antimicrobial resistance in escherichia coli, …

ANTIMICROBIAL RESISTANCE IN ESCHERICHIA COLI, KLEBSIELLA SPP., AND

STAPHYLOCOCCUS AUREUS ISOLATES FROM BOVINE MASTITIS IN CANADA

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

STINANILSSON

In partial fulfillment of requirements

for the degree of

Master of Science

May, 2011

©StinaNilsson, 2011

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ABSTRACT

ANTIMICROBIAL RESISTANCE IN ESCHERICHIA COLI, KLEBSIELLA SPP., AND STAPHYLOCOCCUS AUREUS ISOLATES FROM BOVINE MASTITIS IN CANADA

Stina Nilsson Advisor: University of Guelph, 2011 Dr. P. Boerlin

This study characterized the antimicrobial resistance (AMR) genes present in a

collection of Escherichia coli, Klebsiella spp., and Staphylococcus aureus isolates from

bovine mastitis cases in Canada. Ampicillin-resistant E. coli isolates along with second-

generation cephalosporin resistant Klebsiella spp., any MRSA, and a collection of

penicillin-resistant S. aureus isolates were screened for the presence of AMR genes and,

in the case of S. aureus, also virulence genes. A high diversity of P-lactamase genes was

detected in E. coli and Klebsiella spp. mastitis isolates in comparison to those found in

beef cattle fecal isolates by other researchers, and the ubiquitous blacMY-2 gene was found

in bacteria from mastitis. The P-lactam-resistant S. aureus isolates contained many blaz

gene variants. The first MRSA isolate from bovine mastitis in Canada was identified as

ST8, a sequence type normally associated with humans.

ACKNOWLEDGEMENTS

First and foremost I would like to thank my advisor Dr. Patrick Boerlin for his guidance

and support. His wide knowledge and passion for science has helped encourage and

inspire me over the last two years. I would also like to thank my committee members Dr.

John Prescott, Dr. J. Trenton McClure and Dr. David Kelton for all of their feedback and

direction. Special thanks to our laboratory technicians Gabhan Chalmers and Vivian

Nicholson for all of their assistance in the laboratory. Their patience and positive attitude

helped me succeed in the laboratory. I thank all of the other Boerlin lab members: Matt

Leslie, Jennie Pouget, Fiona Coutinho, Heidi Mascarenhas, Shaun Kernaghan and Walter

Wang for all of the friendships and laughs we had together. I am grateful to Dr. David

Pearl for all of his statistical help. I would also like to thank Philippe Garneau and Dr.

Josee Harel from the Universite de Montreal for allowing me to be a part of their

laboratory for two weeks and helping me to run classical microarrays. Thanks to the

Canadian Bovine Mastitis Research Network (CBMRN) and their funding agencies

including the Natural Science and Engineering Research Council, Alberta Milk, Dairy

Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait

Inc., Dairy Farmers of Canada, Canadian Dairy Network, Agriculture and Agri-Food

Canada, Public Health Agency of Canada, Technology PEI Inc., Universite de Montreal

and the University of Prince Edward Island. Finally, I would like to thank my parents

Anders and Birgitta, and my fiance George for all of their support along the way.

i

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

TABLE OF CONTENTS ii

LIST OF TABLES iv

LIST OF FIGURES vii

LIST OF ABBREVIATIONS viii

DECLARATION OF WORK x

INTRODUCTION 1

CHAPTER ONE: LITERATURE REVIEW 4

1. BOVINE MASTITIS 4

2. ANTIMICROBIALS AND ANTIMICROBIAL RESISTANCE IN MASTITIS ISOLATES 9

3. DEVELOPMENT AND TRANSFER OF RESISTANCE IN MASTITIS BACTERIA...23

4. MASTITIS AND ANTIMICROBIAL RESISTANCE 26

5. DETECTION OF ANTIMICROBIAL RESISTANCE AND ANTIMICROBIAL RESISTANCE GENES IN MASTITIS ISOLATES 35

6. THESIS PROPOSAL OVERVIEW 40

CHAPTER TWO: ANTIMICROBIAL RESISTANCE IN ESCHERICHIA COLI AND KLEBSIELLA SPP. FROM CANADIAN BOVINE MASTITIS ISOLATES. 45

ABSTRACT 45

INTRODUCTION 46

MATERIALS AND METHODS 47

RESULTS 52

DISCUSSION 54

ACKNOWLEDGEMENTS 61

CHAPTER THREE: p-LACTAM RESISTANCE IN STAPHYLOCOCCUS AUREUSFROM CANADIAN BOVINE MASTITIS ISOLATES 71

ABSTRACT 71

INTRODUCTION 72

ii

MATERIALS AND METHODS 73

RESULTS 76

DISCUSSION 78

ACKNOWLEDGEMENTS 83

DISCUSSION AND CONCLUSIONS 92

REFERENCES 97

APPENDIX 1: FREQUENCY OF ANTIMICROBIAL RESISTANCE AND ANTIMICROBIAL RESISTANCE GENES IN ESCHERICHIA COLI, KLEBSIELLA SPP. AND STAPHYLOCOCCUS AUREUS ISOLATES FROM MASTITIS 123

APPENDIX 2: GENE LIST FROM THE AMR-VE AND MRSAIDENTIBAC ARRAYTUBES 147

APPENDIX 3: DETERMINATION OF ANTIMICROBIAL RESISTANCE IN ESCHERICHIA COLI AND KLEBSIELLA SPP. FROM CANADIAN BOVINE MASTITIS ISOLATES A/aPSE-i PLASMID GENES AND POLYMERASE CHAIN REACTION CONDITIONS 157

APPENDIX 4: FREQUENCY OF VIRULENCE GENES IN STAPHYLOCOCCUS AUREUSFROM CANADIAN BOVINE MASTITIS ISOLATES 160

i i i

LIST OF TABLES

Table 1. Antimicrobial susceptibility testing results for ampicillin-resistant E. coli (n=42)

and Klebsiella spp. (n=19) from bovine mastitis in Canada 62

Table 2. Frequency of AMR and integrase genes among 42 ampicillin-resistant E. coli

from bovine mastitis in Canada 64

Table 3. Frequency of AMR and intll genes among multi-resistant and susceptible

Klebsiella spp. isolates from bovine mastitis in Canada 65

Table 4. Agreement between susceptibility testing results and genotypes for E. coli

antimicrobial resistance genes tested 66

Table 5. Associations between P-lactamases and other resistance determinants 67

Table 6. Associations of virulence genes with resistant or susceptible S. aureus isolates

from bovine mastitis 85

Table 7. Number and identity of blaz variants found within farms across Canada 87

Table 8. MLST analysis of fifteen penicillin-resistant and nine susceptible S. aureus

isolates 88

Table 9. Frequency of P-lactam resistance and resistance genes in Escherichia coli from

bovine mastitis milk isolates 123

Table 10. Frenuencv of ^-lactam resistance and resistance °enes in S. aureus from bovine

mastitis milk isolates 126

Table 11. Frequency of P-lactam resistance and resistance genes in Klebsiella spp. from


Table 12. Frequency of tetracycline resistance and resistance genes in E. coli from bovine

mastitis milk isolates 132

iv

Table 13. Frequency of tetracycline resistance and resistance genes in S. aureus from


Table 14. Frequency of tetracycline resistance and resistance genes in Klebsiella spp.

from bovine mastitis milk isolates 135

Table 15. Frequency of aminoglycoside resistance and resistance genes in E. coli from


Table 16. Frequency of aminoglycoside resistance and resistance genes in S. aureus from


Table 17. Frequency of aminoglycoside resistance and resistance genes in Klebsiella spp.


Table 18. Frequency of sulfonamide resistance and resistance genes in E. coli from


Table 19. Frequency of sulfonamide resistance and resistance genes in S. aureus from


Table 20. Frequency of sulfonamide resistance and resistance genes in Klebsiella spp.


