motor (3) 2 2 0 3 3 3 2 2 3 3 3 3

Jilapi (3) 2 0 1 3 1 0 2 1 1 3 2 1

Water (3) 2 0 0 2 0 3 2 1 3 3 1 1

Achar (3) 0 0 0 1 0 0 2 1 0 0 0 2

Amra (3) 0 0 1 0 0 1 2 1 0 1 1 1

Tehari (3) 0 0 1 2 0 0 1 1 0 0 0 0

Roll (3) 0 1 0 2 0 2 1 2 0 2 2 1

Sugarcane juice (3) 2 0 0 2 1 2 0 1 0 2 1 2

Cucumber (3) 1 1 2 2 1 2 2 2 2 1 2 2

*parenthesis: number of samples


Table 3. Antibiotic sensitivity pattern of the isolated bacteria

Isolates

Number of isolates resistant to antibiotics

C (3

0 µg

)

S (1

0 µg

)

CL

(30

µg)

VA (3

0 µg

)

AM

L (1

0 µg

)

CIP

(5 µ

g)

K (3

0 µg

)

AZM

(15

µg)

NV

(30

µg)

NA

(30

µg)

CN

(10

µg)

RD

(5 µ

g)

AM

P (1

0 µg

)

CFM

(5 µ

g)

TE (3

0 µg

)

E (1

5 µg

)

Salmonella spp. (2) 0 1 0 2 1 0 0 2 2 0 1 2 1 0 0 2

Escherichia coli other than O157, 0111, 026 (5)

0 1 1 1 1 0 0 1 1 0 1 1 1 1 1 1

Escherichia coli O157,0111,026 (3) 2 3 2 3 2 3 2 3 3 2 0 3 3 0 3 3

Enterobacter sakazakii (8) 0 3 3 4 4 0 0 5 5 0 1 4 4 1 0 4

Klebsiella spp. (5) 0 4 1 5 4 0 0 5 5 0 0 5 4 0 0 5

Enterobacter cloacae (11) 3 6 3 7 6 0 1 6 7 2 1 7 6 1 2 7

Enterobacter aerogenes (9) 1 4 3 6 5 0 1 5 6 0 1 6 5 0 1 6

Erwinia spp. (7) 2 2 5 6 5 0 3 4 6 3 1 6 6 2 4 6

Aeromonas spp. (5) 2 3 3 3 3 0 2 3 3 2 2 3 3 2 3 3

Kluyvera spp. (4) 1 2 1 2 1 0 1 2 2 2 0 2 1 1 2 2

Serratia spp. (6) 4 2 3 5 5 1 2 5 5 2 0 5 5 1 1 5

Pantoea spp.(6) 3 1 2 4 3 1 1 3 4 2 0 3 3 2 2 5

Citrobacter spp. (7) 1 5 1 6 6 0 0 5 6 1 0 6 5 2 0 6

Raoultella spp.(7) 0 3 2 5 4 0 1 5 5 0 1 5 4 0 0 5

Enterococcus spp. (10) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Yersinia spp. (20) 4 6 13 17 9 8 15 12 8 7 11 2 4 18 3 6

Bacillus spp. (12) 0 4 2 4 0 1 4 6 5 8 4 3 0 8 3 3

Staphylococcus spp.(10) 0 3 1 3 0 0 2 5 3 8 2 1 0 6 1 2

Pseudomonas spp. (15) 10 9 10 10 10 0 10 9 10 10 0 10 10 10 9 10

Listeria spp.( 15) 10 12 11 13 10 2 5 13 12 15 11 8 11 15 13 12

Lactobacillus spp. (6) 5 6 6 6 5 5 6 4 5 6 6 6 6 6 6 5

Weissalla spp. (8) 3 8 4 8 4 3 7 3 3 8 4 7 4 8 3 3

Lactococcus spp. (7) 7 7 5 7 7 5 7 6 7 7 7 7 7 7 7 5

Pediococcus spp. (2) 0 1 0 0 0 0 0 0 0 1 1 1 0 1 0 0

Leuconostoc spp. (1) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

*parenthesis: number of isolates


Antibiotic sensitivity pattern of Pseudomonas

spp. isolates revealed that these strains were resis-

tant to multiple antibiotics (minimum 10 antibiotics

and maximum 14 antibiotics) (Table 3). However, an-

tibiotic sensitivity patterns of Enterococcus isolates

revealed that these strains were not resistant to any

of the 16 antibiotics tested (Table 3). In contrast, iso-

lated Enterococcus spp. was sensitive to vancomycin

(30 µg/milliliter). Antibiotic sensitivity pattern of E.

coli O157, Salmonella Listeria spp and Yersinia spp

isolates revealed that these strains were resistant

to multiple antibiotics (minimum 4 antibiotics and

maximum 12 antibiotics) (Table 3). Furthermore, an-

tibiotic sensitivity patterns of LAB isolates revealed

that these strains were resistant to multiple antibiot-

ics (minimum 9 antibiotics and maximum 16 antibiot-

ics) (Table 3).

DISCUSSION

Food borne illnesses of microbial origin are a

major health problem associated with street foods

(Kaneko et al., 1999; Mensah et al., 1997, 2001, 2002).

The traditional processing methods that were used

in the preparation, inappropriate holding tempera-

ture and poor personal hygiene of food handlers are

some of the main causes of contamination of street

foods (Barro et al., 2006; Mensah et al., 2002). In ad-

dition the foods were not effectively protected from

flies and dust (Bryan et al., 1997; Bryan et al., 1992).

In Bangladesh, street foods are mostly prepared and

processed manually and sold to the public at various

lorry terminals, by the roadside or by itinerant ven-

dors (Mensah et al., 2002). Researchers have inves-

tigated the microbiological quality of street vended

foods in different countries and high bacterial counts

and a high incidence of food borne pathogens in

such foods typically have been reported (Jayasuriya,

1994; Mosupye and Von, 2000; Kubheka et al., 2001;

Hanashiro, 2005; Tendekayi et al., 2008). In this study,

higher than the ICMSF recommended (less than 106

CFU/g) levels of aerobic bacterial populations and

coliform bacterial (less than 11 CFU/g) populations

were observed in most of the samples tested. More-

over potential pathogenic bacteria including E. coli

O157, O111 or O26, Salmonella, Listeria monocy-

togenes, Staphylococcus aureus were detected in

some street food samples. These findings demon-

strate that street foods sold in Dhaka constitute a like-

ly potential hazard to human health. The presence of

higher numbers of Enterobacteriaceae in street food

samples that were cooked or deep oil fried appears

to be a good indicator of post-processing contami-

nation. Contamination of food by enteric pathogens

can occur from inadequate cooking or use of con-

taminated water during preparation and processing,

or improper washing and handling or lack of hygiene

of the vendors. Sometimes the source of the food or

water may also be contaminated. Therefore, access

to running water and health education to the ven-

dors on personal hygiene, food safety and proper

disposal of waste would improve food quality there-

by reducing food borne incidences.

CONCLUSIONS

Of the samples analyzed, almost all the street

foods were found to be heavily contaminated with

coliforms, fecal coliform bacteria and other patho-

gens. Therefore, the inspection authorities are re-

quired to take the necessary steps to make these

products safe for consumers. There are alarming

levels of multi drug resistant (MDR) pathogens pres-

ent in some street food samples. This is a great con-

cern for human health and the regulatory agencies

should take the necessary measures to improve the

food hygiene conditions of street food. The deep

fried street food including shingara, samocha and

jilapi were found microbiologically safe when served

immediately. However, the holding bowls and the

Table 3 Abbreviation: chloramphenicol(C), streptomycin (S), cephalexin (CL), vancomycin (VA), amoxicillin

(AML), ciprofloxacin (CIP), kanamycin (K), azithromycin (AZM), novobiocin (NV), nalidixic acid (NA) gentamicin

(CN), rifampicin (RD), ampicillin (AMP), cefixime (CFM), tetracycline (TE), erythromycin (E).


adjacent areas including the vendor’s personal hy-

giene were not found suitable for providing food

that would be considered safe as these products

were found to be moderately contaminated with

coliforms, fecal coliforms and other pathogenic

bacteria. However, the raw sugercane juice and raw

fresh-cut fruits were contaminated with fecal coli-

forms and other pathogens and pose serious health

problem; because these items are usually eaten raw.

Street foods pose risks of both a physical and chemi-

cal nature given that they are exposed to the road

side and traffic pollution caused from different kinds

of vehicles. It is necessary to investigate the physi-

cal and chemical contamination of the street food

samples and there is a need to develop awareness

in order to avoid physical and chemical hazards.

Street food vendors practiced minimal hygienic and

sanitary practices. The hygienic practices in ques-

tion included food preparation, handling of uten-

sils; a place for food preparation, personal hygiene

and methods of storing cooked food. Due to lack

of proper knowledge and guidance on street food

vending, vendors prepared their foods in explicitly

unhygienic and unsanitary conditions.

Improving the safety of street-vended foods re-

mains a tremendous challenge. The research data

presented here suggested the need of every vendor,

helper or food handler to undergo basic training

in food hygiene before being involved in food re-

tail as street vendors. The food inspectors role is to

ensure that these vendors follow the required rules

for proper hygiene and sanitation. Mass awareness

using electronic media, food hygiene trainers train-

ing, regular monitoring, and Hazard Analysis and

Critical Control Point (HACCP) control measures

should be developed in reducing the safety of street

foods. Nevertheless, the HACCP system is the most

cost-effective approach for assuring food safety at

all stages of the food supply. It will enable the sys-

tematic identification of potential hazards and their

control measures. A HACCP approach also provides

guidance in the selection of enforcement and edu-

cation priorities, rather than general sanitation and

superficial improvements.

ACKNOWLEDGEMENTS

The authors would like to thank Mr. Arafat-al-

Mamun for technical assistance, and Mr. Harun-ur

Rashid and Ms. Emon Sharmin for the laboratory as-

sistance required to complete this task. The authors

would also like to thank the United Nations Univer-

sity, Tokyo, Japan (UNU-ISP) for financial support in

this work.

REFERENCES

Barro, N., A. Bello, S. Aly, C. Ouattara, A, Ilboudo,

and A. Traoré. 2006. Hygienic status and assess-

ment of dishwashing waters, utensils, hands, and

pieces of money from street food processing sites

in Ouagadougou, Burkina Faso. African J. Biotech.

5:1107-1112.

Bryan, F., M. Jermini, R. Schmitt, E. Chilufya, M.

Mwanza, A. Matoba, E. Mfume, and E. Chibiya.

1997. Hazards associated with holding and reheat-

ing foods at vending sites in a small town in Zam-

bia. J. Food Prot. 60:391-398.

Bryan, F., P. Teufel, S. Riaz, S. Roohi, F. Qadar, and

Z. Malik.1992. Hazards and critical control points

of street-vending operations in a mountain resort

town in Pakistan. J Food Prot. 55:701-707.

Kaneko, K., I. Hayashidani, O. Hideki, and K. Yoshim-

itsu. 1999. Bacterial contamination of ready-to- eat

foods and fresh products in retail shops and food

factories. J. Food Prot. 62:644-649.

FAO. 2012. National food safety policy and seminar

entitled Towards a National Food Safety and

Quality Policy - a key note speeches. Available at

http://bdfoodsafety.org/news.php?NewsId=22,

accessed on May 19, 2013.

FAO. 2010. Institutionalization of healthy street food

system in Bangladesh: A pilot study with three

wards of Dhaka City Ccrporation as a model”. Fi-

nal Report PR #7/07. Pages 1-93.

Hanashiro, A., M. Morita, G. R. Matte, M. H. Matte,

and E. A. F. S. Torres. 2005. Microbiological qual-

ity of selected street foods from a restricted area

of Sao Paulo city, Brazil. Food Control 16:439–444.


Jayasuriya, D.C. (1994). Street food vending in Asia:

Some policy and legal aspects. Food Control

5:222-226.

Kubheka, L.C., F.M. Mosupye and H. A. Von 2001.

Microbiological survey of the food vended salad

and gravy in JHB, South Africa. Food Control

2:127-131.

Mensah, P., M. Amar-Klemesu, A. Hammond, and A.

Haruna., .2001. Bacterial contamination on lettuce,

tomatoes, beef and goat meat from metropolitan

Accra. Ghana Medical Journal 35:1-6.

Mensah, P., K. Owusu-Darko D. Yeboah-Manu, A.

Ablordey, F. Nkrumah, and H. Kamiya, 1997. The role

of street vended foods in the transmission of enteric

pathogens. Ghana Medical Journal 33:19-29.

Mensah, P., D. Yeboah-Manu, K. Owusu-Darku, and

A. Ablordey. 2002. Street food in Accra, Ghana:

how safe are they? Bulletin of the World Health

Organization 80:546-556.

Mosupye F.M. and H. A. Von. 2000. Microbiological

hazard identification and exposure assessment of

street food vending in JHB, South Africa. Int. J.

Food Microbial. 61:137-145.

Tambekar, D., V. Jaiswal, D. Dhanorkar, P. Gulhane,

and M. Dudhane. 2008. Identification of micro-

biological hazards and safety of ready-to-eat food

vended streets of Amravati City, India. J. Appl.

Biosci. 7:195 - 201.

Tendekayi, H. G., K. S. Bernard, M. Cabinet, and C.

Dombo. 2008. The microbiological quality of infor-

mally vended foods in Harare, Zimbabwe. Food

Control 19:829-832.

Vidal, R., M. Vidal, R. Lagos, M. Levine and V. Prado.

2004. Multiplex PCR for diagnosis of enteric infec-

tions associated with diarrheagenic Escherichia

coli. J. Clin. Microbiol. 42:1787-1789.




ABSTRACT

Lipids are often used as substrates when measuring growth and lipolytic activity of lipase-producing

bacteria, however, the introduction of non-miscible lipid substrates into aqueous in vitro culture systems

is problematic for generating accurate and consistent results. The objective of the present study was to

develop a digesta-free method for culturing and assaying lipolytic activity of mixed as well as pure popu-

lations of known ruminal lipase-producing bacteria. Accordingly, the inclusion of 0, 11, and 21 g of glass

beads as a solid support matrix in place of digesta was examined. Results showed a significant increase (P

< 0.05) in rate of lipolysis in the incubations containing 11 or 21 g glass beads compared to the non bead

incubations. Activity was also increased (P < 0.05) in a separate study when tubes containing beads were

incubated horizontally rather than vertically. These results indicate that glass beads are a suitable substitute

for rumen digesta when examining lipolytic activity of mixed rumen cultures in vitro. When tested against

pure cultures of the ruminal lipase-producing bacteria Anaerovibrio lipolyticus 5s, Butyrivibrio fibrisolvens

49, Propionibacterium avidum and acnes, addition of glass beads did not significantly increase rates of

free fatty acid release; however, results showed that there was substantial variation between triplicate sets

incubated without glass beads. Standard deviations suggest that the use of glass beads had a tendency

to reduce variability within triplicate sets. Thus, inclusion of glass beads provides a clean and consistent

incubation system for examining lipase activity in vitro.

Keywords: lipolysis, lipase, glass beads, digesta, support matrix, lipid, rumen, enzyme, interfacial activation, microbes

Correspondence: Robin C. Anderson, [email protected]:+1-979-260-9317 Fax:+1-979-260-9332

Development of Non-Forage Based Incubation System For Culturing Ruminal Lipase-Producing Bacteria In Vitro‡

H. D. Edwards1, R. C. Anderson2*, T. M. Taylor1, R. K. Miller1, M. D. Hardin3, N. A. Krueger2, D. J. Nisbet2

1Texas A&M University, College Station, TX 2USDA/ARS, Southern Plains Agricultural Research Center, College Station, TX

3IEH Laboratories & Consulting Group, Lake Forest Park, WA

‡Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does

not imply its approval to the exclusion of other products that may be suitable.



INTRODUCTION

Currently there is a need for the advancement and

development of methods for culturing ruminal lipase-

producing bacteria in order to gain an enhanced in-

sight of the functional role(s) these organisms have

in the rumen. The lipid substrates generally used to

study these bacteria are problematic due to their un-

even dispersion when added to water based media,

generally leading to inconsistent results. Ruminant-

produced foods contain high proportions of saturat-

ed fats, a result of microbial biohydrogenation within

the rumen which rapidly saturates and thus limits the

availability of free unsaturated fatty acids for absorp-

tion and assimilation (Harfoot and Hazlewood, 1997).

Biohydrogenation of mono- or polyunsaturated fatty

acids by ruminal microbes cannot occur unless free

fatty acids (FFA) are first hydrolyzed from their lipid

precursors, a process known as lipolysis.

As reviewed by Lourenço and colleagues (2010)

the ability of ruminal microbes to hydrolyze triglyc-

erides was reported more than 50 years ago (Garton

et al., 1958). Since then, numerous studies have been

conducted to characterize the biological and physi-

cal factors affecting ruminal lipolysis by mixed or

pure populations of ruminal bacteria. For instance,

Hawke and Silcock (1970) have shown that more than

50% of ruminal lipase activity is contained within the

particulate fraction of freshly collected ruminal fluid.

It has been recognized that the enzymatic activity of

lipases is markedly increased in environments that

stabilize the lipid/water interface that occurs at the

point of contact between oil and water (Paiva et al.,

2000). Thus, studies using rumen contents as incu-

bation materials likely provided a solid support that

served to stabilize the lipid/water interface, a phe-

nomenon referred to as interfacial activation (Rao

and Damodaran, 2002). A major limitation to studies

conducted with particulate matter and digesta is that

these materials are not homogenous in size or mi-

crobial composition, which can lead to considerable

variation and experimental error during incubation.

The main objective of this study is to examine the

use of glass beads as a solid support matrix at vary-

ing levels (0, 11, and 21 g) in vitro to determine if they

would serve as an acceptable replacement for rumen

digesta. We hypothesize that the glass beads will in-

crease lipase activity to a greater extent than digesta

by providing a more homogenous support matrix

allowing a consistent dispersion of the oil substrate

and therefore improved interaction between lipase

and oil substrate.


Mixed culture handling procedures

The mixed bacterial populations used in this study

were obtained from fresh rumen contents collected

from a cannulated cow grazing on predominantly

ryegrass (Lolium multiflorum Lam.) pasture. Rumen

fluid and digesta were separated by straining though

a nylon paint strainer (Leyendecker et al., 2004) into

separate pre-warmed insulated containers that had

been flushed with warm water prior to sample col-

lection. The containers were filled completely (ap-

proximately 500 mL), capped and transported to the

lab. Upon arrival at the laboratory, CO2 was bubbled

through the rumen fluid to keep it in an anaerobic

state until its use as a source of bacterial inoculum

(within 30 min of collection). The digesta was kept in

its closed container until distributed under a continu-

ous stream of CO2 to its respective incubation tubes.

The cow was cared for according to procedures ap-

proved by U.S. Department of Agriculture – Agricul-

tural Research Service (USDA-ARS) Southern Plains

Agricultural Research Center’s Animal Care and Use

Committee (Protocol #2010005).

Media preparation

Mixed bacterial populations in fresh ruminal fluid

were cultured in a standard rumen fluid medium con-

taining per liter: 100 mL clarified rumen fluid (Hespell

and Bryant, 1981), 22.5 mg each of K2HPO4 and 22.5

mg KH2PO4 (JT Baker, Mallinckrodt Baker Inc., Phil-

lipsburg, NJ), 45.0 mg (NH4)2SO4, 45.0 mg NaCl, 4.5

mg MgSO4.7H2O, 4.5 mg CaCl2.6H2O and 22.5 mg

CaCl2; 1.0 mL of 0.1% resazurin, 4,000 mg NaHCO3


and 500.0 mg cysteine hydrochloride (JT Baker). For

cultivation of pure cultures of lipase-producing bac-

teria, a standard pure culture medium was prepared

and contained per liter: 292 mg each of K2HPO4 and

KHPO4, 480 mg (NH4)2SO4, 480 mg NaCl, 100 mg

MgSO4•7H2O, 64 mg CaCl2•2H20, 4,000 mg Na2CO3,

600 mg cysteine-HCl, 10 g trypticase (BBL Microbiol-

ogy Systems, Cockeysville, MD), 2.5 g yeast extract

(Difco; Becton, Dickinson and Company, Sparks,

MD), branched-chain fatty acids (1 mmol each of iso-

butyrate, isovalerate, and 2-methylbutyrate), 15.0 mL

of 2.0% glucose stock dissolved in water, hemin, vita-

min mix (20 mg each thiamine, pantothenate, nico-

tinamide, pryridoxine HCl, riboflavin, 1 mg p-amino-

benzoic acid, 0.5 mg biotin, 0.5 mg folic acid, 0.2 mg

vitamin B-12, and 0.5 mg lipoic acid) and trace miner-

als (Cotta and Russell, 1982). All chemicals were pur-

chased from Sigma-Aldrich (Milwaukee, WI) unless

otherwise noted. Media were prepared by boiling to

remove dissolved O2 and then saturated with O2-free

CO2 gas by cooling on ice while under a continuous

flow of 100% CO2. The cooled media were distribut-

ed (6 mL/tube unless otherwise specified) using the

anaerobic Hungate technique as described by Bry-

ant (1972) into 18 x 150 mm glass tubes pre-loaded

with 0, 11, or 21 g soda lime glass beads as indicated

(Fisher Scientific, Pittsburgh, PA) and with 0.1 mL ol-

ive oil for the standard rumen fluid medium and 0.2

mL olive oil for the standard pure culture medium.

The tubes were immediately closed with crimp tops.

After sterilization, the tubes were cooled and stored

at room temperature until inoculation.

Determination of lipolytic activity of mixed rumen bacterial populations in the presence of varying levels of glass beads

Two separate triplicate sets of incubation tubes

for each treatment (0, 11 or 21 g glass beads) were

inoculated (1 mL/tube) with freshly collected ruminal

fluid. One set of tubes for each treatment was col-

lected immediately after inoculation to serve as 0 h

controls and the other set was collected after 48 h

incubation at 39°C while being agitated at 40 rpm in

an Innova™ 4000 – incubator shaker. Zero h controls

were used to determine presence of FFA that may

have been introduced during inoculation, and pro-

vide for proper baseline correction for subsequent

FFA analyses. Biological activity was terminated im-

mediately upon collection of tubes by the addition

of 0.5 mL of concentrated (37%) HCl. Lipolytic activ-

ity was determined by extraction and colorimetric

measurement of FFA accumulation using methods

described by Kwon and Rhee (1986). All subsequent

studies herein described used the same incubation

procedures and measurement of FFA accumulation

unless otherwise specified.

Comparison of incubation orientations and their effects on enzyme activity

Differing tube incubation orientations were com-

pared to identify tube orientation most conducive

to obtaining the highest rate of lipolytic activity.

Standard rumen fluid medium was prepared as pre-

viously described and 6.0 mL distributed to tubes

preloaded with 21 g of glass beads and 0.1 mL olive

oil. Following sterilization, two sets of tubes for each

tube orientation were inoculated with 1.0 mL freshly

collected rumen fluid and then incubated horizon-

tally or vertically under 100% CO2 for 0 or 48 h while

agitating at 40 rpm. Concluding incubation, biologi-

cal activity was stopped and accumulation of FFA

measured.

Comparison of glass beads as a support matrix versus rumen digesta

Five g of freshly collected and squeezed (to elimi-

nate residual rumen fluid) rumen digesta or 21 g

sterile glass beads were added to sterile 18 x 150

mm glass tubes preloaded with 0.1 mL olive oil.

Tubes were then each inoculated with 6 mL of freshly

collected, strained ruminal fluid. Transfer of digesta

and ruminal fluid were done while flushing with 100%

CO2. All tubes were subsequently closed with rubber

stoppers and then collected as either 0 h controls

(three each of tubes prepared with digesta or glass

beads) or after 48 h of horizontal incubation at 39°C

while being agitated at 40 rpm. Immediately upon


collection, biological activity was terminated and ac-

cumulation of FFA was measured.

Free fatty acid accumulation by pure cultures of ruminal lipase-producing bac-teria in the presence or absence of glass beads

Pure cultures of Anaerovibrio lipolyticus 5s and

Butyrivibrio fibrisolvens 49 were obtained from Dr.

Jay Yanke, Agriculture-Agri Food Canada. Strains of

Propionibacterium avidum and Propionibacterium

acnes were previously isolated from the rumen of a

pastured cow (Krueger et al., 2008). For long-term

preservation of pure cultures, bacteria were stored

in 20% anaerobic glycerol at -80ºC. Upon removal

from storage, each bacterial isolate was revived by

two consecutive 24 to 48 h culture transfers, each in

10 mL standard pure culture medium supplemented

with 2.0% pre-sterilized olive oil. Revived cultures

were each inoculated (0.2 mL) into four triplicate

sets of 18 x 150 mm crimp top tubes containing 6 mL

standard pure culture medium plus 0.2 mL added ol-

ive oil and either no (2 sets of triplicate tubes) or 21

g of glass beads (other two sets of triplicate tubes).

Immediately following inoculation two sets of the

triplicate tubes from each bacterial isolate, one set

with and the other without added beads, were acidi-

fied with 0.5 mL of concentrated (37%) HCl to stop

growth and enzyme activity prior to each incubation

series, thus serving as 0 h controls. The remaining

sets of tubes were incubated horizontally for 48 h un-

der above-described conditions and accumulation

of FFA measured.

Statistical analysis

Tests for the effects of the different treatments

were done using a general analysis of variance

(ANOVA) (Statistix v.9.0, Analytical Software, Talla-

hassee, FL). Significant differences between means

were separated and identified by least squares dif-

ferences (LSD) analysis (P < 0.05).

RESULTS

Lipolytic activity of mixed rumen bacte-rial populations in the presence of varying levels of glass beads

When mixed ruminal populations were tested dur-

ing batch culture, a main effect (P = 0.0048) of bead

inclusion was observed on rates of FFA release after

48 h culture. For instance, rates of FFA accumulation

(mean ± SD) were higher (P < 0.05) for broth cultures

grown in tubes containing 11 g of glass beads com-

pletely immersed (88.59 ± 12.93 nmol/mL per h) or

21 g of glass beads just barely covered by the menis-

cus of the broth in the upright tube (174.34 ± 64.48

nmol/mL per h) than for cultures grown in broth with-

out added glass beads (4.49 ± 7.77 nmol/mL per h).

Comparison of reaction tube orienta-tion and its effect on enzyme activity ob-served during incubation

Different tube orientations were compared during

incubation of mixed culture containing 21 g glass

beads (Figure 1, Study 1). There was approximately a

9.5 fold increase in the observed rate of FFA release

by ruminal microorganisms incubated in horizontally

oriented reaction tubes as compared to microbes

incubated in vertically oriented tubes. Mean rates

of FFA release significantly increased from approxi-

mately 12.39 nmol/mL per h to approximately 130.54

nmol/mL per h when tubes were incubated horizon-

tally as compared to vertically (P < 0.05).

Lipolytic activity during incubation of strained rumen fluid containing either glass bead or digesta

The inclusion of glass beads or rumen digesta was

compared to determine if the use of glass beads

would prove to be sufficient to replace rumen di-

gesta as a support matrix for ruminal-lipase pro-

ducing bacteria. Results show that FFA release by

mixed bacterial populations in freshly collected and

strained ruminal fluid was higher (P < 0.05) following


48 h incubation on a bed of glass beads than on a

bed of fresh squeezed rumen digesta (Figure 1,

Study 2). Mean rates of FFA release significantly in-

creased from approximately 90.32 nmol/mL per h to

approximately 336.36 nmol/mL/h when tubes were

incubated with glass beads as compared to rumen

digesta (P < 0.05).

Growth of ruminal lipase-producing bacteria in pure culture incubated in the presence and absence of glass beads

Glass beads as a support matrix for enzyme ac-

tivity was used to characterize major contributors

to ruminal lipolytic activity from amongst tested

bacterial isolates. Results showed that the presence

or absence of glass beads did not (P > 0.05) affect

lipolytic activity of A. lipolyticus 5s, B. fibrisolvens

49, P. avidum, and P. acnes (Table 1). Nevertheless,

although inclusion of glass beads did not result in

statistically significant differences in observed FFA

release amongst tested lipase-producing microor-

ganisms, the presence of the glass beads did result

in numerically higher rates of FFA release for all of

the bacterial organisms tested with the exception

of P. avidum. The variability in rate measurements,

whether expressed as standard deviations or as the

coefficient of variation, also were numerically higher

for tubes not containing glass beads as compared to

the glass bead treatments, with the exception of B.

fibrisolvens 49 (Table 1).

DISCUSSION

It is recognized that the insolubility of lipid sub-

strates and the lack of interfacial activation are major

limitations to the study of lipolytic enzymes in aque-

ous media. For instance, lipolytic activity by mixed

populations of ruminal microbes is known to be in-

0

50

100

150

200

250

300

350

400

450

HorizontalIncubation

VerticalIncubation

Beads (21 g) Digesta (5 g)

Acc

umul

atio

n o

f Fr

ee F

atty

Aci

ds

(nm

ol/

mL

per

h)

a

b

b

a

Study 1 Study 2

Figure 1. Comparison of rates of FFA accumulation by mixed populations of ruminal microbes incubated 48 h at 39°C in two separate incubation studies. Study 1, culture tube sets were incubating vertically or horizontally containing 21 g glass beads and 6.0 mL mixed culture rumen fluid; Study 2, culture tube sets were incubating with 5.0 g rumen digesta or 21.0 g glass beads (each occupying approximately the same volume within the tubes) in the presence of 6.0 ml of mixed culture rumen fluid; values are means ± standard deviation (n = 3). Unlike letters indicate that means differ (P < 0.05).


Tab

le 1

. Co

mp

aris

on

of

rate

s o

f FF

A a

ccum

ulat

ion

and

ass

oci

ated

var

iab

ility

by

pur

e cu

ltur

es o

f lip

ase-

pro

duc

ing

ru

min

al m

icro

bes

incu

bat

ed w

itho

ut a

nd w

ith

add

itio

n o

f g

lass

bea

ds.

a Val

ues

dep

ict

leas

t sq

uare

mea

ns ±

SD

cal

cula

ted

fro

m c

ultu

res

incu

bat

ed in

trip

licat

e. C

oef

ficie

nt o

f var

iatio

n (C

V) i

s p

rese

nted

with

in p

aren

-th

esis

. bC

ultu

res

wer

e in

cub

ated

in 1

8 x

150

mm

crim

p t

op

cul

ture

tub

es c

ont

aini

ng 6

mL

stan

dar

d a

naer

ob

ic m

ediu

m (1

00%

CO

2) w

itho

ut o

r w

ith 2

1 g

g

lass

bea

ds

and

0.2

mL

add

ed o

live

oil

at 3

9°C

for

48 h

with

co

nsta

nt a

gita

tion

(40

rpm

).

Rat

e o

f FF

A a

ccum

ulat

ion

(nm

ol/

mL

per

h (C

V)a )

Ave

rag

e co

effic

ient

of

varia

tion

Incu

bat

ion

cond

i-tio

nsb

Ana

ero

vib

rio li

po

lytic

us

5sB

utyr

ivib

rio fi

bris

olv

ens

49Pr

op

ioni

bac

teriu

m a

vid

umPr

op

ioni

bac

teriu

m

acne

s

With

out

bea

ds

86.7

8 ±

15.

50 (1

8%)

89.9

8 ±

27.

08 (3

0%)

797.

98 ±

237

.31

(30%

)54

.87

± 2

9.69

(54%

)33

%

With

gla

ss b

ead

s10

7.52

± 0

.85

(1%

)16

9.43

± 4

9.57

(29%

)42

2.22

± 5

9.91

(14%

)47

.05

± 1

4.84

(36%

)19

%

P va

lue

0.08

170.

0715

0.05

600.