Table 21. Frequency of macrolide and lincosamide resistance in Staphylococcus aureus


Table 22. Distribution of macrolide-lincosamide-streptogramin (MLS) resistance

determinants in Escherichia coli, Klebsiella spp., and Staphylococcus aureus (Roberts

2008) 146

Table 23. Genes present on the AMR-ve ArrayTube 147

Table 24. Genes present on the MRSA ArrayTube 151

v

Table 25. Genes of interest detected on the blapsE-i plasmid 157

Table 26. Polymerase chain reaction conditions for the detection of the cassette array

containing bla?sE-i i n£ coli isolates from chicken and swine 159

Table 27. Frequency of virulence genes among penicillin-resistant and susceptible &

aureus from bovine mastitis in Canada 160

vi

LIST OF FIGURES

Figure 1. Promoter mutations of the ampC gene 68

Figure 2. blacyw-i dendrogram representing the similarity between restriction profiles of

blacMY-2 plasmids from bovine mastitis E. coli (OVC EC218, OVC EC2415), plasmids

from beef cattle, and representative blacMY-2 plasmids from each of four other replicon

types 69

Figure 3. WapsE-i class 1 integron structure and PCR primer positions 70

Figure 4. Neighbour-Joining plot depicting the diversity of the blaz gene in bovine

mastitis 89

Figure 5. Neighbour-Joining plot depicting the diversity of the MLST sequence types in

bovine mastitis 90

Figure 6. SCCmec type IVc from the bovine mastitis MRSA isolate SA822 90

vii

LIST OF ABBREVIATIONS

AAC AHL AMC AMK AMP AMR ANT APH ATP BLAST CA-MRSA CBMRN CHL CIP CIPARS CLSI CMT CNF CRO DCT DHPS DNA dUTP ESBL ESCMID ETEC EUCAST FOX GEN GI HACCP HGT IM IMI IS KAN MIC MLS MLST MRSA

Aminoglycoside acetyltransferase Animal Health Laboratory Amoxicillin-clavulanic acid Amikacin Ampicillin Antimicrobial resistance Aminoglycoside nucleotidyl transferase Aminoglycoside phosphotransferase Adenosine triphosphate Basic local alignment search tool Community-acquired MethiciUin-resistant Staphylococcus aureus Canadian Bovine Mastitis Research Network Chloramphenicol Ciprofloxacin Canadian Integrated Program for Antimicrobial Resistance Surveillance Clinical and Laboratory Standards Institute Caliornia Mastitis Test Cytotoxin necrotizing factor Ceftriaxone Dry cow therapy Dihydropteroate synthase Deoxyribonucleic acid Deoxyuridine triphosphate Extended spectrum P-lactamase European Society of Clinical Microbiology and Infectious Diseases Enterotoxigenic Escherichia coli The European Committee on Antimicrobial Susceptibility Testing Cefoxitin Gentamicin Genomic island Hazard analysis and critical control points Horizontal gene transfer Intramammary Intramammary Infection Insertion sequence Kanamycin Minimum inhibitory concentration Macrolides-lincosamides-streptogramin Multi-locus sequence typing MethiciUin-resistant Staphylococcus aureus

viii

MUE NA NAL NARMS NCCLS NJ PBP PCR PTSAg RFLP RNA rRNA

sec SCCmec SGI SOX ST STR SXT TET TIO USA

Median unbiased estimate Not available Nalidixic acid National Antimicrobial Resistance Monitoring System National Committee for Clinical Laboratory Standards Neighbour-j oining Penicillin-binding protein Polymerase Chain Reaction Pyrogenic toxin superantigens Restriction fragment length polymorphism Ribonucleic acid Ribosomal ribonucleic acid Somatic cell count Staphylococcal cassette chromosome mec Salmonella genomic island 1 Sulfisoxasole Sequence type Streptomycin Trimethoprim-sulfamethoxazole Tetracycline Ceftiofur United States of America

ix

DECLARATION OF WORK

The work presented in this thesis was performed by me, with the following exceptions:

1) The sampling of the E. coli, Klebsiella spp., and S. aureus isolates was performed

by the Canadian Bovine Mastitis Research Network (CBMRN).

2) The E. coli, Klebsiella spp., and S. aureus isolates were provided and the

susceptibility testing was performed by Dr. J. Trenton McClure and Matt Saab

from the University of Prince Edward Island, Charlottetown, Prince Edward

Island.

3) An additional five ampicillin-resistant E. coli isolates were provided by the

Animal Health Laboratory, University of Guelph.

4) An additional five ampicillin-resistant E. coli isolates were provided by Ministere

de rAgriculture, des Pecheries et de 1'Alimentation du Quebec (MAPAQ).

5) Nineteen E. coli isolates known to include 6/flpsE-i were provided from the

Canadian Integrated Program for Antimicrobial Resistance Surveillance

(CIPARS), Public Health Agency of Canada, Guelph, Ontario.

6) Antimicrobial susceptibility testing of the blacuY-2 positive isolates and the ten E.

coli from MAPAQ and the AHL was performed by the AMR Laboratory,

Laboratory for Foodbome Zoonoses, Public Health Agency of Canada, Guelph,

Ontario.

7) E. coli serotyping was performed by Kim Ziebell and collaborators at the

Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph,

Ontario.

x

8) Plasmid sequencing was performed at the National Microbiology Laboratory,

Winnipeg, Canada.

9) Rep-typing of the blacuY-i plasmids was performed by Gabhan Chalmers.

10) Sequencing by primer-walking on the E. coli blapsE-i positive isolates was

performed by Fiona Coutinho.

11) Help to run the classical slide microarray for the Klebsiella spp. isolates was

provided by Philippe Garneau and Dr. Josee Harel from the Universite de

Montreal.

12) The sequencing reactions were performed at the Guelph Molecular Supercentre,

Laboratory Services Division, University of Guelph, Ontario.

xi

INTRODUCTION

Despite numerous control measures, mastitis, or "inflammation of the mammary

glands", remains the most common disease in dairy cattle and a great problem for milk

production. It often leads to a decreased amount of milk, the production of low quality

milk and to expensive treatments, and, as such, is the most costly disease in the dairy

industry (Philpot and Nickerson 2000). Mastitis is mainly caused by microorganisms,

including Escherichia coli, Staphylococcus aureus and Klebsiella spp., and is responsible

for the majority of antimicrobial use on dairy farms (Bradley 2002), in particular the 0-

lactams. Usage of antimicrobials for both therapy and prevention of bovine mastitis may

lead to antimicrobial resistance (AMR) in agents of mastitis, making treatment less

effective. Selection pressure for the development of AMR occurs in the cow's udder, in

the gastrointestinal tract, on the skin and mucosa as well as in or on humans handling the

animals.

AMR is either intrinsic or acquired, and is often caused by the presence of specific

resistance determinants (Aarestrup 2006). These determinants act by a variety of

mechanisms. For example, resistance to p-lactam antimicrobials is caused mainly by |3-

lactamase enzymes which hydrolyze the P-lactam ring (Walsh 2000). Most AMR genes

are readily transmissible within and between microorganisms and may be selected for by

antimicrobial usage. The major transfer mechanisms for AMR genes are conjugation,

transformation and transduction (Schwarz and Chaslus-Dancla 2001). Thus,

microorganisms from unpasteurized milk may have the ability to spread resistance

determinants through the food chain, potentially resulting in treatment failures in human

infections.

1

Within Canada, little information is currently available regarding the prevalence of

AMR in mastitis bacteria from dairy cattle and no studies have looked at the genetic

determinants responsible for resistance. Very few of the studies done within Canada

provide a cross-Canada viewpoint. Only one study by Sabour and collaborators looking

at resistance profiles from a limited number of S. aureus isolates from bovine mastitis did

so in several provinces (i.e., Ontario, Quebec, and Prince Edward Island) (Sabour et al.

2004). Broader and more in depth investigations of AMR and its determinants would aid

in gaining a better understanding of the epidemiology of resistance in major agents of

bovine mastitis in Canada. The Canadian Bovine Mastitis Research Network sampled a

total of 89 dairy herds between 2007 and 2008 in six different provinces across Canada

(Reyher et al. 2011) and therefore provides the perfect opportunity for such an attempt

and for this project in particular.

In the following thesis, resistance genes from E. coli, Klebsiella spp., and S. aureus

isolates from bovine mastitis across Canada were characterized using molecular

techniques. The objectives of this project were:

1. To identify the resistance genes present in the bovine mastitis isolates with emphasis

on the P-lactamase genes and to analyze associations between resistance genes and

similarities between phenotypic and <*enotvnic identification methods.

2. To characterize P-lactamase genes of particular epidemiological importance and their

genetic environment in E. coli and/or Klebsiella spp., including the blacuY-2 and W#PSE-I

genes.

3. To type any methicillin-resistant S. aureus (MRS A) and to characterize its methicillin-

resistance determinant in order to relate them to other MRSA in a global context.

2

4. To determine the sequence diversity of the blaz genes encoding penicillin-resistance in

Canadian bovine S. aureus isolates and to relate it to other characteristics of the

corresponding strains.