7043


creased when using rumen microbes associated with

digesta or added forage substrate than when using

strained ruminal fluid alone (Dohme et al., 2003; Gar-

ton et al., 1958; Hawke and Silcock, 1970; Krueger et

al., 2010; Shorland et al., 1955; Van Nevel and De-

meyer, 1995). However, the heterogeneous makeup

of these contents introduces considerable variability

into the conduct of such studies as differences in par-

ticle size, chemical composition, stage of digestion

and microbial colonization can markedly affect the

amount of surface area available for contact with the

lipid substrate. For instance, Krueger et al. (2010) re-

ported rates of ruminal lipolysis to be approximately

5,060 nmol FFA liberated/g of undiluted rumen con-

tents per h during a 24 h incubation of 5 g freshly

collected rumen digesta with 0.5 g added olive oil.

Conversely, based on estimates of amounts of lipid

degraded following 24 h incubation of 25 mL freshly

collected strained ruminal fluid (lacking particle-

associated bacteria), with 0.4 g ground forage and

0.125 g added soy oil, approximately 640 nmol FFA

would have been liberated/mL of rumen contents

per h (Dohme et al., 2003). In contrast, based on ac-

cumulations of FFA reported by Van Nevel and De-

meyer (1995) during a 6 h incubation of 10 mL freshly

collected and filtered rumen fluid diluted with 50 mL

buffer containing 0.5 g of a ground concentrate diet

and 0.08 g soy oil, the rate of lipolysis was calculated

to be approximately 170 nmol FFA/mL per h.

In attempt to reduce the variability of FFA lib-

eration observed between the studies, the present

study examined the use of glass beads in several

experiments as a potential replacement for rumen

digesta. Glass beads serve as a more homogenous

support matrix and thus it would be reasonable to

hypothesize that they would provide a more consis-

tent dispersion of lipids in aqueous media than the

heterogeneous, rumen digesta. When mixed ruminal

populations were tested during batch culture, rates

of FFA release after 48 h incubation were 20- and 39-

fold higher (P < 0.05) for cultures incubated in tubes

where the broth medium was in contact with a bed

of glass beads (11 and 21 g beads, respectively) ver-

sus cultures grown in broth medium not containing

beads (4.49 ± 7.77 nmol/mL per h). Results from the

present study demonstrated that rates of FFA accu-

mulation during incubation of 1 mL freshly collected

rumen fluid in 6 mL of a standard aqueous medium

supplemented with 0.1 mL olive oil were lower than

previous research discussed above.

It is possible that the glass beads may provide a

solid support matrix that promotes secretion of the

extracellular lipases by microorganisms growing in

medium by providing surfaces for microbial attach-

ments and/or lipid adsorption. Thus the glass beads

may allow microorganisms and lipids to come into

proximity of one another and their subsequent in-

terfacial activation but also provide an environment

conducive to the growth of lipase-producing bacte-

ria by allowing for their attachment. In support of this

Martinez and Nudel (2002) demonstrated that secre-

tion of lipase produced by Acinetobacter calcoaceti-

cus was stimulated by glass beads. Similarly, adsorp-

tion of lipases to siliconized or hydrocarbon-coated

glass beads has been used to cause interfacial acti-

vation in a variety of lipases (Fernandez-Lafuente et

al., 1998; Ferrato et al., 1997).

Rates of FFA release were also higher for cultures

grown in tubes where beads (11 g) were completely

immersed within the broth medium than for cultures

grown in broth alone. This result was unexpected

because approximately 2 cm of aqueous medium,

on which most but not all of the added oil floated,

remained above the bed-level for tubes where the

beads were completely immersed. This result pro-

vides further support that even though the glass

beads were not necessarily in contact with the oil

their contact with the bacteria may have provided

an environment optimal for bacterial growth and/or

lipase secretion as discussed prior.

Different tube orientations were compared dur-

ing incubation and tubes were agitated in attempt

to increase contact between the medium and lipid

substrate, and microorganisms to increase rates of

observed lipolytic activity. Tube sets containing 21 g

of glass beads were incubated vertically or horizon-

tally and results showed that there was an increase

(P < 0.05) in rates of FFA accumulation (nmol/mL per

h) in reaction tubes incubated horizontally (Figure

1, Study 1). It is likely that the horizontal incubation


provided a greater surface area, free movement of

enzyme and dispersion of oil allowing for increased

interaction resulting in the higher rates of FFA re-

lease for horizontally incubated tubes.

Glass beads and rumen digesta were directly com-

pared by examining both matrices in liquid medium

during incubation. Lipolytic activity was significantly

higher (P < 0.05) for mixed populations of ruminal

microbes incubated in liquid medium containing

glass beads as compared to those incubated in me-

dium containing rumen digesta (Figure 1, Study 2).

The rates of FFA release observed in samples con-

taining digesta-supported microbes in this study

were comparable to those reported by Van Nevel

and Demeyer (1995), thus demonstrating that the

glass beads served as a sufficient replacement for

rumen digesta.

The glass beads were also used in this study to de-

termine their applicability for use in supporting the

characterization of in vitro lipase activity from pure

cultures of ruminal lipolytic microorganisms. Regard-

ing the bacteria used in this study, Anaerovibrio lipo-

lyticus and Butyrivibrio fibrisolvens have long been

recognized as important contributors to ruminal li-

polysis. Propionibacterium avidum and P. acnes are

also known to express lipase activity, though less

is known regarding their contribution to ruminal li-

polysis. Each of the different bacterial strains were

cultured individually in the presence and absence of

glass beads for 48 h. Different from ruminal mixed

cultures, the presence or absence of beads did not

influence (P > 0.05) observed lipase activity by any

of these bacterial strains although the rate of FFA

accumulation by P. avidum was numerically higher

when cultured without beads (Table 1). However, the

standard deviation for the P. avidum cultures incu-

bated without the glass beads was quite high which

lessens the level of confidence in the measured rate.

Conversely, the variability was numerically lower for

the bead than the non bead treatments for all the

bacteria except B. fibrisolvens 49 and coefficients

of variation were numerically lower for all strains.

When averaged across all four strains, the coefficient

of variation was 42% lower for the bead treatments

than for the non bead treatment.

Henderson (1971) demonstrated that the lipase of

A. lipolyticus 5s appears in the medium early in the

life of the culture and the lipase activity was not asso-

ciated with the bacterial cell or fragmented bacteria.

Similar to A. lipolyticus 5s, the lipase for P. avidum

and P. acnes have been shown to also be produced

extracellularly (Greenman et al., 1983). Furthermore,

one function of the lipase secreted by P. acnes has

been shown to possibly aid in colonization, by pro-

moting cell adherence to components such as oleic

acid (Gribbon et al., 1993), thus the glass beads likely

aid in increasing this adhesion and contact with the

oil substrate. However, B. fibrisolvens, differs from

the extracellular lipase-producing rumen bacterium

A. lipolyticus 5s, P. avidum, and P. acnes in that they

produce esterases exhibiting lipase activity that are

cell bound (Lanz and Williams, 1973). The produc-

tion of cell bound esterases instead of an extracel-

lular lipase may suggest that B. fibrisolvens does not

require a support matrix due to the esterases being

already supported by the cell itself. This is consis-

tent with results in Table 1, showing that unlike the

other bacteria the standard deviation is higher for

B. fibrisolvens 49 with the bead treatment than the

fluid treatment. This suggests that the glass beads

may be effective at reducing variability between rep-

licates providing a consistent incubation system for

obtaining reproducible results for extracellularly pro-

duced lipase but not when the enzyme is cell bound.

Conversely, the inclusion of glass beads in mixed

cultures did have a marked effect on lipolytic activ-

ity for mixed ruminal microbes. The differences in

characterization between pure cultures and mixed

cultures with and without the glass beads may sug-

gest that while species of ruminal microbes grown

in pure culture contribute appreciably to cumulative

lipolytic activity in the rumen, the identities of highly

active, extracellular lipase producing, rumen bacte-

rial genera/species has yet to be made.

CONCLUSIONS

The introduction of glass beads markedly in-

creased in vitro lipolytic activity in mixed culture


incubation over rumen digesta, most likely due to

increased interfacial activation due to a more ho-

mogeneous support matrix. However, the presence

of beads as a support matrix for individual cultures

of lipase-producing bacteria examined in this study

did not appear to have a significant effect on lipo-

lytic activity, leaving potential for the identification

of other highly active contributors to rumen lipolysis.

The results of these studies may be utilized both for

the development or improvement of methods for

culturing lipase-producing bacteria, and for gaining

further insight into the bacteria responsible for the

majority of lipolytic activity in the rumen.

ACKNOWLEDGEMENTS

This research was supported in part by USDA, Co-

operative State Research, Education and Extension

Service (CSREES) grant number 2009-51110-05852.

The expert technical assistance of Jackie Kotzur

(USDA/ARS, College Station, TX) is greatly appreci-

ated.

REFERENCES

Bryant, M. P. 1972. Commentary on the Hungate

technique for culture of anaerobic bacteria. Am. J.

Clin. Nutr. 25: 1324-1328.

Cotta, M. A., and J. B. Russell. 1982. Effect of pep-

tides and amino acids on efficiency of rumen bac-

terial protein synthesis in continuous culture. J.

Dairy Sci. 65: 226-234.

Dohme, F., V. Fievez, K. Raes, and D. I. Demeyer.

2003. Increasing levels of two different fish oils

lower ruminal biohydrogenation of eicosapentae-

noic and docosahexaenoic acid in vitro. Anim. Res.

52: 309-320.

Fernandez-Lafuente, R., P. Armisen, P. Sabuquillo, G.

Frenandez-Lorente, and J. M. Guisan. 1998. Immo-

bilization of lipase by selective adsorption on hydro-

phobic supports. Chem. Phys. Lipids 93: 185-197.

Ferrato, F., F. Carrlere, L. Sarda, and R. A. Verger.

1997. Critical reevaluation of the phenomenon in-

terfacial activiation. In: B. Rubin and E. A. Dennis

(eds.) Methods in Enzymology No. 286 Part B. Aca-

demic Press, New York.

Garton, G. A., P. N. Hobson, and A. K. Lough. 1958.

Lipolysis in the rumen. Nature 182: 1511-1512.

Greenman, J., K. T. Holland, and W. J. Cunliffe.

1983. Effects of pH on biomass, maximum specific

growth rate and extracellular enzyme production

by three species of cutaneous propionibacteria

grown in continuous culture. J. Gen. Microbiol.

129: 1301-1307.

Gribbon, E. M., W. J. Cunliffe, and K. T. Holland. 1993.

Interaction of Propionibacterium acnes with skin

lipids in vitro. J. Gen. Microbiol. 139: 1745-1751.

Harfoot, C. G., and G. P. Hazlewood. 1997. Lipid me-

tabolism in the rumen. In: P. N. Hobson and C. S.

Stewart (eds.) The Rumen Microbial Ecosystem p

382-426. Chapman & Hall, London, UK.

Hawke, J. C., and W. R. Silcock. 1970. The in vitro

rates of lipolysis and biohydrogenation in rumen

contents. Biochim. Biophys. Acta 218: 201-212.

Henderson, C. 1971. A study of the lipase produced

by Anaerovibrio lipolytica, a rumen bacterium. J.

Gen. Microbiol. 65: 81-89.

Hespell, R. B., and M. P. Bryant. 1981. The genera

Butyrivibrio, Succinvirbio, Succinimonas, Lachno-

spira, and Selenomonas. In: M. P. Starr, H. Stolp, H.

G. Trüper, A. Barlows and H. G. Schlegel (eds.) The

Prokaryotes. A Handbook on Habitats, Isolation,

and Identification of Bacteria No. II. p 1002-1021.

Springer-Verlag, Berlin.

Krueger, N. A., R. C. Anderson, T. R. Callaway, T.

S. Edrington, and D. J. Nisbet. 2008. Isolation of

prominent lipolytic rumen bacteria. J. Anim. Sci.

86, E-Suppl. 2/J. Dairy Sci., Vol. 91, E. Suppl. 1: 87.

Krueger, N. A., R. C. Anderson, L. O. Tedeschi, T. R.

Callaway, T. S. Edrington, and D. J. Nisbet. 2010.

Evaluation of feeding glycerol on free-fatty acid

production and fermentation kinetics of mixed

ruminal microbes in vitro. Bioresour. Technol. 101:

8469-8472.

Kwon, D., and J. Rhee. 1986. A simple and rapid

colorimetric method for determination of free fatty

acids for lipase assay. J. Am. Oil. Chem. Soc. 63:

89-92.


Lanz, W. W., and P. P. Williams. 1973. Characteriza-

tion of esterases produced by a ruminal bacterium

identified as Butyrivibrio fibrisolvens. J. Bacteriol.

113: 1170-1176.

Leyendecker, S. A., T. R. Callaway, R. C. Anderson,

and D. J. Nisbet. 2004. Technical note on a much

simplified method of collecting ruminal fluid using

a nylon paint strainer. J. Sci. Food Agri. 84: 387-389.

Lourenço, M., E. Ramos-Morales, and R. J. Wallace.

2010. The role of microbes in rumen lipolysis and

biohydrogenation and their manipulation. Animal

4: 1008-1023.

Martinez, D. A., and B. C. Nudel. 2002. The improve-

ment of lipase secretion and stability by addition

of inert compounds into Acinetobacter calcoaceti-

cus cultures. Can. J. Microbiol. 48: 1056-1061.

Paiva, A. L., V. M. Balcão, and F. X. Malcata. 2000.

Kinetics and mechanisms of reactions catalyzed by

immobilized lipases. Enzyme Microb. Technol. 27:

187-204.

Rao, C. S., and S. Damodaran. 2002. Is interfacial

activation of lipases in lipid monolayers related to

thermodynamic activity of interfacial water? Lang-

muir 18: 6294-6306.

Shorland, F. B., R. O. Weenink, and A. T. Johns. 1955.

Effect of the rumen on dietary fat. Nature 175:

1129-1130.

Van Nevel, C., and D. I. Demeyer. 1995. Lipolysis and

biohydrogenation of soybean oil in the rumen in

vitro: inhibition by antimicrobials. J. Dairy Sci. 78:

2797-2806.




ABSTRACT

Saccharomyces cerevisiae boulardii is frequently used as a dietary supplement to promote intestinal

health and reduce the impact of growth of enteric pathogens in livestock, including cattle and swine. Citrus

by-products are also fed as dietary supplements that have the additional benefit of inhibiting the growth

of enteric pathogens. Previous research identified that supplementation of Saccharomyces boulardii to

feed containing citrus pulp significantly reduced the average daily gain of weanling pigs challenged with

Salmonella enterica, suggesting citrus pulp reduces the effectiveness of Saccharomyces boulardii. To in-

vestigate this possibility, an in vitro analysis was conducted on the activity of Saccharomyces boulardii in

swine fecal microbial media supplemented with citrus pulp. Citrus pulp inclusion reduced (P < 0.01) popu-

lations of Saccharomyces boulardii within 48 h post-exposure, suggesting that this product may exhibit

antifungal properties. Co-incubation of Salmonella with Saccharomyces boulardii reduced populations of

both microbes; inclusion of citrus pulp did not lead to a further reduction of yeast populations in the co-

culture. The cell lysate from Saccharomyces boulardii was also found to provide a carbon source that was

utilizable by Escherichia coli, but not Salmonella. Together, these results suggest that citrus pulp reduces

the viability of Saccharomyces boulardii and that the subsequent effects of this interaction on enterics are

varied. Though further research is needed to determine how citrus pulp influences the activity of Saccharo-

myes boulardii in vivo, these data strongly suggest caution should be exercised in providing citrus pulp to

livestock being fed diets supplemented with live yeast probiotics.

Keywords: probiotics, citrus pulp, Salmonella Typhi, Escherichia coli, E. coli O157:H7, Saccharomyces boulardii, enterics, swine, feed supplement, antifungal

Correspondence: Janet R. Donaldson, [email protected], Tel: +1 662 325 9547; Fax +1 662 325 7582

Effect of Citrus Pulp on the Viability of Saccharomyces boulardii in the Presence of Enteric Pathogens †

J. G. Wilson1, T. C. McLaurin1, J. A. Carroll2, S. Shields-Menard1, T. B. Schmidt3, T. R. Callaway4, and J. R. Donaldson1

1Department of Biological Sciences, Mississippi State University, Mississippi State, MS2Livestock Issues Research Unit, U. S. Department of Agriculture, Agriculture Research Service, Lubbock, TX

3Animal Science Department, University of Nebraska, Lincoln, NE4Food and Feed Safety Research Unit, U. S. Department of Agriculture, Agriculture Research Service, College Station, TX

†Mandatory Disclaimer: “Proprietary or brand names are necessary to report factually on available data; however, the USDA neither

guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, or

exclusion of others that may be suitable.” USDA is an equal opportunity provider and employer.



INTRODUCTION

The microbial communities associated with the

gastrointestinal (GI) tract of animals can be altered

in response to changes in environment, food con-

sumption or exercise (Chaucheyras-Durand and Du-

rand, 2010). This can cause severe distress, which is

problematic particularly in livestock. Abrupt changes

in the gastrointestinal microbial community due to

lifestyle or environmental changes can lead to acido-

sis, increased colonization of pathogens, and other

harmful effects. In order to reduce these deleterious

effects, probiotics have often been administered to

livestock (Chaucheyras-Durand and Durand 2010;

Siragusa and Ricke, 2012). Probiotics are microor-

ganisms that provide a benefit to the host by improv-

ing health and growth. The mechanisms by which

probiotics function are varied and debated, but are

primarily attributed to a competitive ability to pre-

vent pathogens from having access to colonization

sites within the host and also to prevent pathogens

from acquiring nutrients (Boirivant and Strober, 2007;

Rolfe, 2000). These changes alter the GI population

and influence immune parameters and responses,

which ultimately improve growth efficiency (Isolauri

et al., 2001; Vanbelle et al.,1990).

Saccharomyces cerevisiae subtype boulardii is a

probiotic yeast that has been extensively studied in

relation to preventing or alleviating intestinal dis-

tress (Rolfe, 2000). Along with pathogen inhibitory

effects, evidence suggests Saccharomyces cerevi-

siae helps to stabilize the rumen microbial commu-

nity, which may decrease the risk of acidosis in ru-

minants (Chaucheyras-Durand et al., 2005, Newbold

et al., 1996; Nisbet and Martin, 1991). Furthermore,

weanling pigs provided a diet supplemented with

Saccharomyces boulardii had an improved average

daily weight gain (ADG) and reduced mortality as-

sociated with endotoxemia (Collier et al., 2011).

Citrus pulp is a by-product produced from citrus

processing and is used as a low cost alternative car-

bohydrate source in livestock diets, particularly in cit-

rus producing regions of the United States and South

America (Ariza et al., 2001, Bampidis and Robinson

2006). Previous studies have reported that citrus by-

product feeds also act as antimicrobial agents against

the enteric pathogens Escherichia coli O157:H7 and

Salmonella enterica (Callaway et al., 2008, Fett and

Cooke 2003). This antimicrobial activity is likely attrib-

uted to the essential oils associated with these citrus

products, including, but not limited to citrullene, lin-

alool, and limonene (Nannapaneni et al., 2008).

Citrus products have been reported to promote

the growth of Bacillus subtilis (Sen et al., 2011), which

indicates that supplementation of diets with citrus

by-products may promote growth of certain micro-

organisms within the GI tract. However, depending

upon the source, citrus by-products can also have

inhibitory effects on the probiotic Bifidobacterium

bifidum (Sendra et al., 2008). Carroll and colleagues

have reported that weanling pigs provided a diet

supplemented with both Saccharomyces boulardii

and citrus by-products experienced a decline in ADG

post-exposure to Salmonella (unpublished results),

suggesting an undesirable interaction occurred be-

tween the yeast and pathogen in the gut. The aim of

the current study was to analyze the interaction be-

tween Saccharomyces boulardii and enteric bacteria

to determine if the viability of Saccharomyces bou-

lardii is altered in the presence of citrus pulp using

an in vitro swine fecal microbial fermentation system.


Microbial strains and growth conditions

Escherichia coli O157:H7 (ATCC 43895) and Sal-

monella enterica ssp. Typhi (ATCC 6539) were rou-

tinely cultured in the general culture medium tryp-

tic soy broth (TSB) at 37°C. E. coli and S. Typhi were

transformed with the plasmid pXEN-13 to allow for

selection onto TSB supplemented with 100 µg/ml

ampicillin (TSB amp) as previously described by our

group (Free et al., 2012). Saccharomyces cerevisiae

ssp. boulardii was obtained from a commercial sup-

plier (Saccharomyces cerevisiae I-1077, Lallemand

Animal Nutrition). Saccharomyces boulardii was

routinely cultured in yeast peptone dextrose media

(YPD, Sigma-Aldrich) at 37°C.


Survival in fecal growth medium

Fecal samples were collected from pigs at the

Leveck Animal Research Center at Mississippi State

University (Mississippi State, MS). Fecal medium was

prepared essentially as previously described (Free

et al., 2012; Russell and Martin, 1984). Briefly, 33.3g

of fresh feces were vortex-mixed in 33.3mL of sterile

water prior to addition to 1L of base medium. The

fecal medium was incubated overnight at 39°C in a

shaker incubator. The following day 5.0 g of ground

citrus pulp (Texas Citrus Exchange, Mission, TX) was

added to 100 mL of fecal growth medium.

Bacterial cultures were grown overnight at 39°C

in 5 mL TSB amp. Cultures were then diluted 1:100

and allowed to grow for 4 h to reach log phase (Op-

tical density, OD600 approximately 0.4), at which time

cultures were centrifuged for 5 min at 10,000 x g to

remove antibiotics. Resulting cell pellets were re-

suspended in an equal volume of freshly prepared

swine fecal fluid media supplemented with either

0% or 5% citrus by-products. Cultures were subse-

quently incubated at 39°C for 48 h. For enumeration

of yeast, aliquots were diluted in 1X phosphate buff-

ered saline (1X PBS) and plated on YPD agar supple-

mented with 100 U/mL of penicillin and 100 µg/mL

streptomycin and 0.25 µg/mL fungizone (Invitrogen).

Cultivation trials confirmed that this medium did

not inhibit the growth of Saccharomyces boulardii.

For enumeration of bacteria, samples were serially

diluted in 1X PBS and subsequently plated onto nu-

trient agar supplemented with 100 µg/mL ampicillin

(NA amp). Plates were incubated at 37°C and colony

forming units (CFU) were enumerated after 24 to 48

h of incubation. A minimum of three independent

replicates was performed for each strain and condi-

tion tested.

Scanning electron microscopy

Cultures of Saccharomyces boulardii were grown

for 24 h at 37°C in YPD. Cultures were allowed to in-

cubate for an additional 48 h at 39°C in the presence

(or absence) of 5% citrus pulp. Cells were pelleted at

10,000 x g for 5 min and fixed in 2.5% glutaraldehyde

in 0.1M sodium cacodylate buffer for a minimum of

16 h. Samples were then prepared for observation

as previously described (Merritt et al. 2010). Samples

were viewed using a JOEL 6500F field emission scan-

ning electron microscope (JOEL Ltd, Tokyo, Japan).

A minimum of 20 cells was examined.

Survival in Saccharomyces boulardii ly-sate

Cultures of E. coli, S. Typhi, and Saccharomyces

boulardii were grown overnight at 37°C as described

in the previous section. Cells were then pelleted,

washed with 1X PBS, and resuspended in mineral

salts media (MSM) lacking a carbon source (Alvarez

et al., 1996). Saccharomyces boulardii cultures were

lysed via sonication (Fisher Scientific Sonic Dismem-

brator Model 120; setting 3, 30 sec pulse; Pittsburgh,

PA) and filtered through a 0.22 µm syringe filter.

Salmonella and E. coli were diluted 1:100 in 0.2mL

of fresh MSM supplemented with either 20% fil-

trate from Saccharomyces boulardii or 2% glucose.

Growth was monitored by OD600 readings over a 24

h period with a Biotek Synergy HT microplate reader

(Biotek, Winooski, VT). A minimum of three indepen-

dent replicates was performed.

Statistical analysis

The fold change (log10 Ntreated CFU/mL / log10 Noriginal

CFU/mL) and log10 CFU/mL of yeast and bacterial

populations were analyzed as means across each

treatment. Data were analyzed by analysis of vari-

ance (ANOVA) using the Glimmix procedures of SAS

(version 9.2, 2013, Institute, Inc, Cary, NC), with sig-

nificance declared at P < 0.05.

RESULTS AND DISCUSSION

Citrus pulp reduces the viability of Sac-charomyces boulardii in vitro

Saccharomyces boulardii was grown in swine fe-

cal microbial fluid and viability was assessed over a


48 h growth period. Viability decreased by 0.85 log10

CFU/mL (from 7.95 to 7.10 log10 CFU/mL) after 48 h

in this cultivation medium (P = 0.005; Table 1). In the

presence of 5% citrus pulp, viability was reduced by

1.65 log10 after 48 h (7.97 to 6.31 log10 CFU/mL, P <

0.0001). This was an approximate 10% reduction be-

yond what was attributed to the medium alone. This

suggests that citrus pulp can directly impact yeast

viability.

To further analyze this interaction with citrus pulp,

Saccharomyces boulardii was incubated in YPD

broth in the presence (or absence) of 5% citrus pulp

and the integrity of the cell walls were assessed by

scanning electron microscopy (Figure 1). Alterations

in the cell wall morphology were evident in yeast

treated with citrus pulp, indicating that citrus pulp

introduces damage into the cell wall of Saccharo-

myces boulardii. Together, these data suggest that

citrus by-products may exhibit slight fungicidal ac-

tivity, or that the mechanism by which the products

were processed confers this activity to the product.

The essential oils from citrus products are known to

Table 1. Fold change of Saccharomyces cerevisiae boulardii (SCB) populations (Log10 CFU/mL) cultured with citrus pulp (CP), Salmonella typhi and/or Escherichia coli O157:H7

12 h 24 h 48 h

SCB 0.99 a, x 0.96 a, x 0.89 a, y

+CP 0.95 a, c, x 0.89 a, b, x 0.79 b, y

+Salmonella 0.99 a, x 0.86 b, y 0.80 b, z

+Salmonella +CP 0.99 a, x 0.84 b, y 0.83 a, b, y

+E. coli 1.14 b, x 1.09 c, x 0.96 c, y

+E. coli +CP 1.11 b, c, x 0.99 a, y 0.84 a, b, d, z

a,b,c Means within a column sharing a common superscript are not different. Significance declared at P < 0.05.x,y,z Means within a row sharing a common superscript are not different. Significance declared at P < 0.05.

Figure 1. Citrus pulp introduces alterations into the cell surface of S. boulardii. S. boulardii was cultured in the absence (A) or presence (B) of citrus pulp for 24 h and samples were subsequently analyzed by scanning electron microscopy. Scale bars represent 1µm.


exhibit antifungal, as well as antibacterial activities

(Caccioni et al., 1998; Cvetnia and Vladimir-Knee-

via, 2004). A study using Saccharomyces cerevisiae

reported that while not all of the oils from citrus

products eradicated Saccharomyces cerevisiae, all

seemed to have an inhibitory effect (Belletti et al.,

2004). This suggests that the presence of any citrus

essential oils may directly alter the overall effective-

ness of live yeast probiotics.

Interactions between Saccharomyces boulardii and Salmonella

To investigate the possibility that the reduced vi-

ability of Saccharomyces boulardii in the presence

of citrus pulp would alter the competitive activity of

this probiotic against Salmonella, Saccharomyces

boulardii and S. Typhi were cultured concurrently in a

swine fecal microbial fermentation system and viabil-

ity for both microbes was assessed over a 48 h pe-

riod. The addition of S. Typhi reduced the viability of

Saccharomyces boulardii by 14% within 24 h (8.23 to

7.05 log10 CFU/mL, P = 0.005) and by 20% within 48

h (8.23 to 6.55 log10 CFU/mL, P = 0.005; Table 1). Co-

cultivation of S. Typhi and Saccharomyces boulardii

reduced populations of S. Typhi by 8% within 24 h

(7.03 to 6.43 log10 CFU/mL, P = 0.003) and by 17%

within 48 h (7.03 to 5.86 log10 CFU/mL, P < 0.0001;

Table 2). Since in a co-culture condition the reduc-

tions in populations of Saccharomyces boulardii

were more severe than those of S. Typhi within 24 h

(P = 0.04), it is possible that S. Typhi utilizes nutrients

in the fecal fluid media first or may be more efficient

at utilization of nutrients in a mixed culture. This data

warrants further investigation.

The addition of citrus pulp decreased popula-

tions of Salmonella as expected based on a previ-

ous study (Callaway et al., 2008). Within 48 h post

exposure, populations of S. Typhi were reduced by

16% in the presence of citrus pulp (6.80 to 5.73 log10

CFU/mL, P < 0.0001). Populations also decreased by

17% within 48 h of cultivation in the presence of Sac-

charomyces boulardii (7.04 to 5.87 log10 CFU/mL, P

< 0.0001). However, in the presence of both citrus

pulp and Saccharomyces boulardii, populations of S.

typhi were reduced by 12% within 24 h (P = 0.0585)

and by 21% within 48 h (P < 0.0001; Table 2).

These data indicate that a combination of cit-

rus pulp and Saccharomyces boulardii might lead

to an enhanced lysis of S. Typhi. Though this is a

promising result, it does not necessarily correlate

with a beneficial synergy in vivo. A previous study

found that the combination of Saccharomyces bou-

lardii and citrus pulp reduced the ADG of weanling

pigs following Salmonella infections (Carroll et al.,

unpublished results). Therefore, an alternative in-

terpretation of these data could suggest that the

enhanced lysis of S. Typhi from the combination of

Table 2. Fold change of Salmonella typhi populations (Log10 CFU/mL) cultured with citrus pulp (CP) and/or Saccharomyces cerevisiae boulardii (SCB).

a,b Means within a column sharing a common superscript are not different. Significance declared at P < 0.05.x,y,z Means within a row sharing a common superscript are not different. Significance declared at P < 0.05.

12 h 24 h 48 h

Salmonella 0.94 0.92 0.88 a

+CP 1.01 x 0.94 y 0.84 a, b, z

+SCB 0.99 x 0.91 y 0.83 a, b, z

+SCB+CP 0.94 x 0.88 x 0.79 b, y


Saccharomyces boulardii and citrus pulp may lead

to an increase in cytotoxin release. This must be

taken into account when analyzing potential anti-

microbial compounds in vivo and warrants further

investigation.

Interactions between Saccharomyces boulardii and E. coli O157:H7

Since enhanced reductions of S. Typhi popula-

tions were observed in the presence of citrus pulp

and Saccharomyces boulardii, Escherichia coli

O157:H7 was also examined to determine whether

this effect would extend to other gram-negative

bacteria. Populations of E. coli O157:H7 remained

stable in the fecal growth medium during the 48 h

(P = 0.7; Table 3). Reductions in E. coli populations

in the presence of citrus pulp were only observed

after 48 h (7.78 to 6.01 log10 CFU/mL reduction, P

< 0.001). Saccharomyces boulardii did not reduce

populations of E. coli, but the combination of Sac-

charomyces boulardii and citrus pulp reduced pop-

ulations of E. coli by 22% within 48 h (8.43 to 6.60

log10 CFU/mL reduction, P = 0.0076; Table 3). These

results indicate that the presence of Saccharomy-

ces boulardii does not affect the viability of E. coli

O157:H7 and that even in a mixed culture the ef-

fects are due to the presence of citrus pulp-related

factors.

Cell lysate of Saccharomyces boular-dii as a potential carbon source for other microorganisms

Variations in the growth analysis of Saccharomyces

boulardii populations may be due to the reduced vi-

ability of Saccharomyces boulardii in the presence of

citrus pulp. This reduction in viability could have po-

tentially two effects on the other microorganisms in

the system: 1) removes competition for nutrients, or

2) provides an additional source of nutrients that can

be utilized by other microorganisms in the system.

To determine whether it was possible that lysed Sac-

charomyces boulardii could provide an additional

source of nutrients to enteric bacteria, Saccharomy-

ces boulardii cells were lysed and the filter-sterilized

lysate was analyzed as a potential carbon source.