3

CHAPTER ONE: LITERATURE REVIEW

1. BOVINE MASTITIS 1.1 Introduction

Considered to be the most costly disease in dairy cattle, mastitis or 'inflammation

of the mammary gland', accounts for huge economic losses in the dairy industry (Philpot

and Nickerson 2000). Mastitis is found in 10-50% of cow quarters at any given time. It is

mostly subclinical in nature and difficult to detect. Clinical intra-mammary infections

(IMIs) are easier to detect but less frequent (Kim and Heald 1999). Mastitis can be caused

by physical injury or chemical irritation of the udder, as well as by many different

microorganisms including bacteria, yeasts and algae (Bradley 2002). Over 80% of IMIs

are caused by the following microorganisms: Escherichia coli, Staphylococcus aureus,

Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis (Bradley

2002). Of these bacteria, S. aureus and S. agalactiae are considered contagious pathogens

adapted to survive within the host. In contrast, the Enterobacteriacae, including E. coli

and Klebsiella spp., are classified as environmental bacteria which act as opportunistic

pathogens (Bradley 2002). The Enterobacteriacae inflict damage through the release of

toxins and cell surface structures leading to an increase in somatic cell count (SCC)

(Philpot and Nickerson 2000).

Mastitis may also play a role in public health. As the microorganisms spread, the

SCC in the milk increases causing production of low quality milk which usually has

higher bacterial counts even after pasteurization (Philpot and Nickerson 2000).

Furthermore, because most antimicrobial applications on the dairy farm are due to

mastitis, humans drinking unpasteurized milk may be exposed to microorganisms

4

carrying antimicrobial resistance genes which may cause treatment problems for human

infections.

1.2 Subclinical Mastitis

Overview. A normal mammary gland produces milk with a SCC of less than 50,000

cells/mL. Cows with an excess of 50,000 cells/mL milk, bacteria in the milk, and

decreased production of milk but no other visible signs of disease are considered to have

subclinical mastitis (Harmon 1994, Chebel 2007). Subclinical mastitis frequently goes

unnoticed and is a major cause of economic loss through decreased milk production

(Philpot and Nickerson 2000). Although subclinical and clinical mastitis can be caused

by the same pathogens, chronic subclinical mastitis is mostly caused by contagious

pathogens, in particular by S. aureus (Harmon 1994).

Diagnosis of Subclinical Mastitis. Due to the lack of clinical signs, subclinical mastitis

is diagnosed by measuring the SCC and by repeated bacteriological culture of the same

causative agent (Erskine et al. 2003). Subclinical mastitis is usually detected during

monthly SCC counts done by a milk-recording organization (Philpot and Nickerson

2000). A California Mastitis Test (CMT) is normally used to test the SCC at the cow-side

(Harmon 1994).

Treatment of Subclinical Mastitis. Treatment of subclinical mastitis is typically carried

out by the infusion of antimicrobials into the udder. Because subclinical mastitis is not

life-threatening and does not result in rapid gland loss, a bacterial culture can be

performed before antimicrobial treatment, enabling bacterial identification and

antimicrobial susceptibility testing for a more targeted and appropriate treatment strategy

(Erskine et al. 2003).

5

Overall, antimicrobial treatment of subclinical mastitis is variably effective and

only slightly increases cure rates (Wilson et al. 1999). However, this depends on the

causative agent. S. agalactiae is generally susceptible to most intramammary

antimicrobials, and is relatively easy to treat with P-lactams. Therapy results in a cure rate

of 70-90% (Erskine et al. 2003) compared to rates as low as 27% without treatment

(Wilson et al. 1999). P-lactams are also used for treating S. aureus infections. However,

S. aureus infections are often chronic, with abscess formation, resulting in poor drug

distribution within the mammary gland. This, along with more frequent antimicrobial

resistance and the ability of S. aureus to survive the host immune response in various

ways, results in significantly lower cure rates (Erskine et al. 2003). Cure rates of 43%

have been reported for S. aureus mastitis after treatment, which do not differ significantly

from the self-cure rates of untreated cases (Wilson et al. 1999). However, targeted

therapy based on susceptibility testing can help increase cure rates. Overall, the decision

to treat a cow with subclinical mastitis should depend upon a number of factors including

which microorganism is causing the infection, antimicrobial susceptibility of the

pathogen, stage of lactation, and health of the cow.

1.3 Clinical Mastitis

Overview. Clinical mastitis is typically associated with abnormal milk comppsition,

appearance, and/or inflammation of the mammary gland (Chebel 2007). Severe clinical

mastitis, most often caused by coliforms, is defined as an IMI which includes systemic

involvement whereas mild clinical mastitis is described as an IMI with abnormal milk, no

systemic signs and with or without local signs of inflammation (Erskine et al. 2003,

Roberson 2003). Coliform mastitis generally starts with a large multiplication of

6

microorganisms that are then phagocytosed (Erskine et al. 2003). Thus, clinical mastitis

caused by coliforms is frequently not diagnosed until after peak bacterial numbers and

antimicrobial therapy may come too late to be effective.

Diagnosis of Clinical Mastitis. Unlike subclinical mastitis, the signs of clinical

mastitis are readily visible. A preliminary diagnosis without bacteriological culture, for

acute mastitis, is normally based upon the clinical signs and known pathogens within the

herd (Sanford et al. 2006).

Treatment of Clinical Mastitis

Severe Clinical Mastitis. Due to the systemic effects of severe clinical mastitis,

supportive care with administration of fluids, anti-inflammatories and frequent stripping

of the affected quarter, is the first response to treating this type of IMI. Intramammary

antimicrobials are typically not an effective treatment solution because the inflammatory

response itself often quickly eliminates the microorganisms (Roberson 2003). For this

reason, approximately 40% of severe clinical mastitis cultures provide no growth and

another 40% show the presence of coliforms (Roberson 2003). For the remaining 20%,

the causative agent is a Gram-positive microorganism and antimicrobial agents targeted

towards Gram-positive bacteria are utilized (Sanford et al. 2006).

Mild Clinical Mastitis. Mild clinical mastitis is not an immediate danger to the cow

and bacteriological samples of the milk can be obtained and analysed before treatment.

Only if the microorganism is Gram-positive is antimicrobial treatment is recommended.

7

1.4 Epidemiology of Mastitis

Contagious Mastitis. The major pathogens of contagious mastitis are S. aureus,

Streptococcus agalactiae and Mycoplasma, especially M. bovis. These microorganisms

are normally found on the cow udder and teat skin. Furthermore, certain S. aureus strains

have a higher chance of survival on the teat skin due to the presence of particular

virulence factors (Piccinini et al. 2009). S. aureus is often transmitted from cow to cow

through milking machines, hands, udder cloths and teat cup liners (Singh et al. 2006).

Thus, strict hygiene and proper maintenance of milking equipment are imperative in the

control of S. aureus mastitis.

Environmental Mastitis. The major coliforms involved in environmental mastitis are

Escherichia spp., Klebsiella spp., and Enterobacter spp. (Hogan and Smith 2003). These

bacteria are not normally transferred from cow udder to cow udder, but reside in the

environment and are mainly of fecal origin. More specifically, coliforms are found in

bedding material and manure, which are the two primary sources of infection (Hogan and

Smith 2003). Risk factors involved in the onset of the infection include high temperature

and humidity (Hogan and Smith 2003). Although most coliform mastitis is not peracute,

environmental mastitis accounts for approximately 70% of all peracute cases, of which

those caused by Klebsiella spp. are among the most dangerous (Hogan and Smith 2003).

In a study of the incidence rate of clinical mastitis in Canada, the most common mastitis

pathogen causing clinical mastitis was S. aureus (21.7%), followed by E. coli (17.6%), S.

uberis (13.3%), coagulase-negative staphylococci (10.1%), and Klebsiella spp. (9.1%)

(Olde Riekerink et al. 2008).

8

Mastitis Prevention. In order to prevent both contagious and environmental mastitis in

Canada, mastitis preventation practices have been adopted. More specifically, a Canadian

Quality Milk Program has been developed by the Dairy Farmers of Canada using a

Hazard Analysis and Critical Control Points (HACCP) approach. This plan includes:

proper milking hygiene, including the use of individual towels; for each cow, pre-dipping

of teats in disinfectants before milking; proper adjustment and maintenance of milking

machines to avoid milk reflux; and teat dipping after milking. Finally, subclinical mastitis

must be detected and treated promptly, and cows with chronic infections should be culled

because these cows often harbour contagious organisms which can easily be spread

through the herd (Philpot and Nickerson 2000). Systematic dry cow therapy is another

preventative measure which aids in eradicating or reducing contagious mastitis from a

herd. Additional elements in the prevention of environmental mastitis include choice of

bedding material and quality of drinking water (Sandholm et al. 1995). The best way to

ensure that mastitis is not a major problem within a dairy herd is through the

implementation of a broad palette of preventative measures. The use of an antimicrobial

agent is only one among the numerous components of mastitis control strategies and in

order to maintain their effectiveness, both for veterinary and human medicine, particular

care should be taken to keep their use ̂ rudent.

2. ANTIMICROBIALS AND ANTIMICROBIAL RESISTANCE IN

MASTITIS ISOLATES

2.1 Organisms of Interest

Escherichia colu Members of the genus Escherichia are straight Gram-negative

motile rods and belong to the Enter-obacteriaceae family (Brenner et al. 2001).

9

Escherichia coli is the most important commensal within its genus and colonizes the

lower ileum and large intestine of most vertebrates. It is a dominant facultative anaerobic

organism found in feces (Songer and Post 2005). Although most strains are non

pathogenic, E. coli is a major cause of ruminant mastitis, canine pyometra, urinary tract

infection, as well as septicemia and enteritic infections in mammals (Songer and Post

2005). Pathogenic E. coli strains are classified into intestinal and extra-intestinal

pathogens. Extra-intestinal pathogenic E.coli can cause opportunistic systemic and local

infections such as septicemia, encephalitis in newborns, mastitis, urinary tract infections,

and many others.

In a study by Lipman and collaborators, 20 mastitis isolates from the Netherlands

were tested for the presence of toxins and adhesins. Four of these strains produced F17

fimbriae and only one strain produced a CNF toxin (Lipman et al. 1995). Wenz and

collaborators also found that only 10% of their clinical mastitis isolates from the United

States contained a virulence gene and no association with severity of disease was

detected (Wenz et al. 2006). An absence of specific virulence genes in E. coli associated

with bovine mastitis has also been identified in other studies (Shpigel et al. 2008, Suojala

et al. 2011). Environmental E. coli generally contaminate in and around the teat orifice

and make their way into the lumen (Gyles et al. 2004). Subsequently, bacteria rapidly

spread until neutrophils migrate to the gland and remove the microorganisms by

phagocytosis (Erskine et al. 2003). This releases endotoxin, which activates both the

inflammatory and immune response cascades and thus results in the visible symptoms of

clinical mastitis.

10

Klebsiella spp. Klebsiella spp. are nonmotile, facultatively anaerobic,

encapsulated organisms (Brenner et al. 2001). These bacteria are commonly found in the

environment in surface water, sewage, soil and plants as well as on the mucosal surfaces

of mammals. The infections caused by Klebsiella spp. are generally opportunistic, and

Klebsiella pneumoniae has been known to cause bovine mastitis, equine metritis, navel

ill/joint ill and neonatal septicemia in foals, calves and kids (Songer and Post 2005). As

an agent of bovine mastitis, Klebsiella spp., mainly K pneumoniae and K. oxytoca, are

difficult to treat due to their poor response to antibiotic therapy as well as the severity of

infection with a fatality rate as high as 80% (Paulin-Curlee et al. 2008). Out of 16

Klebsiella spp. isolates collected from clinical cases, 15 were K. pneumoniae and only

one was K oxytoca (Munoz et al. 2007). Little information regarding the specific

virulence factors found in Klebsiella spp. from mastitis is available. Gundogan and Yaker

isolated Klebsiella spp. including K pneumoniae from milk products in Turkey and

determined that 67% produced siderophores, and 72% produced hemolysin (Gundogan

and Yakar 2007). The significance of these virulence factors in the pathogenesis of

mastitis is difficult to evaluate because the microorganisms from this study might have

originated from the processing of the dairy products and not directly from the mastitic

i-»r\ii7C5 -rviilV v\_r vv o n i i i A . .

Staphylococcus aureus. Staphylococcus spp. are Gram-positive, aerobic cocci

(Brenner et al. 2001). These organisms are mainly present on the skin and less frequently

in the throat, mouth, nose, mammary glands and the intestinal tract of mammals (Kloos

1980). Most infections caused by staphylococci occur when the epithelial barrier is

breached. S. aureus is the major pathogenic species of this genus and can cause

11

septicaemia and a variety of purulent infections in many animal species. It is the major

and most ubiquitous coagulase-positive Staphylococcus found in animals and humans

(Songer and Post 2005).

In bovine mastitis, S. aureus enters the teat, colonizes the teat canal and

establishes in the secretory tissue by adhesion to the ductular and alveolar mammary-

gland epithelial cells (Gyles et al. 2004). Once attached, the microorganism begins to

multiply and release cytotoxigenic substances leading to neutrophil infiltration. The

cytotoxigenic substances released by S. aureus in the mammary gland include, among

others, the a- and P-haemolysins as well as a toxin of the leukocidin family and the

coagulase enzyme (Haveri et al. 2007). These exotoxins induce an inflammatory

response, deactivate the immune system and degrade tissues providing additional

nutrients for bacterial growth. Also, 60-70% of S. aureus strains isolated from bovine

mastitis produce pyrogenic toxin superantigens (PTSAg) which depress the immune

system and may aid in the persistence of subclinical mastitis (Haveri et al. 2007). Not all

of the virulence factors involved in bovine mastitis are currently understood. However,

persistant IMI strains seem to regularly carry the sed and sej virulence genes which

encode staphylococcal enterotoxins and the P-lactamase gene blaz (Haveri et al. 2007).

Another study conducted in Italy, mainly detected the presence of seg, set, sent, sen, and

seo enterotoxins (Piccinini et al. 2010). Specific S. aureus strains are able to resist

phagocytosis by neutrophils and thus are found within epithelial cells, neutrophils and

macrophages (Gyles et al. 2004). Aggregation of neutrophils and S. aureus in the

mammary gland leads to obstruction of lobules and eventually involution. Necrotic foci

can form which often develop further into abscesses.

12

There has also been evidence of an adaptation of some S. aureus strains to the

bovine udder. Specific multilocus sequence types and genotypes have been associated

only with bovine mastitis indicating adaptation of these specific strains. These sequence

types have also been shown to be 2.5 times more difficult to cure than sequence types

that are not bovine specific (van den Borne et al. 2010).

2.2 Antimicrobials

Introduction. Antimicrobial drugs inhibit the growth or kill bacteria by taking advantage

of structural differences between a host and a parasite. Many antimicrobial agents

(including antibiotics) are derived from substances produced by naturally occurring

bacteria or fungi (Walsh 2000). The most common types of naturally occurring

antimicrobials used in medicine include the penicillins and cephalosporins which are

produced by fungi as well as Streptomyces strains. Presently, beside a number of minor

agents, the major classes of antimicrobials in existence and clinical use are the

aminoglycosides, the macrolide-lincosamide-streptogramin family, the pMactams

(cephalosporins and penicillins), the quinolones, the tetracyclines, the phenicols, and the

polymyxins (Giguere et al. 2006). Their activities rely on five major mechanisms of

action: inhibition of cell wall synthesis (P-lactams, bacitracin, vancomycin), damage to

cell membrane function (polymyxins), hindrance of nucleic acid synthesis (quinolones),

inhibition of protein synthesis (aminoglycosides, phenicols, tetracyclines), and inhibition

of folic acid synthesis (sulfonamides) (Walsh 2000).

Antimicrobial Action. The synthesis of peptidoglycan is a major step in cell-wall

biosynthesis targeted by drugs that inhibit transpeptidase enzymes which connect

adjacent peptide strands (Walsh 2000). Another group of target enzymes for cell-wall

13

biosynthesis are the transglycosylases which act on glycan strands and are able to extend

these sugar chains through new peptidoglycan incorporation. Antimicrobials act as

pseudo-substrates by acylating the active sites of the above mentioned enzymes.

Subsequent failure to make peptidoglycan links lead to a weaker cell wall and bacterial

lysis, along with other mechanisms, which result in cell death but have not yet been

characterized precisely (Walsh 2000). Of the major antimicrobial groups used to treat

mastitis, the P-lactams act by this mechanism (Giguere et al. 2006). P-lactams hinder

peptidoglycan synthesis by inhbiting transpeptidase or other peptidoglycan-active

enzymes called penicillin-binding proteins (PBPs) by strong covalent binding.

Antimicrobials which affect protein synthesis inhibit important steps or cause

misreading and the production of nonsense products in the initiation, elongation and

termination or protein assembly by the ribosome. The tetracyclines block the elongation

phase by binding to a site on the 30S ribosomal subunit which interferes with the binding

of the aminoacyl transfer RNA to the RNA-ribosome complex (Wax et al. 2008). The

lincosamides and macrolides, including erythromycin, bind the 5 OS ribosomal subunit,

preventing transpeptidation (Bryskier 2005). The aminoglycosides, including

streptomycin, act by binding the 30S ribosomal subunit which causes a misreading

resulting in non-sense proteins and in protein synthesis interruption ^Vakulenko and

Mobashery 2003).

Certain antimicrobials interfere with DNA replication and repair by targeting

DNA topoisomerases. DNA topoisomerases are the enzymes responsible for uncoiling

the double-stranded DNA after each round of replication. Quinolone antimicrobials act

by forming a complex with the topoisomerase IV and DNA gyrase, leading to the

14

production of double-stranded breaks and setting off the SOS repair system (Bryskier

2005).