Cultures of E. coli O157:H7 or S. Typhi were grown in

MSM supplemented with either glucose or Saccha-

romyces boulardii lysate. MSM without the addition

of a carbon source did not support growth of either

E. coli or S. Typhi; the addition of glucose to this me-

dium did allow for growth of both microorganisms

(data not shown). Surprisingly, E. coli O157:H7, but

not S. Typhi, utilized the lysate from Saccharomyces

boulardii as a carbon source (Figure 2). Though the

growth was minimal, this could potentially allow for

sustainability of the population as Saccharomyces

boulardii are reduced by citrus pulp.

Table 3. Fold change of Escherichia coli O157:H7 populations (Log10 CFU/mL) cultured with citrus pulp (CP) and/ or Saccharomyces cerevisiae boulardii (SCB).

12 h 24 h 48 h

E. coli 1.02 0.99 0.98 a

+CP 1.03 x 0.98 x 0.77 b, y

+SCB 1.00 0.99 0.98 a

+SCB+CP 0.93 x 0.87 x, y 0.78 b, y

a,b Means within a column sharing a common superscript are not different. Significance declared at P < 0.05.x,y Means within a row sharing a common superscript are not different. Significance declared at P < 0.05.


In co-cultures with Saccharomyces boulardii and

E. coli O157:H7, citrus pulp may affect E. coli ini-

tially. This is evident by an increase in populations

of Saccharomyces boulardii and a decrease in E.

coli populations (Tables 1 and 3). However, as expo-

sure increased, the populations of Saccharomyces

boulardii decreased (through lysis with citrus pulp

by-products). The populations of E. coli did not de-

crease to the same level as cultures in the presence

of citrus pulp alone. The co-culture data, along with

the ability of E. coli O157:H7 to utilize Saccharomy-

ces boulardii lysate as a carbon source, suggests that

extended exposure to citrus pulp would decrease

populations of Saccharomyces boulardii, which may

potentially lead to a stabilization of populations of E.

coli O157:H7.

CONCLUSIONS

These findings suggest that caution must be ex-

tended when providing live yeast in combination

with citrus by-products as the antimicrobial factors

of the supplements may result in undesirable growth

of enteric pathogen populations. Further research is

needed to determine how this relationship alters the

gastrointestinal microbiome in vivo.

ACKNOWLEDGEMENTS

The authors would like to thank Ms. Amanda Law-

rence at the Mississippi State University Institute for

Imaging and Analytical Technologies for her assis-

tance with the electron microscope. This work was

funded through the Mississippi Agricultural and For-

estry Experiment Station Special Research Initiatives

Grant and through the Mississippi State University

Shackoul’s Honors College Undergraduate Research

Fellowship (to TCM).

REFERENCES

Alvarez, H. M., F. Mayer, D. Fabritius and A. Stein-

buchel. 1996. Formation of intracytoplasmic lipid

inclusions by Rhodococcus opacus strain PD630.

Arch. Microbiol. 165:377-86.

Figure 2. Escherichia coli O157:H7, but not Salmonella typhi, can utilize the cell lysate from S. boulardii as a sole carbon source. Salmonella typhi and E. coli O157:H7 were cultured in minimal media lacking carbon supplemented with S. boulardii cell lysate. Growth was monitored over a 24 h period by the OD600. Values represent the average of three independent replicates.

0

0.1

0.2

0.3

0.4

0.5

0 3 6 9 12 15 18 21 24

OD

600

Time (h)

E. coli

Salmonella typhi


Ariza, P., A. Bach, M. D. Stern and M. B. Hall. 2001. Ef-

fects of carbohydrates from citrus pulp and hominy

feed on microbial fermentation in continuous cul-

ture. J. Anim. Sci. 79:2713-8.

Bampidis, V. A. and P. H. Robinson. 2006. Citrus by-

products as ruminant feeds: A review. Animal Feed

Sci. and Tech. 128:175-217.

Belletti, N., M. Ndagijimana, C. Sisto, M. E. Guerzoni,

R. Lanciotti and F. Gardini. 2004. Evaluation of the

antimicrobial activity of citrus essences on Saccha-

romyces cerevisiae. J. Agric. Food Chem. 52:6932-8.

Boirivant, M. and W. Strober. 2007. The mechanism

of action of probiotics. Curr. Opin. Gastroenterol.

23:679-92.

Caccioni, D. R. L., M. Guizzardi, D. M. Biondi, A. Ren-

da and G. Ruberto. 1998. Relationship between

volatile components of citrus fruit essential oils

and antimicrobial action on Penicillium digitatum

and Penicillium italicum. Int. J. Food Microbiol.

43:73-79.

Callaway, T. R., J. A. Carroll, J. D. Arthington, C. Pratt,

T. S. Edrington, R. C. Anderson, M.L. Galyean, S.

C. Ricke, P. Crandall and D. J. Nisbet. 2008. Citrus

products decrease growth of E. coli O157:H7 and

Salmonella typhimurium in pure culture and in fer-

mentation with mixed ruminal microorganisms in

vitro. Foodborne Pathog. Dis. 5:621-7.

Chaucheyras-Durand, F. and H. Durand. 2010. Pro-

biotics in animal nutrition and health. Benef. Mi-

crobes. 1:3-9.

Chaucheyras-Durand, F., S. Masseglia and G. Fonty.

2005. Effect of the microbial feed additive Saccha-

romyces cerevisiae CNCM I-1077 on protein and

peptide degrading activities of rumen bacteria

grown in vitro. Curr. Microbiol. 50:96-101.

Collier, C. T., J. A. Carroll, M. A. Ballou, J. D. Starkey

and J. C. Sparks. 2011. Oral administration of Sac-

charomyces cerevisiae boulardii reduces mortal-

ity associated with immune and cortisol responses

to Escherichia coli endotoxin in pigs. J. Anim. Sci.

89:52-8.

Cvetnia, Z. and S. Vladimir-Kneevia. 2004. Antimicro-

bial activity of grapefruit seed and pulp ethanolic

extract. Acta. Pharmaceut. 54:243-250.

Fett, W. F. and P. H. Cooke. 2003. Reduction of Esch-

erichia coli O157:H7 and Salmonella on laborato-

ry-inoculated alfalfa seed with commercial citrus-

related products. J. Food Prot. 66:1158-65.

Free, A. L., H. A. Duoss, L. V. Bergeron, S. A. Shields-

Menard, E. Ward, T. R. Callaway, J. A. Carroll, T.

B. Schmidt and J. R. Donaldson. 2012. Survival of

O157:H7 and non-O157 serogroups of Escherichia

coli in bovine rumen fluid and bile salts. Foodborne

Path. Dis. 9:1010-14.

Isolauri, E., Y. Sutas, P. Kankaanpaa, H. Arvilommi

and S. Salminen. 2001. Probiotics: effects on im-

munity. Am. J. Clin. Nutr. 73:444S-450S.

Merritt, M. E., A. M. Lawrence and J. R. Donaldson.

2010. Comparative study of the effect of bile on the

Listeria monocytogenes virulent strain EGD-e and

avirulent strain HCC23 Arch. Clinical Micro. 1:4-9.

Nannapaneni, R., A. Muthaiyan, P. G. Crandall, M. G.

Johnson, C. A. O’Bryan, V. I. Chalova, T. R. Calla-

way, J. A. Carroll, J. D. Arthington, D. J. Nisbet and

S. C. Ricke. 2008. Antimicrobial activity of com-

mercial citrus-based natural extracts against Esch-

erichia coli O157:H7 isolates and mutant strains.

Foodborne Pathog. Dis. 5:695-9.

Newbold, C. J., R. J. Wallace and R. M. McIntosh.

1996. Mode of action of the yeast Saccharomyces

cerevisiae as a feed additive for ruminants. Br. J.

Nutr. 76:249-61.

Nisbet, D. J. and S. A. Martin. 1991. Effect of a Sac-

charomyces cerevisiae culture on lactate utilization

by the ruminal bacterium Selenomonas ruminan-

tium. J. Anim. Sci. 1991.4628-4633.

Rolfe, R. D. 2000. The role of probiotic cultures in the

control of gastrointestinal health. J. Nutr. 130:396S-

402S.

Russell, J. B. and S. A. Martin. 1984. Effects of various

methane inhibitors on the fermentation of amino

acids by mixed rumen microorganisms in vitro. J.

Anim. Sci. 59:1329-38.

Sen, S., S. L. Ingale, J. S. Kim, K. H. Kim, Y. W. Kim,

C. Khong, J. D. Lohakare, E. K. Kim, H. S. Kim, I. K.

Kwon and B. J. Chae. 2011. Effect of supplementa-

tion of Bacillus subtilis LS 1-2 grown on citrus-juice

waste and corn-soybean meal substrate on growth

performance, nutrient retention, caecal microbiol-

ogy, and small intestinal morphology of broilers.


Asian-Austral J. Anim. Sci. 24:1120-7.

Sendra, E., P. Fayos, Y. Lario, J. Fernandez-Lopez, E.

Sayas-Barbera and J. A. Perez-Alvarez. 2008. Incor-

poration of citrus fibers in fermented milk contain-

ing probiotic bacteria. Food Microbiol. 25:13-21.

Siragusa, G. R. and S. C. Ricke. 2012. Probiotics as

pathogen control agents for organic meat produc-

tion. In Organic meat production and processing.

Ricke S. C., E. J. Van Loo, M. G. Johnson and C. A.

O’Bryan, ed. (John Wiley & Sons, Inc., New York).

pp 331-349.

Vanbelle, M., E. Teller and M. Focant. 1990. Probiot-

ics in animal nutrition: a review. Arch. Tierernahr.

40:543-67.




ABSTRACT

Antibiotic resistance in food animals has become an important issue for public health safety. The genes

that code antibiotic resistance often enter the feedlot environment via feces and have the potential to be

transferred through agroecosystems and into the food chain, either directly in their original bacterial host

or via horizontal gene transfer. The objective of this study was to determine the distribution of erythromy-

cin resistance genes associated with beef cattle excretions and ascertain whether these genes are enriched

in areas of feedlot pens with high deposition of fecal material over time. The spatial distribution of manure

accumulation was determined using georeferenced electromagnetic induction (EMI) readings at two times

and EMI directed soil sampling. Feedlot surface samples from high- and low-manure accumulation zones

were compared. The data indicated that 14 months of manure accumulation did not result in an increase

in erm(B) positive feedlot soils, and the distribution of erm(B) genes was not correlated with areas of high

manure deposition within the pens.

Keywords: Antibiotic resistance, resistance, antibiotic resistance gene, manure, erythromycin,

ermB, feedlot pen, cattle, PCR, food animals

INTRODUCTION

Erythromycin, a macrolide antibiotic commonly

used to treat infections in humans, is on the World

Health Organization’s list of antimicrobial agents

that are critical to human health (World Health Or-

Correspondence: Lisa Durso, [email protected]: +1 -402-472-9622 Fax: +1-402-437-5712

ganization, 2007). Related macrolides (Tulathromy-

cin (Draxxin), Tilmicosin (Micotil), and Tylosin (Tylan))

have been used in cattle to treat respiratory disease,

pneumonia, metritis, mastitis, and foot rot (Smith

Thomas, 2009). Tylosin is also used as a feed additive

for cattle to prevent liver abscesses, and as part of a

mineral supplement to help control pinkeye (Smith

Thomas, 2009). Bacteria can develop resistance to

macrolide antibiotics by encoding a suite of more

Persistence of erythromycin resistance gene erm(B) in cattle feedlot pens over time‡

A. R. Mantz 1, D. N. Miller2, M. J. Spiehs3, B. L. Woodbury3, and L. M. Durso2

1 Department of Biological Systems Engineering, University of Nebraska, 223 L. W. Chase Hall, P. O. Box 830726, Lincoln, NE 68583, USA

2USDA, ARS, 137 Keim Hall, UNL-East Campus, Lincoln, NE 68583, USA3USDA, ARS, Meat Animal Research Center, State Spur 18D, Clay Center, NE 68933, USA

‡Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not

imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.



than 30 erythromycin ribosomal methylase (erm)

genes (Roberts et al., 1999). The erm genes can be

found in both commensal and pathogenic bacteria,

including Gram-positive and -negative species (De

Leener et al., 2004; Roberts, 2004; Bae et al., 2005;

Chen et al., 2007; Dogan et al., 2005).

In bacteria from cattle, resistance to tylosin is en-

coded by erm genes and erm(B) is the most com-

mon (Jost et al., 2004). For example, in a survey of

U.S. livestock systems, the erm(B) gene was found

to represent 56% of the total erm genes in bovine

manure samples (Chen et al., 2007). The erm(B)

gene has been reported in Campylobacter from

cattle feedlots (Bae et al., 2005), Enteroccocci from

pastured cattle (Anderson et al., 2008), in livestock

manure and pre-harvest production systems (Chen

et al., 2007), and pen floor fecal samples from feed-

lot heifers (Jacob et al., 2008). Many studies screen

for the erm(B) gene from pathogenic and commen-

sal bacterial isolates, but this strategy does not al-

low for the assessment of non-target, unculturable

bacteria. Since one of the primary concerns associ-

ated with antibiotic resistance in agricultural settings

is the horizontal gene transfer from animals to hu-

mans, whole community DNA needs to be screened

in order to assess the entire reservoir of antibiotic

resistance genes present in a sample (Isaacson and

Torrence, 2002). One element that contributes to hu-

man health risk associated with antibiotic resistance

genes from agricultural settings is the persistence

of the genes over time (Unc and Goss, 2004). A lon-

gitudinal study demonstrated that the erm(B) gene

could persist in fecal samples from cattle in field con-

ditions for over 150 days (Alexander et al., 2011).

In commercial cattle feedlot operations, feces are

continually deposited onto the pen surface and accu-

mulate until they are removed by scraping, typically

once a year. Identification of zones within the feed-

lot that are enriched for antibiotic resistance genes

would allow for targeted sampling and remediation

efforts. The large size and spatial heterogeneity of

the feedlot pen presents challenges for sample col-

lection. Typically, cattle in pens tend to congregate

in certain areas, resulting in zones of high manure

accumulation in the pen. Previous studies identi-

fied correlations between electromagnetic induction

(EMI) readings and areas of high manure deposition

(Woodbury et al., 2009; Eigenberg et al., 2010).

We hypothesized that the incidence of erm(B)

genes in the feedlot were a consequence of excre-

tion from the animal and would be concentrated in

areas with high manure enrichment. To test this hy-

pothesis we examined cattle feedlot pens that were

allowed to accumulate manure for 14 months. Feed-

lot pen surface samples were collected based on dif-

ferences in manure accumulation, delineated using

EMI sampling methods and were evaluated using a

conventional PCR-based erm(B) assay of total com-

munity DNA samples.


Sampling

In order to ensure that samples were collected

from areas representing the continuum of manure

deposition, pens were mapped for EMI and sample

sites were co-located with selected EMI values us-

ing the spatial response surface sampling design

(RSSD) program contained in the USDA-ARS ESAP

(ECe Sampling Assessment and Prediction) software

package (Lesch et al., 2000).

Feedlot surface material samples were taken from

ten feedlot pens (each 30 m by 60 m) at the U.S.

Meat Animal Research Center, in conjunction with

a previously described study (Spiehs et al., 2012).

Half of the pens contained animals receiving a nor-

mal, controlled diet of dry-rolled corn and half of the

pens contained animals receiving a diet containing

14 – 35% wet distillers grains plus solubles (WDGS)

(levels changed based upon the age of the cattle in

the pens). All pens have a concrete apron adjacent

to the feeding area and water areas along the lat-

eral sides of the pens with a mound in the center.

Following EMI mapping, twelve sample sites were

identified in each pen, as described above, and GPS

coordinates were recorded. In general, the feedlot

pens have a gradient slope at 2% declination from

the feeding area down to the bottom of the pen


where aged manure and liquids accumulate (Wood-

bury et al., 2009). During the 13-month study, the

pens were not cleaned and two separate groups

of cattle were fed in the pens, each containing 32

mixed-breed finishing steers per pen. The first set

was exchanged for the second between September

18th and 22nd, 2009.

A total of 240 feedlot surface material samples

were collected (120 from June 2009, 120 from August

2010). Grab samples of the feedlot surface material

were collected from the surface (0 to 10 cm depth),

placed in 3.8-L plastic bags, and held on ice during

transport to the laboratory. Aliquots of the samples

were stored at -20ºC until DNA extractions could be

performed. The remaining feedlot surface material

was immediately dried in a forced-air oven at 100ºC

for 24 hours, ground, and analyzed for moisture con-

tent, volatile solids content, nutrients, and soil pH as

previously described by Spiehs et al. (2012).

DNA Extraction and Quantification

Feedlot surface soil samples were extracted

as previously described by Miller et al. (1999), and

purified using a Wizard® DNA Purification System

(Promega, Madison, WI). DNA concentrations were

determined using fluorometry. Calibration stan-

dards were created using diluted λ DNA (Quant-iT™

PicoGreen® dsDNA Assay Kit) at concentrations of

1 µg mL-1, 10 µg mL-1, 100 µg mL-1, and 1000 µg mL-

1, and PicoGreen® was diluted to 1:200 with 1xTE.

The standards, mixed with the diluted PicoGreen®,

were used to make a linear standard curve for cali-

bration. Samples were prepared by mixing 5 µL of

sample, 45 µL of 1xTE, and 50 µL of diluted Pico-

Green®. Samples were allowed to rest under alumi-

num foil for 5 minutes and then the fluorescence was

measured. To verify fluorometric results, a subset of

samples (three samples from each set of 30) was also

screened on 1.5% agarose gels using established

mass standards. Gels were stained for 10 minutes in

an ethidium bromide solution, destained for 25 min-

utes in distilled water, and visualized on a UV transil-

luminator (Ultraviolet Productions, Upland, CA).

Polymerase Chain Reaction

A polymerase chain reaction (PCR) assay was

performed for the detection of the erm(B) gene

using primers developed by Böckelmann et al.

(2009). The forward and reverse primer sequences

were 5’-GGATTCTACAAGCGTACCTTGGA-3’ and

5’-GCTGGCAGCTTAAGCAATTGCT-3’, respectively.

The amplification reactions were made with 0.25 µL

each of forward and reverse primers (1:100 concen-

tration), 11 µL PCR grade water, 12.5 µL Jumpstart

Red TAQ ReadyMix (Sigma-Aldrich, St. Louis, MO),

and 1 µL diluted sample (1:100 concentration). Posi-

tive and negative controls were run for every assay.

Thermocycling was performed using a PTC-100 Pel-

tier thermocycler (Bio-Rad, Hercules, CA). The cy-

cles were set at 95°C for 2 minutes, then 35 cycles

repeating through 95°C for 30 seconds, 60°C for 45

seconds and 72°C for 1 minute, then finally 72°C for

7 minutes. Samples were run on an agarose gel for

45 minutes on 145 V, stained with ethidium bromide

for 10 minutes, and then destained with distilled wa-

ter for 20 minutes. Gels were photographed using

a Kodak Gel Logic 100 Imaging System (Carestream

Health, Inc., Rochester, NY).

Statistical Analysis

The ANOVA and Logistic procedures available in

SAS Analysis program version 9.2 (SAS Inst., Cary,

NC) were used to determine the effect of diet treat-

ment, date of sampling, and pen location on erm(B)

prevalence and used to determine differences be-

tween soil parameters related to erm(B) status. Dif-

ferences were considered significant at P ≤ 0.05

and were considered tendencies when the P-values

ranged from P = 0.05 to P < 0.10.

RESULTS AND DISCUSSION

The persistence and distribution of the antibi-

otic resistance gene erm(B) was examined in cattle

feedlot pens over a 14 month period. Data indicate

no differences in the incidence of erm(B) over the


course of the study, regardless of animal diet (Table

1). Initial samples from June 2009 revealed a high

prevalence of the gene in the pens, with 76% (n=91)

of the samples testing positive for the erm(B) gene.

In August 2010, fourteen months later, 81% (n=97) of

the feedlot surface material samples were positive

for the erm(B) gene. Thus, despite fourteen months

of manure deposition, the prevalence of the gene

in the feedlot soil samples showed no statistical

change.

Since the initial source of erm(B) genes detected

in feedlot pen soil is likely to be the fecal deposition,

the locations of high manure deposition within the

pen were assumed to be the locations where erm(B)

would most likely be detected. Mapping of the pens

with EMI allows for the identification of regions in

the pen with signatures characteristic of high ma-

nure deposition (Woodbury et al., 2009). Thus, if

Table 1. Prevalence of erm(B) positive samples based upon diet fed, pen location, and sample date

June 2009 August 2010

Diet* Pen Mound† Edge Mound Edge0% WDGS 307 60.0% (n = 5)‡ 71.4% (n = 7) 83.3% (n = 6) 50.0%, (n = 6)

309 50.0% (n = 4) 100.0% (n = 8) 100.0% (n = 4) 87.5% (n = 8)

311 80.0% (n = 5) 85.7% (n = 7) 50.0% (n = 2) 70.0% (n = 10)

313 50.0% (n = 2) 90.0% (n = 10) 75.0% (n = 4) 62.5% (n = 8)

315 71.4% (n = 7) 100.0% (n = 5) 100.0% (n = 3) 100.0% (n = 9)

Ave 62.3% 89.4% 81.7% 74.0%

35% WDGS 308 40.0% (n = 5) 85.7% (n = 7) 100.0% (n = 3) 88.9% (n = 9)

310 25.0% (n = 4) 62.5% (n = 8) 100.0% (n = 4) 87.5% (n = 8)

312 80.0% (n = 5) 71.4% (n = 7) 75.0% (n = 4) 62.5% (n = 8)

314 75.0% (n = 4) 75.0% (n = 8) 100.0% (n = 3) 88.9% (n = 9)

316 83.3% (n = 6) 100.0% (n = 6) 50.0% (n = 4) 87.5% (n = 8)

Ave 60.7% 78.9% 85.0% 83.1%

0% vs 35% WDGS P diff 0.905 0.244 0.813 0.404

Overall Ave 61.5%A§ 84.2%B 83.3%B 78.5%B

*Diet indicates either a corn-based diet excluding wet distillers grains plus solubles (0% WDGS) or a diet including up to 35% WDGS. †Mound indicates sample from the central mound and edge indicates the lower area surrounding the mound. ‡Number of samples in each cell classified as either mound or edge. Twelve total samples per pen. §Means with different letters within a row are significantly different at P < 0.05.


the erm(B) genes were concentrated in areas of high

manure deposition, we would have expected to find

more erm(B) positive samples in areas with high EMI

readings. Our data did not support this theory at

either the initial or final sample collection (Figure 1),

as erm(B) positive and negative results were scat-

tered indiscriminately across low and high EMI read-

ing samples. Pen size and orientation may impact

cattle behavior (Wilson et al., 2010), and therefore

manure accumulation, so further evaluation of other

pens with different designs is warranted.

Next, the ecology of the feedlot pen was con-

sidered, including pen design and animal behavior.

Cattle feedlot pens are generally outdoors and ex-

posed to the elements. Often there is a mound lo-

cated in the pen to provide a dry area for the cattle

during wet weather (Woodbury et al., 2001). Cattle

are non-randomly distributed in the pens and even

though cattle movement can mix surface material

across the feedlot pen, distinct zones can develop

where fecal organisms are fortified (Woodbury et al.,

2009). Subtle differences were detected in erm(B)

gene prevalence between pen sites (mound versus

edge) based upon the date (Table 1). Initial preva-

lence of erm(B) on the mound in June 2009 was less

than the prevalence of erm(B) in pen edge samples

in June 2009, and the prevalence differed (P = 0.016)

from both mound and pen edge samples in August

2010. The prevalence of erm(B) in the mound ver-

sus the pen edge, however, did not differ from one

another in August 2010. A comparison of the over-

all prevalence in 2009 to 2010 (75.8% and 80.8%, re-

spectively) showed no difference (P= 0.271).

A variety of feedlot surface properties were eval-

uated to determine if any had an effect on erm(B)

distribution in the cattle feedlot pen (Table 2). Our

data did not support the idea that the erm(B) genes

are distributed across the entire feedlot pen over

time. Both erm(B) positive and negative surface

samples were compared for each sampling date and

location (mound versus edge) within the pen. Sig-

nificant differences were observed between erm(B)

positive and negative samples for VS, total N, pH,

and ECa. However, for most surface parameters on

a particular date and location, there were no differ-

ences between erm(B) positive and erm(B) negative

samples. Furthermore, when a significant difference

was observed between erm(B) positive and negative

samples in one set of circumstances (pen location

and date), that difference was not significant for any

of the other set of date and location combinations.

For instance, surface pH for June 2009 in pen edge

samples was lower in erm(B) positive compared to

erm(B) negative samples, but there were no differ-

ences in pH for these mound or edge samples in Au-

gust 2010 or in the mound samples for June 2009.

There were no clear linkage between erm(B) and abi-

otic environmental parameters of the feedlot surface

material such as temperature and pH (Table 2).

In this study, results are based on PCR assays and

therefore are not capable of detecting whether vi-

able antibiotic resistant microorganisms are present

in the environment, only whether a specific gene is

present in the environment. However, since there is

concern that antibiotic resistance genes from ani-

mal production settings may impact human health

via horizontal gene transfer (Brabban et al., 2005;

Colomer-Lluch et al., 2011a; b; Hawkey and Jones,

2009), the gene-based information is relevant when

considering issues of public health. An organism

does not need to be alive to contribute an antibiotic

resistance gene. The mechanism used by genes to

move through the environment to impact humans

remains unclear.

The addition of antibiotic resistant bacteria to the

feedlot surface is attributed to animal feces, but af-

ter the fecal bacteria leave the gastrointestinal tract

(GIT), they are exposed to a drier, more oxygenated

soil environment that quickly inactivates or kills many

gut microorganisms. The bacterial community found

on the feedlot surface material has been shown to

be very distinct from the composition of the individ-

ual animal’s GIT (Durso et al., 2011). So, even though

the original source of the erm(B) genes is assumed

to be fecal bacteria, once excreted from animals the

biological components of feces, such as the erm(B)

genes, display distribution and persistence patterns

that are different from those of the chemical and

physical components of the fecal material.

Finally, it must be noted that antibiotic resistance


Figure 1. Mapping of feedlot pens at two time points. Results of electromangnetic induction (EMI) maps of feedlot pens are displayed on a color scale. High EMI readings have been previ-ously correlated with areas of high manure deposition (Woodbury et al., 2009; Eigenberg et al., 2010). Results of the erm(B) screening locations are displayed using red dots to indicate of erm(B) positive samples and black dots to indicate erm(B) negative samples.

307 308 309 310 311

312 313 314 315 316

308 309 310 311

312 313 314 315 316

Jun

e 2

00

9A

ug

ust

20

10

erm(B) positive

erm(B) negative

307


Table 2. Feedlot pen surface composition at the edge and mound areas based upon the detec-tion of erm(B) at the beginning and end of the feedlot feeding trial. Bold numbers indicate data that was found to be statistically relevant.

June 2009 August 2010

erm(B) Positive

erm(B) Negative

erm(B) Posi-tive

erm(B) Negative

Pen Edge

Moisture, % 19.7 17.3 20.3 19.4

Volatile Solids, % 20.1 17.6 17.0 15.1

Total S, g/kg DM 2.4 2.6 2.5 2.3

Total N, g/kg DM 6.7 5.8 8.4 7.2†

Total P, g/kg DM 3.2 3.1 3.8 3.5

Total K, g/kg DM 9.8 10.3 9.2 8.5

Soil temperature, ºC 30.0 28.9 33.2 33.4

Surface temperature, ºC 43.3 39.2 43.2 41.3

Soil pH 7.7 8.1* 7.4 7.5

Shallow ECa‡, mS/m 171.2 192.0 171.1 138.8*

Deep ECa, mS/m 165.1 173.6 174.4 149.6

Pen Mound

Moisture, % 12.3 10.7 14.6 15.2

Volatile Solids, % 13.6 13.0 13.0 9.5*

Total S, g/kg DM 1.4 1.3 1.7 1.3†

Total N, g/kg DM 5.3 4.4 6.1 4.3*

Total P, g/kg DM 2.3 2.1 2.8 2.2†

Total K, g/kg DM 8.8 8.2 8.0 6.8

Soil temperature, ºC 30.0 30.8 33.1 33.6

Surface temperature, ºC 43.5 44.9 42.7 42.6

Soil pH 7.5 7.5 7.3 7.3

Shallow ECa, mS/m 124.5 121.3 119.2 102.0

Deep ECa, mS/m 135.0 134.6 127.6 107.5

*Means with a different letter within a row for a particular sample time differ at P < 0.05.

†Indicates a tendency (0.05<P < 0.1) for the erm(B) positive and negative samples to differ for that par-ticular sample date.

‡Apparent electrical conductivity as measured by Woodbury et al. (2009)


is complex, encompassing many different classes of

drugs and mechanisms of resistance. The dynamics

of erythromycin resistance, as coded for by erm(B),

is not necessarily the same as the dynamics of other

macrolide antibiotic resistance genes. There is not

currently enough information to determine how dif-

ferent kinds of antibiotic resistance genes persist

and move through agroecosystems, or how the data

collected here for erm(B) relates to distribution and

persistence of other antibiotic resistance genes.

Previous studies strongly support the idea that the

composition of resistance genes in any particular

habitat is a reflection of the species of bacteria that

are commonly found in each environment (Durso et

al., 2012; Patterson et al., 2007).

In conclusion, erm(B) genes were not enriched in

feedlot soils despite 14 months of manure accumu-

lation. Locations of high manure deposition were

not the same as the locations of the erm(B) gene

and the gene was not associated with specific feed-

lot pen zones. The dynamics of antibiotic resistance

in cattle feedlot pens is likely dependent on the spe-

cific antibiotic resistance gene being studied, and is

likely influenced by a number of biological, physical,

and chemical parameters of the soil.

ACKNOWLEDGEMENTS

Thanks are extended to Jennifer McGhee for pro-

viding technical assistance and guidance, as well as

Alan Kruger, Todd Boman, John Holman, Dale Jans-

sen, and Sue Wise for data collection and processing.

REFERENCES

Alexander, T.W., J.L. Yanke, T. Reuter, E. Topp, R.R.

Read, B.L. Selinger, and T.A. McAllister. 2011. Lon-

gitudinal characterization of antimicrobial resis-

tance genes in feces shed from cattle fed different

subtherapeutic antibiotics. BMC Microbiol. 11:19.

Anderson, J.F., T.D. Parrish, M. Akhtar, L. Zurek, and

H. Hirt. 2008. Antibiotic resistance of enterococci

in American Bison (Bison bison) from a nature pre-

serve compared to that of enterococci in pastured

cattle. Appl. Environ. Microbiol. 74:1726-1730.

Bae, W., K.N. Kaya, D.D. Hancock, D.R. Call, Y.H.

Park, and T.E. Besser. 2005. Prevalence and an-

timicrobial resistance of thermophilic Campylo-

bacter spp. from cattle farms in Washington state.

Appl. Environ. Microbiol. 71:169-174.

Böckelmann, U., H.H. Dörries, M.N. Ayuso-Gabella,

M. Salgot de Marçay, V. Tandoi, C. Levantesi, C.

Masciopinto, E. Van Houtte, U. Szewzyk, T. Wint-

gens, and E. Grohman. 2009. Quantitative PCR

monitoring of antibiotic resistance genes and

bacterial pathogens in three European artificial

groundwater recharge systems. Appl. Environ. Mi-

crobiol. 75:154-163.

Brabban, A.D., E. Hite, and T.R. Callaway. 2005. Evo-

lution of foodborne pathogens via temperate bac-

teriophage-mediated gene transfer. Foodborne

Pathog. Dis. 2, 287-303.

Chen, J, Z. Z. Yu, F.C. Michel, Jr., T. Wittum, and M.