Antimicrobials Used for Mastitis Treatment

Introduction. Antimicrobials are used to both treat and prevent mastitis. These agents

are administered primarily through the intramammary route but the parenteral route is

also used for severe clinical mastitis. For mild to moderate clinical mastitis as well as

subclinical mastitis, intramammary infusion is the most common method of

administration. In Canada, the following antimicrobials are registered for use by

intramammary infusion: cephapirin (first generation cephalosporin), erythromycin

(macrolide), penicillin (P-lactam), penicillin-novobiocin, pirlimycin (lincosamide),

streptomycin (aminoglycoside) in combination with penicillin and polymyxin B

(polymyxin), and ceftiofur (third generation cephalosporin) (Canadian Animal Health

Institute 2009). Most antimicrobials approved for treatment of clinical mastitis therapy

are focused on Gram-positive microorganisms and are administered intra-mammary.

Dry cow therapy (DCT) is another major area of antimicrobial usage. The

following antimicrobials are registered for DCT in Canada: cephapirin, cloxacillin (0-

lactam), erythromycin, and penicillin-novobiocin. The aim of DCT is to eradicate

existing infections and prevent new ones from forming (Sandholm et al. 1995). DCT is

performed at the end of lactation and aids in the prevention of both environmental and

contagious mastitis. Directly after drying off, a cow's udder begins to involute and new

infections can appear or existing ones can become more severe if no preventative care is

taken until the udder has finished involution (Sandholm et al. 1995). DCT is effective

because the antimicrobial remains in the udder in large concentrations because of its

15

formulation, enhancing and extending its therapeutic effectiveness. The antimicrobials

utilized have typically been directed towards Gram-positive organisms because

contagious mastitis is more prevalent and these pathogens are able to survive for long

periods of time within the udder (Dingwell et al. 2003).

P-lactams. The P-lactam antimicrobials represent a major class of drugs which are

used to treat both Gram-negative and Gram-positive infections. P-lactams include the

penam penicillins, cephalosporins, carbapenems, and the monobactams (Poole 2004).

They are named after a bicyclic P-lactam ring shared by both the penicillins and

cephalosporins. Some P-lactams do not contain this ring, including the carbapenems and

monobactams, making them resistant to many P-lactamase enzymes (Wax et al. 2008).

The carbapenems and monobactams have not been approved for use in veterinary

medicine. P-lactamase inhibitors (clavulanic acid) are used in veterinary medicine but are

not often used in herbivores because, similar to oral P-lactams, they can disrupt the

normal flora leading to diarrhea; there is no approved product for food animals in North

America (Giguere et al. 2006).

Penam Penicillins. The penam penicillins were the first antimicrobials used for

therapeutic purposes. They are categorized into six different groups. The benzyl

penicillins are in group one; the orally absorbed benzyl penicillins of group two include

procaine, benzathine and phenoxymethyl penicillin. They have high activity against

Gram-positive microorganisms; however, resistance caused by P-lactamases is

widespread in S. aureus (Bryskier 2005).

The antistaphylococcal isoxazolyl penicillins of group three include cloxacillin,

methicillin and oxacillin, which are resistant to S. aureus penicillinase. Thus, their main

16

usage in mastitis therapy is for the treatment and prevention (DCT) of staphylococcal

mastitis (Giguere et al. 2006). Group four, extended-spectrum penicillins (ampicillin,

amoxicillin), have greater activity towards Gram-negatives but are marginally less active

against Gram-positives and are susceptible to most |3-lactamases. The expensive

antipseudomonal penicillins are used only for the treatment of Pseudomonas infections

and are not used in the field of mastitis. The p-lactamase-resistant penicillins of group six

(temocillin) are not used for treatment in animals (Giguere et al. 2006).

Cephalosporins. Cephalosporins are derived from Cephalosporin C which is

naturally produced by the organism Cephalosporium acremonium. They are semi

synthetic and contain a 7-aminocephalosporanic acid nucleus as well as the characteristic

P-lactam ring (Hornish and Kotarski 2002). Cephamycins (cefotetan, cefoxitin), also

related to the cephalosporins, are derived from Streptomyces spp. and do not contain 7-

aminocephalosporanic acid. Cephalosporins act by the same mechanism as penam

penicillins and are highly active against most Gram-positive bacteria and some Gram-

negative microorganisms. Cephalosporins are categorized into first, second, third and

fourth generations based upon their chronological appearance and their spectrum of

activity. These agents have been further classified into seven groups based upon their

activity, oral usage and stability to P-lactamases (Hornish and Kotarski 2002). Group one

first generation cephalosporins (cephalothin, cefacetrile, cephapirin) have a high activity

towards Gram-positives including p-lactamase producing S. aureus and moderate activity

against Gram-negatives. These agents are mainly used for treating and preventing

mastitis caused by Gram-positives. Group two oral first generation antimicrobials are not

used for treatment of ruminants due to a potential disruption of their normal flora. Group

17

three second-generation cephalosporins (cefaclor, cefoxitin, cefotetan) are stable against

a variety of P-lactamases but are not used frequently due to their cost. Group four are the

third-generation parenteral cephalosporins (cefotaxime, ceftriaxone, ceftiofur) which

have high antibacterial activity towards Gram-positives and Enterobacteriacae as well as

resistance towards P-lactamases. These are used for the treatment of life-threatening

Gram-negative infections including bovine pneumonia, and parenteral treatment of severe

coliform mastitis. The fifth group is the fourth-generation parenteral cephalosporins

(cefepime, cefpirome, cefquinome) which have high activity towards Gram-negatives.

Cefquinome is currently used in Europe and Japan to treat bovine respiratory disease as

well as coliform mastitis (Giguere et al. 2006).

P-lactamase Inhibitors, Carbapenems, and Monobactams. The carbapenems and

monobactams are only labeled for use in human medicine. However, some P-lactamase

inhibitors (clavulanic acid, sulbactam) have been introduced into veterinary medicine in

combination with aminopenicillins (Giguere et al. 2006). The P-lactamase inhibitors

occupy the active site of P-lactamases irreversibly and therefore allow the antimicrobial

agent to act upon its target without disruption. Clavulanic acid has high activity against

plasmid mediated P-lactamases as well as chromosomal penicillinases but little affinity

for chromosomal cephalosporinases such as cephamycinases of the AmpC family

(Saudagar et al. 2008).

Tetracyclines. The tetracyclines are classic broad-spectrum antimicrobials with

activity against both Gram-negative and Gram-positive microorganisms (Giguere et al.

2006). They include semi-synthetic agents but all are derived from Streptomyces spp.

These antimicrobials are used widely in both swine and ruminants, both for therapy and

18

prevention. They are used parenterally for cases of severe (toxic) clinical mastitis because

they do cross the blood-mammary gland barrier, have moderate activity to mastitis

pathogens, including coliforms, and are relatively inexpensive.

Aminoglycosides. The aminoglycosides are large molecules with amino acid group

side chains which are used to treat aerobic Gram-negative and staphylococcal infections.

They include, among others, streptomycin, dihydrostreptomycin, kanamycin, neomycin,

gentamicin, tobramycin, and amikacin. The toxicity of aminoglycosides limits their

clinical use mainly to the treatment of severe sepsis caused by Gram-negative

microorganisms and topical applications (Giguere et al. 2006). However, streptomycin is

currently labelled for use as an intramammary infusion in Canada.

Sulfonamides. Sulfonamides are the oldest broad-spectrum antibacterial agents.

The major sulfonamides used include sulfadiazine, sulfamethoxazole, sulfadimethoxine,

sulfadiazine, and sulfisoxazole. They are synthetic derivatives from sulphanilamide and

act by interfering with folic acid biosynthesis. More specifically, the sulfonamides

compete against PABA for the dihydropteroate synthetase active site. Trimethoprim-

sulfonamides act sequentially to inhibit enzyme systems involved in the synthesis of

tetrahydrofolic acid (Giguere et al. 2006). In Canada, trimethoprim-sulfonamides are

commonly used parentally in cases of severe clinical mastitis suspected to be caused by

coliforms due to its spectrum of activity and its ability to penetrate the blood-mammary

gland barrier.

Macrolides. The macrolides are a family of mainly bacteriostatic antimicrobials

characterized by a 12- to 16-membered lactone ring (Bryskier 2005). They are classified

based upon the number of carbon atoms in the lactone ring and the following molecules

19

are currently used for veterinary clinical practice: erythromycin, tylosin, spiramycin,

tilmicosin, and tulathromycin (Giguere et al. 2006). Erythromycin has good activity

towards Gram-positive aerobes along with tylosin and spiramycin, while tilmicosin and

tulathromycin inhibit various Gram-positives and certain Gram-negative microorganisms.