Morrison. 2007. Development and application

of Real-time PCR assays for quantification of erm

genes conferring resistance to macrolides-lincos-

amides-sterptogramin B in livestock manure and

manure management systems. Appl. Environ. Mi-

crobiol. 73:4407-4416.

Colomer-Lluch, M., L. Imamovic, J. Jofre, and M.

Muniesa. 2011a. Bacteriophages carrying antibi-

otic resistance genes in fecal waste from cattle,

pigs, and poultry. Antimicrob. Agents Chemother.

55:4908–4911. doi: 10.1128/AAC.00535-11.

Colomer-Lluch, M., J. Jofre, and M. Muniesa. 2011b.

Antibiotic resistance genes in the bacteriophage

DNA fraction of environmental samples. PLoS One

6, e17549. doi: 10.1371/journal.pone.0017549.

De Leener, E., A. Martel, A. Decostere, and F. Haese-

brouck. 2004. Distribution of the erm (B) gene, tet-

racycline resistance genes, and Tn1545-like trans-

posons in macrolide- and lincosamide-resistant

enterococci from pigs and humans. Microb. Drug

Resist. 10:341-345.

Dogan, B., Y. H. Schukken, C. Santisteban, and K.

J. Boor. 2005. Distribution of serotypes and anti-

microbial resistance genes among Streptococcus

agalactiae isolates from bovine and human hosts.


J. Clin. Microbiol. 43:5899-5906.

Durso, L.M., G.P. Harhay, T.P. Smith, J.L. Bono, T.Z.

DeSantis, and M.L. Clawson. 2011. Bacterial com-

munity analysis of beef cattle feedlots reveals that

pen surface is distinct from feces. Foodborne Pat-

hog. Dis. 8:647-649. doi: 10.1089/fpd.2010.0774.

Durso, L.M., D.N. Miller, and B.J. Wienhold. 2012.

Distribution and quantification of antibiotic re-

sistant genes and bacteria across agricultural

and non-agricultural metagenomes. PLOS ONE

10.1371/journal.pone.0048325

Eigenberg,, R.A., B.L. Woodbury, J.A. Nienaber, M.J.

Spiehs, D.B. Parker, and V.H. Varel. 2010. Soil con-

ductivity and multiple linear regression for preci-

sion monitoring of beef feedlot manure and run-

off. J. Environ. & Eng. Geophys. 15:175-183.

Hawkey, P.M. and A.M. Jones. 2009. The changing

epidemiology of resistance. J Antimicrob Cher-

mother 64:i3-i10. doi: 10.1093/jac/dkp256.

Isaacson, R.E. and M.E. Torrence. 2002. The role of

antibiotics in agriculture. American Academy of

Microbiology, Washington, DC.

Jacob, M.E., Z.D. Paddock, D.G. Renter, K.F. Lech-

tenberg, and T.G. Nagaraja. 2010. Inclusion of

dried or wet distillers’ grains at different levels in

diets of feedlot cattle affects fecal shedding of

Escherichia coli O157:H7. Appl. Environ. Microbiol.

76:7238-7242. doi: 10.1128/AEM.01221-10.

Jost, B. H., H. T. Trinh, J. G. Songer, and S. J. Billing-

ton. 2004. A second tylosin resistance determinant,

Erm B, in Arcanobacterium pyogenes. Antimicrob.

Agents Chemother. 48:721-727.

Lesch, S.M., J.D. Rhoades, and D.L. Corwin. 2000.

ESAP-95 version 2.10R: User manual and tutorial

guide. Research Rep. 146. USDA–ARS, G.E. Brown,

Jr. Salinity Laboratory, Riverside, CA.

Miller, D.N., J.E. Bryant, E.L. Madsen, and W.C.

Ghiorse. 1999. Evaluation and optimization of

DNA extraction and purification procedures for soil

and sediment samples. Appl. Environ. Microbiol.

65:4715-4724.

Patterson, A. J., R. Colangeli, P. Spigaglia, and K.P.

Scott. 2007. Distribution of specific tetracycline

and erythromycin resistance genes in environmen-

tal samples assessed by macroarray detection.

Environ. Microbiol. 9: 703–715. doi: 10.1111/j.1462-

2920.2006.01190.x

Roberts, M.C., J. Sutcliffe, P. Courvalin, L.B. Jenses,

J. Rood, and H. Seppala. 1999. Nomenclature

for macrolide and macrolide-lincosamide-streto-

gramin B resistance determinants. Antimicrob.

Agents Chemother. 43:2823-2830.

Roberts, M.C. 2004. Distribution of macrolide, lincos-

amide, streptogramin, ketolide and oxazolidinone

(MLSK) resistance genes in Gram-negative bacte-

ria. Curr. Drug Targets Infect. Disord. 4:207-215.

Smith Thomas, H. 2009. The Cattle Health Hand-

book: preventative care, disease, treatments and

emergency procedures. Editors R. Boyd-Owens,

S. Guare, D. Burns. Storey Publishing, North Ad-

ams, MA , pp38-39.

Spiehs, M.J., D.N. Miller, B.L. Woodbury, R.A. Eigen-

berg, V.H. Varel, and D.B. Parker. 2012. Effect of

feeding wet distillers grains with solubles to beef

cattle on air and manure quality. Appl. Eng. Ag-

ricul. 28:423-430.

Unc, A. and M.J. Goss. 2004. Transport of bacteria

from manure and protection of water resources.

Appl. Soil Ecol. 25:1-18.

Wilson, S.C., R.C. Dobos, and L.R. Fell. 2010. Spec-

tral analysis of feeding and lying behavior of cattle

kept under different feedlot conditions. J. Appl.

Anim. Welfare Sci. 8:13-24.

Woodbury, B.L., D.N. Miller, J.A. Nienaber, and R.A.

Eigenberg. 2001. Seasonal and spatial variations of

denitrifying enzyme activity in feedlot soil. Trans.

ASAE 44:1635-1642.

Woodbury, B.L., S.M. Lesch, R.A. Eigenberg, D.N.

Miller, and M.J. Spiehs. 2009. Electromagnetic in-

duction sensor data to identify areas of manure ac-

cumulation on a feedlot surface. Soil Sci. Soc. Am.

J. 73:2068-2077.

World Health Organization. 2007. Critically impor-

tant antimicrobials for human medicine : catego-

rization for the development of risk management

strategies to contain antimicrobial resistance due to

non-human antimicrobial use : report of the second

WHO Expert Meeting, Copenhagen, 29-31 May.

http://www.who.int/foodborne_disease/resistance/

antimicrobials_human.pdf. Accessed May, 2013.


Shiga Toxin-Producing Escherichia coli (STEC) Ecology in Cattle and Management Based Options for Reducing Fecal Shedding T. R. Callaway, T. S. Edrington, G. H. Loneragan, M. A. Carr, D. J. Nisbet

39

Can Salmonella Reside in the Human Oral Cavity?S. A. Sirsat

30

Growth of Acetogenic Bacteria In Response to Varying pH, Acetate Or Carbohydrate Concentration

R. S. Pinder, and J. A. Patterson

6

Independent Poultry Processing in Georgia: Survey of Producers’ PerspectiveE. J. Van Loo, W. Q. Alali, S. Welander, C. A. O’Bryan, P. G. Crandall, S. C. Ricke

70

ARTICLES

Greenhouse Gas Emissions from Livestock and PoultryC. S. Dunkley and K. D. Dunkley

17

REVIEW




VOLUME 3 ISSUE 1


Linoleic Acid Isomerase Expression in Escherichia coli BL21 (DE3) and Bacillus sppS. Saengkerdsub

145

Current and Near-Market Intervention Strategies for Reducing Shiga Toxin-Producing Escherichia coli (STEC) Shedding in Cattle.

T. R. Callaway, T. S. Edrington, G. H. Loneragan, M. A. Carr, and D. J. Nisbet

103

Potential for Rapid Analysis of Bioavailable Amino Acids in Biofuel Feed Stocks D. E. Luján-Rhenals, and R. Morawicki

121

Isolation and Initial Characterization of Acetogenic Ruminal Bacteria Resistant to Acidic ConditionsP. Boccazzi and J. A. Patterson

129

ARTICLESConsumers’ Interest in Locally Raised, Small-Scale Poultry in GeorgiaE. J. Van Loo, W. Q. Alali, S. Welander, C. A. O’Bryan, P. G. Crandall, and S. C. Ricke

94




VOLUME 3 ISSUE 2

REVIEW


Antimicrobial Activity of Red Clover (Trifolium pratense L.) Extract on Caprine Hyper-Am-monia-Producing Bacteria M. D. Flythe, B. Harrison, I. A. Kagan, J. L. Klotz, G. L. Gellin, B. M. Goff, G. E. Aiken

176

Suitability of Various Prepeptides and Prepropeptides for the Production and Secretion of Heterologous Proteins by Bacillus megaterium or Bacillus licheniformis

S. Saengkerdsub, R. Liyanage, J. O. Lay Jr.

230

Utility of Egg Yolk Antibodies for Detection and Control of Foodborne SalmonellaP. Herrera, M. Aydin, S. H. Park, A. Khatiwara and S. Ahn

195

ARTICLES

Vibrio Densities in the Intestinal Contents of Finfish from Coastal Alabama J.L. Jones, R.A. Benner Jr., A. DePaola, and Y. Hara-Kudo

186

BRIEF COMMUNICATIONS




Potential for Dry Thermal Treatments to Eliminate Foodborne Pathogens on Sprout SeedsT. Hagger and R. Morawicki

218

REVIEW

VOLUME 3 ISSUE 3


Development of Non-Forage Based Incubation System For Culturing Ruminal Lipase-Pro-ducing Bacteria In VitroH. D. Edwards, R. C. Anderson, T. M. Taylor, R. K. Miller, M. D. Hardin, N. A. Krueger, D. J. Nisbet

293

Prevalence of Foodborne Pathogens and Spoilage Microorganisms and their Drug Resis-tant Status in Different Street Foods of DhakaZ. Tabashsum, I. Khalil, Md. N. Uddin, A.K.M. M. Mollah, Y. Inatsu and Md. L. Bari

281

Effect of Citrus Pulp on the Viability of Saccharomyces boulardii in the Presence of Enteric Pathogens

J. G. Wilson, T. C. McLaurin, J. A. Carroll, S. Shields-Menard, T. B. Schmidt, T. R. Callaway, and J. R. Donaldson

303

Persistence of erythromycin resistance gene erm(B) in cattle feedlot pens over timeA. R. Mantz, D. N. Miller, M. J. Spiehs, B. L. Woodbury, and L. M. Durso

312

ARTICLES

The Role of Cellular Prion Proteins (PrPC) on Neuronal Brucella InfectionsM. Aydin, D. F. Gilmore, S. Erdogan, V. Duzguner, and S. Ahn

268




VOLUME 3 ISSUE 4


MANUSCRIPT SUBMISSION

Authors must submit their papers electronically

([email protected]). According to instruc-

tions provided online at our site: www.afabjournal.

com. Authors who are unable to submit electroni-

cally should contact the editorial office for assistance

by email at [email protected].

INSTRUCTIONS TO AUTHORS

• Aerobic microbiology

• Aerobiology

• Anaerobic microbiology

• Analytical microbiology

• Animal microbiology

• Antibiotics

• Antimicrobials

• Bacteriophage

• Bioremediation

• Biotechnology

• Detection

• Environmental microbiology

• Feed microbiology

• Fermentation

• Food bacteriology

• Food control

• Food microbiology

• Food quality

• Food Safety

• Foodborne pathogens

• Gastrointestinal microbiology

• Microbial education

• Microbial genetics

• Microbial physiology

• Modeling and microbial kinetics

• Natural products

• Phytoceuticals

• Quantitative microbiology

• Plant microbiology

• Plant pathogens

• Prebiotics

• Probiotics

• Rumen microbiology

• Rapid methods

• Toxins

• Veterinary microbiology

• Waste microbiology

• Water microbiology

CONTENT OF MANUSCRIPT

We invite you to consider submitting your re-

search and review manuscripts to AFAB. The jour-

nal serves as a peer reviewed scientific forum for to

the latest advancements in bacteriology research

on Agricultural and Food Systems which includes

the following fields:


With an open access publication model of this

journal, all interested readers around the world can

freely access articles online. AFAB publishes origi-

nal papers including, but not limited to the types

of manuscripts described in the following sections.

Papers that have been, or are scheduled to be, pub-

lished elsewhere should not be submitted and will

not be reviewed. Opinions or views expressed in pa-

pers published by AFAB are those of the author(s)

and do not necessarily represent the opinion of the

AFAB or the editorial board.

MANUSCRIPT TYPES

Full-Length Research Manuscripts

AFAB accepts full-length research articles con-

taining four (4) figures and/or tables or more. AFAB

emphasizes the importance of sound scientific ex-

perimentation on any of the topics listed in the focus

areas followed by clear concise writing that describes

the research in its entirety. The results of experi-

ments published in AFAB must be replicated, with

appropriate statistical assessment of experimental

variation and assignment of significant difference.

Major headings to include are: Abstract, Introduc-tion, Materials and Methods, Results, Discussion (or Results and Discussion), Conclusion, Acknowl-edgements (optional), Appendix for abbreviations (optional), and References.

Manuscripts clearly lacking in language will be re-

turned to author without review, with a suggestion

that English editing be sought before the paper is

reconsidered. AFAB offers a fee based language

service upon request. Please contact [email protected] for more information about our fees

and services.

Rapid Communications

Under normal circumstances, AFAB aims for re-

ceipt-to-decision times of approximately one month or less. Accepted papers will have priority for publi-

cation in the next available issue of AFAB. However,

if an author chooses or requires a much more rapid

peer review, the journal editorial office has the capa-

bility to shorten the review timing to one week or less.

Any type of manuscript whether it be a full length

manuscript, brief communication or review paper can

be submitted as a rapid communication. There will be

additional costs for processing and page charges will

be double the normal rate. Authors who choose this

option must select Rapid Communications as the pa-

per type when submitting the paper and the editors

will judge whether a rapid review is possible and let

the author know immediately.

Brief Communications

Brief communications are short research notes giv-

ing the results of complete experiments but are con-

sidered less comprehensive than full-length articles

with three (3) figures and/or tables or less. Manuscripts

should be prepared with the same subheadings as full

length research papers. The running head above the

title of the paper is “Brief Communications.”

Unsolicited Review Papers

Review papers are welcome on any topic listed in

the focus section and have no page limits. Reviews

are assessed the same pages charges as all other

manuscripts. All AFAB guidelines for style and form

apply. Major headings to include are: Abstract, In-troduction, Main discussion topics and appropri-ate subheadings, Conclusions, Acknowledgements (optional) and References. Review papers shorter

than 20 pages of double spaced text and references

will be considered mini-reviews with the subhead-

ing above the title on the first page. The running

head above the title of the paper is either “Review”

or “Mini-review”.

Solicited Review Papers

Solicited reviews will have no page limits. The

editor-in-chief will send invitations to the authors

and then review these contributions when they are

submitted. Nominations or suggestions for potential

timely reviews are welcomed by the editors or edito-


rial board members and should be sent to submit@

afabjournal.com. There will be no page charges for

solicited review papers but the solicitation must origi-

nate from the editor-in-chief or one of the editors. Re-

quests from authors will automatically be classified as

unsolicited review papers. The running head above

the title of the paper will be “Invited Review.”

Conference and Special Issues Reviews

AFAB welcomes opportunities to publish papers

from symposia, scientific conference, and/or meet-

ings in their entirety. Conference organizers need

simply to contact AFAB at [email protected]

and a rapid decision is guaranteed. If in agreement,

the conference organizers must guarantee delivery

of a set number of peer reviewed manuscripts within

a specified time and submitted in the same format

as that described for unsolicited review papers. Con-

ference papers must be prepared in accordance with

the guidelines for review articles and are subject to

peer review. The conference chair must decide

whether or not they wish to serve as Special Issue

Editor and conduct the editorial review process. If

the conference chair/organizer chooses to serve as

special issue editor, this will involve review of the pa-

pers and, if necessary, returning them to the authors

for revision. The conference organizer then submits

the revised manuscripts to the journal editorial of-

fice for further editorial examination. Final revisions

by the author and recommendations for acceptance

or rejection by the chair must be completed by a

mutually agreed upon date between the editor and

the conference organizer. Manuscripts not meeting

this deadline will not be included in the published

symposium proceedings but if submitted later can

still be considered as unsolicited review papers. Al-

though offprints and costs of pages are the same

as for all other papers, the symposium chair may be

asked to guarantee an agreed upon number of hard

copies to be purchased by conference attendees. If

the decision is not to publish the symposium as a

special issue, the individual authors retain the right

to submit their papers for consideration for the jour-

nal as ordinary unsolicited review manuscripts.