Erythromycin is administered by the intramammary route and is used for both lactating

and dry-cow therapy because of its wide Gram-positive spectrum and short withdrawal

time (Giguere et al. 2006).

Lincosamides. The lincosamides are active against many Gram-positives,

anaerobic bacteria and some mycoplasma. Lincomycin derivatives include clindamycin

and pirlimycin (Bryskier 2005). Pirlimycin is used for intramammary treatment (Giguere

et al. 2006).

2.3 Antimicrobial Resistance in Mastitis Isolates

E. coll Resistance patterns for mastitic E. coli differ between countries and this

may be due to varying herd management practices as well as treatment strategies.

Worldwide, resistance percentages for ampicillin range from 0% to 34% in Europe and

from 22% to 98% in the United States (Table 9) (Erskine et al. 2002, Lanz et al. 2003,

Lehtolainen et al. 2003, Makovec and Ruegg 2003, Srinivasan et al. 2007, Hendriksen et

al. 2008). In a study by Erskine and collaborators in the United States, resistance to

ampicillin and cephalothin increased annually; in the United States between 1994 and

2000 however, resistance to other antimicrobials did not change (Erskine et al, 2002). A

few strains which are resistant to extended spectrum P-lactams (ESB) have recently been

found and are important because of their increased resistance to multiple antimicrobials

(Locatelli etal. 2009).

20

Klebsiella spp. There has been minimal research regarding Klebsiella spp. and

their antimicrobial resistance in cases of mastitis. Erskine et al. (2002) found that almost

all Klebsiella spp. were resistant to ampicillin, and approximately 30% were resistant to

tetracycline. Additionally, the number of isolates resistant to ceftiofur increased over time

(Table 11). A Korean study of 54 Klebsiella spp. isolates found 92% to be resistant to

ampicillin, 59% to streptomycin and 42% resistant to tetracycline (Nam et al. 2009).

Their ability to produce extended spectrum P-lactamases (ESBL's) makes Klebsiella spp.

a major public health concern. Klebsiella spp. often produce chromosomally-encoded p-

lactamases namely 6/asHV and 6/ATEM variants which confer resistance to ampicillin,

amoxicillin, carbenicillin, and ticarcillin (Livermore 1995).

S. aureus. With the invention of milking machines as well as the eradication of S.

agalactiae, S. aureus became the most prevalent causal agent of mastitis. Following the

massive usage of penicillin in the 1950s, the number of penicillin-resistant S. aureus

increased (Aarestrup 2006). This contrasts with S. agalactiae, which has remained fully

susceptible to penicillin and can be eradicated successfully (Onile 1985). Worldwide

resistance of S. aureus to penicillin ranges from 3% to 69% (Table 10) (Erskine et al.

2002, Makovec and Ruegg 2003, Hendriksen et al. 2008, Gentilini et al. 2000, Pitkala et

al. 2004, R.ajala-Schultz et al. 2004, Moroni et al. 2006). Makovec and Ruegg tested the

susceptibility of various S. aureus isolates from mastitic milk between the years 1994 and

2001. They found an overall decrease in the resistance of S. aureus towards penicillin

from 49% in 1994 to 30% in 2001 (Makovec and Ruegg 2003). Erskine et al. (2002) also

found a decrease in S. aureus resistance to penicillins and postulated that it was the

consequence of a more conservative use of antimicrobials, predominantly due to

21

increased research, education, as well as general restrictions opposing the over-use of

antimicrobials. Resistance to other antimicrobials including tetracyclines, macrolides,

phenicols and aminoglycosides, has also been documented at low frequencies (Tables 13,

16, and 21). Although uncommon, methicillin (oxacillin)-resistant S. aureus (MRSA) has

also been detected in bovine mastitis. Despite the frequent use of cloxacillin for treatment

of bovine mastitis the presence of MRSA is still remarkably rare in bovine mastitis and

most isolates originate in humans or livestock other than dairy cattle. A number of factors

may explain this rarity, including the separation of the udder environment from the rest of

the cow which does not facilitate the acquisition of resistance determinants, the high local

concentrations of penicillins which may overcome methicilin resistance, and the fact that

most MRSA strains emerged in humans and were then transferred to other species

(Martel et al. 1995). MRSA were detected in 2.5% of masitis isolates from Korea from

1997 to 2004 and 4.7% of isolates from Korea from 2003 to 2009, and 12% of isolates in

2001 (Lee 2003, Moon et al. 2007, Nam et al. 2011). In some countries, MRSA seem to

be emerging in bovine mastitis isolates. A Belgian study recently found 9.3% of 118 S.

aureus isolates from subclinical and clinical mastitis were MRSA and a German study

identified 25 MRSA isolates from bovine mastitis isolated between 2008 and 2009

(Fessler et al. 2010, Vanderhaeghen et al. 2010). A group from Switzerland found that

1.4% (Huber et al. 2010) of isolates were MRSA, 0.7% (Haenni et al. 2011) of isolates

were identified as MRSA in France and 17.2% (Turkyilmaz et al. 2010) in Turkey. There

are no published articles of MRSA in bovine mastitis in North America to date. MRSA

has become a major problem because such strains are frequently resistant not only to

most p-lactam antibiotics, but also to many other antimicrobials, including tetracyclines,

22

aminoglycosides, macrolides, and Uncosamides (Voss and Doebbeling 1995). MRS A

from animals plays a potential role in public health due to its poor response to therapy

and risk of introduction into the food chain (White and McDermott 2001).

3. DEVELOPMENT AND TRANSFER OF RESISTANCE IN MASTITIS

BACTERIA

Antimicrobials exert a selective pressure on bacterial populations resulting in the

emergence of mechanisms to escape from their inhibitory effects (Schwarz and Chaslus-

Dancla 2001). Resistance to antimicrobials can be either intrinsic or acquired. Intrinsic

resistance is a genus or species-specific property, while acquired resistance is a strain-

specific property where bacteria have acquired resistance genes by horizontal transfer or

mutations in pre-existing genes (Carry et al. 2003, Aarestrup 2006). As evident from the

variety of TEM beta-lactamase enzyme variants described below, even minor mutations

may lead to significant changes in resistance. In ruminants, the udder is well-separated

from the rest of the animal and is a sterile environment making it difficult for contagious

mastitis pathogens to acquire resistance determinants when in the udder, therefore

reducing the likelihood of resistance development during the course of existing mastitis

(Martelefa/. 1995).

3.1 Horizontal Gene Transfer and Antimicrobial Resistance

Horizontal gene transfer (HGT) can occur through conjugation, transformation or

transduction (Schwarz and Chaslus-Dancla 2001). Microorganisms under stress can enter

a mutator state which increases the frequency of mutations and the ability of bacteria to

acquire new genes through HGT (Foster 2007). Therefore, under stress, including when

23

infecting a host, bacteria may be more likely to develop resistance. Conjugation involves

the transfer of a plasmid or conjugative transposon by close contact. Transformation is

the transfer of naked DNA into recipient competent cells, and in transduction

bacteriophages inject their DNA into a bacterium (Schwarz and Chaslus-Dancla 2001).

Once transferred the DNA must be stabilized and expressed within the recipient on either

a plasmid or through incorporation into the chromosomal genome.

3.2 Bacterial Genetic Elements Involved in Transfer of Antimicrobial Resistance

Plasmids. Plasmids are self-replicating extra-chromosomal elements which range in

size from 2 to 2400 kb (Lewis et al. 2002). They frequently carry genes for antimicrobial

resistance, virulence and other dispensable functions. Conjugative plasmids promote their

own transfer because they encode genes required for cell-to-cell transfer. Mobilizable

plasmids do not carry the genes responsible for coupling of the cells prior to transfer and

require help from conjugative plasmids (Bennett 2008). Plasmids also function as the

major vectors for the movement of transposons and integrons between bacteria. Plasmids

readily acquire resistance genes through recombinational events of their various common

genes. Antimicrobial resistance plasmids are associated with both Gram-positive and

Gram-negative pathogens and commensals and often carry multiple AMR genes.

Transposons and Integrons. Transposons are small (2-20 kb) "jumping gene"

systems which can incorporate one or several resistance genes. They usually contain

insertion sequences (IS) at either end (Harbottle et al. 2006). Transposons are able to

transpose from one chromosomal site to another or to a plasmid location, thus enabling

HGT of originally chromosomal resistance genes and integrons (Fluit and Schmitz 1999).

24

Integrons are genetic elements which contain two conserved segments flanking a

central region within which gene cassettes are inserted. Through the accumulation of

multiple cassettes, they are frequently involved in resistance to multiple antimicrobial

agents. They are mobile when associated with plasmids or transposons (Lewis et al.

2002).

Genomic Islands. Genomic islands (GIs) are discrete segments of DNA ranging from

10 to 200 kb which play a major role in evolution. Some GIs are mobile, often contain

insertion sequences or transposons and most are capable of integrating into the

chromosome of a host, excising and then transferring to another host (Juhas et al. 2009).

GIs of importance in relation with antimicrobial resistance include the staphylococcal

cassette chromosome mec (SCCmec) which harbours methicillin-resistance in MRSA as

well as the Salmonella genomic island 1 which contains, among others, the blapsE-i P-

lactamase gene (Juhas et al. 2009).

3.3 Basic Resistance Mechanisms

There are four basic mechanisms of antimicrobial resistance, the first of which

prevents the antimicrobial agent from reaching its target. This is carried out by a decrease

in the antimicrobial's ability to penetrate the bacterial cell by mutations causing reduced

expression, structural alteration or removal of porins (Schwarz and Chaslus-Dancla

2001). Secondly, antimicrobials can be removed from the cell by the use of efflux pumps.

Resistance genes coding for efflux proteins have been found on plasmids, transposons as

well as gene cassettes. Many encoded efflux pumps only export a narrow range of related

substrates. However, there are also pumps which transport multiple drugs with similar

structures or even completely different drugs (Wax et al. 2008). Another major resistance

25

mechanism is the alteration or degradation of the antimicrobial, causing its inactivation.

A variety of enzymes are known to cause such modifications on aminoglycosides by

transferring acetyl, adenyl or phosphoric groups onto the antimicrobial. These

modifications inhibit the binding of the agent to its target, thus reducing its antimicrobial

activity (Schwarz and Chaslus-Dancla 2001). Other enzymes including hydrolases and

esterases degrade antimicrobial structure often leading to its inactivation. P-lactamases

are prime examples of such enzymes that act by hydrolyzing the P-lactam ring of P-

lactam antimicrobials. The last basic mechanism involves modifications of antimicrobial

targets. The target site can be chemically modified blocking the antimicrobial agent's

ability to access the target. This is the case with macrolides for which the peptidyl

transferase loop of the ribosome is methylated by Erm methyl transferases, lowering the

affinity of all macrolide drugs for RNA (Walsh 2000). Also, the target may be protected

by specific proteins which inhibit the antimicrobial from binding. Finally, the organism

may acquire or activate alternative pathways. In the case of the sulfonamides, mutational

changes in the chromosomal gene which encodes dihydropteroate synthase (DHPS) or

the acquisition of new DHPS variants may lead to decreased affinity for sulfonamides

and restoration of a previously blocked folate biosynthesis pathway (Skold 2001).

4. MASTITIS AND ANTIMICROBIAL RESISTANCE

4.1 Beta-lactam Resistance and Mastitis. Even though the overall resistance to

certain P-lactam antimicrobials appears to be decreasing, resistance to more potent

members of this class has emerged. These trends include an emergence of MRSA as

described above. Also, of concern are the ESBLs in Enterobacteriaceae because of the

limited therapy option they leave when present and the potential implications for public

26

health (Bradford 2001). A recent study in Italy found 2 E. coli isolates that harboured

CTX-M1 and 3 isolates that harboured TEM-type enzymes with an ESBL profile

(Locatelli et al. 2009). Another study by the same group found a Klebsiella spp. isolate

from bovine mastitis which harboured the CTX-M1 gene (Locatelli et al. 2010). More

research is required in regards to MRSA and ESBLs in bovine mastitis. There is a

surprising lack of susceptibility testing data available for Canada, or at least a surprising

lack of reporting of such resistance from major centres doing resistance testing.

Major P-lactam Resistance Genes. In Gram-negative bacteria, resistance to P-lactams

results mainly from the production of P-lactamases and low permeability of Gram-

negative cell membranes, whereas in Gram-positives it is caused by P-lactamases and the

production of a low-affinity PBP (shown in MRSA where the mecA gene encodes PBP

2a) (Poole 2004).

p-lactamases are classified using both the Ambler and Bush-Jacoby-Medeiros

classification systems. The Bush-Jacoby-Medeiros system classifies P-lactamases based

on their substrates and inhibitors into groups 1-4 (Bush et al. 1995). The Ambler method

classifies P-lactamases into groups A-D, where class B is metal dependent and A, C and

D are metal independent enzymes (Ambler 1980).

The major chromosomal R-lactamases found in EnterobacteHaceae from food

animals are the AmpC-type p-lactamases. These belong to Ambler class C and group one

of Bush-Jacoby-Medeiros (Bush et al. 1995). The most frequent plasmid-mediated P-

lactamases found in bacteria from food animals are CMY, TEM, SHV, CTX-M and OXA

(Li et al. 2007). Although apparently infrequent in E. coli and Klebsiella spp., PSE-1 is

also of interest because of its relation to the genomic island SGI-1 of Salmonella

27

Typhimurium DTI04. A study from Hong Kong found only 3.2% of ampicillin-resistant

E. coli isolates from the bile, blood and urine of humans produced PSE-1 (Ling et al.

1994).

Many Enterobacteriaceae, such as E. coli, produce only small amounts of

chromosomally encoded AmpC that do not usually confer clinically significant resistance

levels. However, promoter alterations can result in elevated AmpC production and a

higher resistance level towards penicillins, monobactams and cephalosporins (Livermore

1995). Chromosomal AmpC p-lactamases in E. coli are considered un-inducible because

they are not linked to any regulators influencing their expression (Li et al. 2007).

The CMY P-lactamases encoded by MCICMY are of the same class as the

chromosomal AmpC. However, they are plasmid-mediated and expressed constitutively

at higher levels (Li et al. 2007). CMY p-lactamases hydrolyse extended-spectrum

cephalosporins and cephamycins, and are resistant to P-lactamase inhibitors. These

enzymes include CMY-2, which has been found in extended-spectrum cephalosporin-

resistant E. coli and Salmonella of animal origin in Canada (Allen and Poppe 2002).

Ceftiofur resistance in E. coli isolates may be associated with the production of the CMY

enzyme.

The TEM-type and SHV-type enzymes are the two largest families of plasmid-

encoded P-lactamases of class A and group 2 (Bradford 2001).These P-lactamases are the

main enzymes found in both Klebsiella spp. and E. coli from animals and are encoded by

the blajEM and blasm genes (Li et al. 2007). There are currently (2009) 174 different

TEM variants (Jacoby and Bush 2009). TEM-1 is the most common enzyme which

confers resistance to penicillins (Bradford 2001). TEM-1 appeared after the introduction

28

of ampicillin in clinical settings in the 1960s and is found in numerous human and animal

pathogens. Most TEM-type variants other than TEM-1 have the ability to hydrolyze

extended-spectrum third generation cephalosporins and are ESBLs (Poole 2004).

However, in contrast to AmpC enzymes, they are susceptible to P-lactamase inhibitors.

The TEM-3 variant was the first to display these ESBL characteristics and is able to

hydrolyze oxyimino-cephalosporins including cefotaxime and ceftazidime (Bradford

2001). Little research regarding TEM variants in mastitis has been done. Recently,

Locatelli et al. found that three clinical mastitis E. coli isolates displaying the ESBL

phenotype out of five isolates contained WATEM genes (Locatelli et al. 2009). Thus, new

TEM variants may be emerging in mastitis and should be closely monitored. The SHV-

type enzymes are penicillinases which emerged after the introduction of extended-

spectrum cephalosporins. They were first described as a chromosomal P-lactamase in

Klebsiella spp. but are most common as a plasmid-encoded enzyme in E. coli (Bradford

2001). The SHV enzymes have fewer derivates than TEM-type variants; however, most

possess the ESBL phenotype and hydrolyze ceftazidime and cefotaxime (Livermore

1995).

The CTX-M enzymes belong to class A group 2be and confer high resistance

towards aminopenicillins, carboxypenicillins. ureidopenicillins and narrow-spectrum first

and second generation cephalosporins (Li et al. 2007). Specific ESBL derivatives can

also confer resistance towards some third and fourth generation cephalosporins (Poole

2004). The plasmids containing CTX-M often carry the WATEM genes as well as other

resistance genes and these enzymes have been found in both food and companion animals

(Li et al. 2007). In a recent European study, two out of five E. coli clinical mastitis

29

isolates displaying the ESBL phenotype contained the blacrx-u gene (Locatelli et al.

2009). Similarly to TEM and SHV, CTX-M enzymes are usually not resistant to |3-

lactamase inhibitors.

The OXA-type pMactamases belong to class A group 2d and confer resistance

towards penicillins, cloxacillin, extended spectrum |3-lactams and sometimes fourth

generation cephalosporins (Poole 2004). Some OXA enzyme variants confer ESBL

activity; however, none have been found in mastitis isolates to date. Several of them also

show resistance to pMactamase inhibitors.