Book Reviews

AFAB publishes reviews of books considered to

be of interest to the readers. The editor-in-chief ordi-

narily solicits reviews. Book reviews shall be prepared

in accordance to the style and form requirements of

the journal, and they are subject to editorial revision.

No page charges will be assessed solicited reviews

while unsolicited book reviews will be assigned the

regular page charge rate.

Opinions and Current Viewpoints

The purpose of this section will be to discuss, cri-

tique, or expand on scientific points made in articles

recently published in AFAB. Drafts must be received

within 6 months of an article’s publication. Opinions

and current perspectives do not have page limits.

They shall have a title followed by the body of the

text and references. Author name(s) and affiliation(s)

shall be placed between the end of the text and list

of references. If this document pertains to a par-

ticular manuscript then the author(s) of the original

paper(s) will be provided a copy of the letter and of-

fered the opportunity to submit for consideration a

reply within 30 days. Responses will have the same

page restrictions and format as the original opinion

and current viewpoint, and the titles shall end with

“Opinions.” They will be published together. Letters

and replies shall follow appropriate AFAB format

and may be edited by the editor-in-chief and a tech-

nical editor. If multiple letters on the same topic are

received, a representative set of opinions concern-

ing a specific article will be published. A disclaimer

will be added by the editorial staff that the opinion

expressed in this viewpoint is the authors alone and

does not necessarily represent the opinion of AFAB

or the editorial board.

COPYRIGHT AGREEMENT

The copyright form is published in AFAB as space

permits and is available online (www.afabjournal.com).


AFAB grants to the author the right of re-publication

in any book of which he or she is the author or edi-

tor, subject only to giving proper credit to the original

journal publication of the article by AFAB. AFAB re-

tains the copyright to all materials accepted for pub-

lication in the journal. If an author desires to reprint

a table or figure published from a non-AFAB source,

written evidence of copyright permission from an au-

thority representing that source must be obtained by

the author and forwarded to the AFAB editorial office.

PEER REVIEW PROCESS

Authors will be requested to provide the names

and complete addresses including emails of five (5) potential reviewers who have expertise in the research

area and no conflict of interest with any of the authors.

Except for manuscripts designated as Rapid Commu-

nication each reviewer has two (2) weeks to review

the manuscript, and submit comments electronically

to the editorial office. Authors have three (3) weeks

to complete the revision, which shall be returned to

the editorial office within six (6) weeks after which the

authors risk having their manuscript removed from

AFAB files if they fail to ask the editorial office for

an extension by email. Deleted manuscripts will be

reconsidered, but they will have to be processed as

new manuscripts with an additional processing fee as-

sessed upon submission. Once reviewed, the author

will be notified of the outcome and advised accord-

ingly. Editors handle all initial correspondence with

authors during the review process. The editor-in chief

will notify the author of the final decision to accept or

reject. Rejected manuscripts can be resubmitted only

with an invitation from the editor or editor-in chief. Re-

vised versions of previously rejected manuscripts are

treated as new submissions.

PRODUCTION OF PROOFS

Accepted manuscripts are forwarded to the edito-

rial office for technical editing and layout. The manu-

script is then formatted, figures are reproduced, and

author proofs are prepared as PDFs. Author proofs

of all manuscripts will be provided to the correspond-

ing author. Author proofs should be read carefully and

checked against the typed manuscript, because the

responsibility for proofreading is with the author(s).

Corrections must be returned by e-mail. Changes

sent by e-mail to the technical editor must indicate

page, column, and line numbers for each correction

to be made on the proof. Corrections can also be

marked using “track changes” in Microsoft Word or

using e-annotation tools for electronic proof correc-

tion in Adobe Acrobat to indicate necessary chang-

es. Author alterations to proofs exceeding 5% of the

original proof content will be charged to the author. All

correspondence of proofs must be agreed to by the

editorial office and the author within 48 hours or proof

will be published as is and AFAB will assume no re-

sponsibility for errors that result in the final publication.

PUBLICATION CHARGES

AFAB has two publication charge options: conven-

tional page charges and rapid communication. The

current charge for conventional publication is $25 per printed page in the journal. There is no additional

charge for the publication of pages containing color

images, micrographs or pictures. For authors who

wish to have their papers processed as a rapid com-

munication, authors will pay the rapid communication

fee when proofs are returned to the editorial office

in addition to twice the conventional page charges.

Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one

week and $100 per journal page.

HARD COPY OFFPRINTS

If you are wishing to obtain a physical hard copy of

the AFAB journal, offprints are available in any quan-

tity at an additional charge: $100/page for black-white

and $150/page for color prints. You may order your

offprints at any time after publication on our website.

Scientific conference organizers may be expected to

agree to a set number of offprints as a part of their

agreement with AFAB.


MANUSCRIPT CONTENT REQUIREMENTS

Preparing the Manuscript File

Manuscripts must be written in grammatically

correct English. AFAB offers a fee based language

service upon request ([email protected]).

Manuscripts should be typed double-spaced, with

lines and pages numbered consecutively. All docu-

ments must be submitted in Microsoft Word (.doc or

.docx, PC or Mac). All special characters (e.g., Greek,

math, symbols) should be inserted using the sym-

bols palette available in this font. Tables and figures

should be placed in separate sections at the end of

the manuscript (not placed in the text). Failure to fol-

low these instructions will cause delays of the pro-

cessing and review of the manuscript.

Title Page

At the very top of the title page, include a title of

not more than 100 characters. Format the title with

the first letter of each word capitalized. No abbre-

viations should be used. Under the title, the authors

names are listed. Use the author’s initials for both first

and middle names with a period (full-stop) between

initials (e.g., W. A. Afab). Underneath the authors, a

list affiliations must be listed. Please use numerical

superscripts after the author’s names to designate

affiliation. If an authors address has changed since

the research was completed, this new information

must be designated as “Current address:”. The cor-

responding author should be indicated with an aster-

isk e.g., * Corresponding author. The title page shall

include the name and full address of the correspond-

ing author. Telephone and e-mail address must also

be provided for the corresponding author, and email-addresses must be provided for all authors.

Editing

Author-derived abbreviations should be defined

at first use in the abstract and again in the body of

the manuscript. If abbreviations are extensive au-

thors may need to provide a list of abbreviations

at the beginning of the manuscript. In vivo, in vitro

and bacterial names must be italicized (obligatory).

Authors must avoid single sentence paragraphs and

merge such paragraphs appropriately. Authors must

not begin sentences with “Figure or Table shows…”

as these are inanimate objects and cannot “show”

anything. When number are reported in text or in ta-

bles, always put a zero in front of decimal numbers:

“0.10” instead of “.10”.

MANUSCRIPT SECTIONS

Abstract

The abstract provides an abridged version of the

manuscript. Please submit your abstract on a sepa-

rate page after the title page. The abstract should

provide a justification of your work, objectives, meth-

ods, results, discussion and implications of study or

review findings . Your abstract must consist of com-

plete sentences without references to other work or

footnotes and must not exceed 250 words. On the

same page as your abstract, please provide at least ten (10) keywords to be used for linking and index-

ing. Ideally, these keywords should include signifi-

cant words from the title.

Introduction

The introduction should clearly present the foun-

dation of the manuscript topic and what makes the

research or the review unique. The introduction

should validate why this topic is important based on

previously published literature, and the relevance of

the current research. Overall goals and project ob-

jectives must be clearly stated in the final sentence

of the last paragraphs of the introduction.

Materials and Methods

Information on equipment and chemicals used

must include the full company name, city, and state

(country if outside the United States or Province if

in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].


Variability, Replication, and Statistical Analysis

To properly assess biological systems indepen-

dent replication of experiments and quantification

of variation among replicates is required by AFAB.

Reviewers and/or editors may request additional

statistical analysis depending on the nature of the

data and it will be the responsibility of the authors

to respond appropriately. Statistical methods com-

monly used in the bacteriology do not need to be

described in detail, but an adequate description

and/or appropriate references should be provided.

The statistical model and experimental unit must

be designated when appropriate. The experimen-

tal unit is the smallest unit to which an individual

treatment is imposed. For bacterial growth stud-

ies, the average of replicate tubes per single study

per treatment is the experimental unit; therefore,

individual studies must be replicated. Repeated

time analyses of the same sample usually do not

constitute independent experimental units. Mea-

surements on the same experimental unit over time

are also not independent and must not be consid-

ered as independent experimental units. For analy-

sis of time effects, assess as a rate of change over

time. Standard deviation refers to the variability

in the biological response being measured and is

presented as standard deviation or standard error

according to the definitions described in statistical

references or textbooks.

Results

Results represent the presentation of data in

words and all data should be described in same

fashion. No discussion of literature is included in

the results section.

Discussion

The discussion section involves comparing the

current data outcomes with previously published

work in this area without repeating the text in the

results section. Critical and in-depth dialogue is

encouraged.

Results and Discussion

Results and discussion can be under combined or

separate headings.

Conclusions

State conclusions (not a summary) briefly in one

paragraph.

Acknowledgments

Acknowledgments of individuals should include

institution, city, and state; city and country if not U.S.;

and City or Province if in Canada. Copies being re-

viewed shall have authors’ institutions omitted to re-

tain anonymity.

References

a) Citing References In Text

Authors of cited papers in the text are to be pre-

sented as follows: Adams and Harry (1992) or Smith

and Jones (1990, 1992). If more than two authors of

one article, the first author’s name is followed by the

abbreviation et al. in italics. If the sentence structure

requires that the authors’ names be included in pa-

rentheses, the proper format is (Adams and Harry,

1982; Harry, 1988a,b; Harry et al., 1993). Citations to a

group of references should be listed first alphabeti-

cally then chronologically. Work that has not been

submitted or accepted for publication shall be listed

in the text as: “G.C. Jay (institution, city, and state,

personal communication).” The author’s own un-

published work should be listed in the text as “(J.

Adams, unpublished data).” Personal communica-

tions and unsubmitted unpublished data must not

be included in the References section. Two or more

publications by the same authors in the same year

must be made distinct with lowercase letters after

the year (2010a,b). Likewise when multiple author ci-

tations designated by et al. in the text have the same

first author, then even if the other authors are differ-

ent these references in the text and the references

section must be identified by a letter. For example


“(James et al., 2010a,b)” in text, refers to “James,

Smith, and Elliot. 2010a” and “James, West, and Ad-

ams. 2010b” in the reference section.

b) Citing References In Reference Section

In the References section, references are listed in

alphabetical order by authors’ last names, and then

chronologically. List only those references cited in the

text. Manuscripts submitted for publication, accepted

for publication or in press can be given in the refer-

ence section followed by the designation: “(submit-

ted)”, “(accepted)’, or “(In Press), respectively. If the

DOI number of unpublished references is available,

you must give the number. The year of publication fol-

lows the authors’ names. All authors’ names must be

included in the citation in the Reference section. Jour-

nals must be abbreviated. First and last page num-

bers must be provided. Sample references are given

below. Consult recent issues of AFAB for examples

not included in the following section.

Journal manuscript:

Examples:

Chase, G., and L. Erlandsen. 1976. Evidence for a

complex life cycle and endospore formation in the

attached, filamentous, segmented bacterium from

murine ileum. J. Bacteriol. 127:572-583.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van

Doesburg, and A. J. M. Stams. 2009. A typical

one-carbon metabolism of an acetogenic and

hydrogenogenic Moorella thermioacetica strain.

Arch. Microbiol. 191:123-131.

Book:

Examples:

Hungate, R. E. 1966. The rumen and its microbes

Academic Press, Inc., New York, NY. 533 p.

Book Chapter:

Examples:

O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.

Assessing consumer concerns and perceptions

of food safety risks and practices: Methodologies

and outcomes. In: S. C. Ricke and F. T. Jones. Eds.

Perspectives on Food Safety Issues of Food Animal

Derived Foods. Univ. Arkansas Press, Fayetteville,

AR. p 273-288.

Dissertation and thesis:

Maciorowski, K. G. 2000. Rapid detection of Salmo-

nella spp. and indicators of fecal contamination

in animal feed. Ph.D. Diss. Texas A&M University,

College Station, TX.

Donalson, L. M. 2005. The in vivo and in vitro effect

of a fructooligosacharide prebiotic combined with

alfalfa molt diets on egg production and Salmo-

nella in laying hens. M.S. thesis. Texas A&M Uni-

versity, College Station, TX.

Van Loo, E. 2009. Consumer perception of ready-to-

eat deli foods and organic meat. M.S. thesis. Uni-

versity of Arkansas, Fayetteville, AR. 202 p.

Web sites, patents:

Examples:

Davis, C. 2010. Salmonella. Medicinenet.com.

http://www.medicinenet.com/salmonella /article.

htm. Accessed July, 2010.

Afab, F. 2010, Development of a novel process. U.S.

Patent #_____

Author(s). Year. Article title. Journal title [abbreviated].

Volume number:inclusive pages.

Author(s) [or editor(s)]. Year. Title. Edition or volume (if

relevant). Publisher name, Place of publication. Number

of pages.

Author(s) of the chapter. Year. Title of the chapter. In:

author(s) or editor(s). Title of the book. Edition or vol-

ume, if relevant. Publisher name, Place of publication.

Inclusive pages of chapter.

Author. Date of degree. Title. Type of publication, such

as Ph.D. Diss or M.S. thesis. Institution, Place of institu-

tion. Total number of pages.


Abstracts and Symposia Proceedings:

Fischer, J. R. 2007. Building a prosperous future in

which agriculture uses and produces energy effi-

ciently and effectively. NABC report 19, Agricultural

Biofuels: Tech., Sustainability, and Profitability. p.27

Musgrove, M. T., and M. E. Berrang. 2008. Presence

of aerobic microorganisms, Enterobacteriaceae and

Salmonella in the shell egg processing environment.

IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)

Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Op-

tions and risks by using diagnostic gene chips. Pro-

gram and abstracts book , The 8th Biennieal Con-

gress of the Anaerobe Society of the Americas. p.

86 (Abstr.)

Data Presentation in Tables and Figures

Figures and tables to be published in AFAB must

be constructed in such a fashion that they are able

to “stand alone” in the published manuscript. This

means that the reader should be able to look at

the figure or table independently of the rest of the

manuscript and be able to comprehend the experi-

mental approach sufficiently to interpret the data.

Consequently, all statistical analyses should be very

carefully presented along with variation estimates

and what constitutes an independent replication

and the number of replicates used to calculate the

averages presented in the table or figure.

Each table and figure must be on a separate

page in the submitted paper. In addition, you will

need to submit all data for charts, tables and

figures in native format when possible (e.g., Mi-

crosoft Excel, Powerpoint). Photographs should

be submitted as high-resolution (600 dpi) .jpg or

tif. files. All figures should be clearly presented with

well defined axis and units of measurement. Sym-

bols, lines, and bars must be made distinct as “stand

alone” black and white presentations. Stippling,

dashed lines etc. are encouraged for multiple com-

parison but shades of gray are discouraged. Color

images, micrographs, pictures are recommended

and there is no additional fee for their submission.

AFAB Online Edition is Now Available!

www.AFABjournal.com

• Free Access

• Print PDFs

• Flip Through Issues

• Search Article Archives

• Order Reprints

• Submit a Paper

Online Publication: www.AFABjournal.com

afab volume 3 issue 4

Documents

university of houston

hangcornell university

kirkhamkansas state

kwon university of arkansas

dunkley university of

inaugural issue

crandalluniversity of

rickeuniversity of arkansas