The enzymes from S. aureus have a particular affinity for penicillin as a substrate

and have been found frequently in isolates of bovine origin. These enzymes are also of

group 2a and are encoded by the blaZ-blal-blal gene cluster. BlaZ is the actual 0-

lactamase, Blal is a repressor and BlaRl is a signal transducer. In a study of S. aureus

mastitis isolates in the United States, 100% of the S. aureus mastitis isolates resistant to

penicillin G contained the blaz gene (Haveri et al. 2005). The blaZ-blal-blal genes are

most often clustered together and are normally located on the chromosome although they

have also been found on plasmids. Olesen et al. found 16 out of 105 bovine S. aureus

isolates contained the blaz gene on a plasmid while it was located on the in chromosome

the other isolates (Olesen et al. 2004). The bla.Z gene has also been detected in

transposons. There is no Canadian data on blaz.

4.2 Tetracyclines

The major resistance mechanisms to tetracyclines include ribosome target

protection, efflux pumps, and enzymatic inactivation of the antimicrobial (Schnappinger

and Hillen 1996). Currently, there at least 25 tetracycline resistance (tet) genes and three 30

oxytetracycline (ptr) genes known, which all confer resistance to tetracycline and

doxycycline (Roberts 2002). Seventeen tet genes and one otr gene code for efflux pumps.

These pumps are either multi-drug or specific transporters. Drug-specific efflux genes

include tetA-E, G, H, K, O and otrB. Resistance genes found to encode resistance through

ribosomal protection include the tetM, O-Q, S, and otr A genes (Roberts 2002).

Due to widespread resistance, tetracyclines are not used on a regular basis for

clinical treatment except for particular infections such as those caused by obligate

intracellular bacteria. For example, in the United States approximately 25% of E. coli

isolates from mastitis were resistant to tetracycline and 44% of these carried both tet(A)

and tet(C) while the rest contained only tet(C) (Srinivasan et al. 2007). Another study of

over 8,000 mastitis isolates from the United States found similar resistance frequencies to

tetracyclines, with particularly high frequency in E. coli, Klebsiella spp., and S. aureus, in

the ranges of 37%, 30% and 9%, respectively (Makovec and Ruegg 2003). Multiple

European studies carried out using E. coli isolates found varying tetracycline resistance

ranging from 14% to 37% (Table 12) (Lanz et al. 2003, Lehtolainen et al. 2003,

Hendriksen et al. 2008). Also, two European studies found S. aureus resistance ranging

from 5-9% (Pitkala et al. 2004, Hendriksen et al. 2008). A Canadian study revealed

differences in the antimicrobial resistance of S. aureus isolates to tetracycline before

(2%) drying off and after (6%) drying off (Leslie et al. 2003).

4.3 Aminoglycosides

Aminoglycoside resistance can occur through various mechanisms, including

decreased antimicrobial uptake, modification of the ribosome, antimicrobial efflux and

enzymatic modification of aminoglycosides (Vakulenko and Mobashery 2003). The

31

major mechanism of resistance to aminoglycosides is by enzymatic modification of the

amino groups of these antimicrobials. The main enzymes causing this modification are

aminoglycoside phosphotransferases (APHs), aminoglycoside acetyltransferases (AACs),

and aminoglycoside nucleotidyltransferases (ANTs) (Vakulenko and Mobashery 2003).

In a study by Srinivasan et al. (2007) in the United States approximately 40% of

E. coli isolates from mastitis were resistant to streptomycin. Twenty percent of these

carried the strA, strB, and aadA genes and 56% contained the aadA gene alone. The strA

and strB genes encode APHs and the aadA gene encodes an AAC. In Switzerland a

similar study found 22% of acute mastitis isolates were resistant to streptomycin. Of

these, 36% contained strA, strB, and aadA, and 48% contained aadA only (Lanz et al.

2003). Although resistance was less frequent in Switzerland, similar gene ratios were

present in both countries. In Europe, resistance of E. coli mastitis isolates to streptomycin

ranges from 9-22% and 16% resistance to kanamycin has been detected (Table 15) (Lanz

et al. 2003, Lehtolainen et al. 2003, Pitkala et al. 2004, Hendriksen et al. 2008). E. coli

aminoglycoside resistance is more frequent in the United States than in Europe.

Approximately 1-7% of S. aureus isolates have been shown to be resistant to

streptomycin and 16% to kanamycin in Europe (Table 16) (Lehtolainen et al. 2003,

Pitkala et al. 2004, Hendriksen et al. 2008). Overall, aminoglycoside resistance is

generally more frequent in E. coli isolates compared to S. aureus isolates, reflecting the

difference in the sources of these bacteria.

4.4 Sulfonamides

Resistance to sulfonamides is most frequently the result of the acquisition of

plasmid-mediated drug-resistant variants of the chromosomal target enzyme

32

dihydropteroate synthase (Skold 2001). Three genes sull, sul2 and sul3 encode drug-

resistant dihydropteroate synthases, although suB is not frequently found in bacteria from

cattle. The sull gene is normally linked to other resistance genes in integrons, while sull

is often found together with the streptomycin-resistance determinants strAlstrB on incQ

or PBP1-related plasmids. Chromosomal resistance to sulfonamides is also known to

occur occasionally by mutational changes in folP, lowering the dihydropteroate synthase

affinity towards sulfonamides (Skold 2001).

In the study of Srinivasan and collaborators, approximately 34% of the E. coli

isolates were resistant to sulfisoxazole and 2% of these isolates carried both sull and sul2

while 27% carried sull and 22% carried sul2 and 49% did not carry either sull or sul2

(Srinivasan et al. 2007). In the United States, Makovec and Ruegg found only 16% of E.

coli isolates and 11% of Klebsiella spp. isolates from subclinical and clinical mastitis

cases were resistant to sulfisoxazole (Makovec and Ruegg 2003). Lanz and collaborators

found 22% of isolates from acute mastitis in Switzerland were resistant to sulfonamides.

Thirteen percent of these contained sull, 57% sul2, and 30% both. Other studies revealed

resistance percentages for E. coli ranging from 9 to 16% for sulfisoxazole and 0-5% for

S. aureus (Tables 18 and 19) (Makovec and Ruegg 2003, Hendriksen et al. 2008,

Bengtsson et al. 2009). Trimethoprim-sulfonamide resistance in E coli ranged from 0-

56.9%) in Europe and was 3.8% in the United States (Makovec and Ruegg 2003,

Hendriksen et al. 2008)

4.5 Macrolides

Although the macrolides, lincosamides and streptogramins (MLS) are structurally

unrelated some macrolide-resistance genes code for resistance towards two or even all

33

three members of the MLS group because their binding sites overlap (Roberts 2008).

Resistance is mediated by the presence of rRNA methylases, efflux pumps, and

inactivating enzymes which are normally encoded by mobile genetic elements. rRNA

methylases represent the most common mechanism of resistance to these antimicrobials

and act by methylating adenine residue(s) preventing the binding of the antimicrobial;

they are encoded by erythromycin resistance methylase (erm) genes (Roberts 2002).

Resistance by rRNA methylases can either be constitutive, where bacteria show high-

level resistance to all MLS antimicrobials or inducible where bacteria susceptible to MLS

antimicrobials express resistance only after exposure. Also, efflux genes coding for

transport proteins including ATP, and major facilator transporters have been found along

with modifying enzymes including two esterases, two hydrolases, seven transferases, and

three phosphorylases (Table 22) (Roberts 2002).

Although resistance to many antimicrobials is decreasing in mastitis isolates,

macrolide resistance has been shown to be increasing. Both Makovec and Ruegg and

another U.S. study found approximately 7% of S. aureus isolates to be resistant to

erythromycin (Erskine et al. 2002, Makovec and Ruegg 2003). European and South

American studies have found similar resistances ranging from 0-11% for erythromycin

(Table 16) (Gentilini et al. 2000, Pitkala et al. 2004, Hendriksen et al. 2008, Bengtsson et

al. 2009). Currently, there is little information regarding the specific genotypes of these

isolates.

34

4.6 Lincosamides

The mechanisms of resistance to lincosamides are the same as described above for

the macrolides. Resistance to lincosamides appears to be infrequent in mastitis isolates;

however, induction tests are not frequently conducted and resistance rates may be

underestimated. An Argentinian study found no resistance towards lincosamides out of

206 S. aureus strains collected from 1996 to 1998 (Gentilini et al. 2000). In the United

States, lincomycin resistance was found to range from 2.1 to 4.8% for pirlimycin in S.

aureus isolates (Table 21) (Erskine et al. 2002, Makovec and Ruegg 2003).

5. DETECTION OF ANTIMICROBIAL RESISTANCE AND

ANTIMICROBIAL RESISTANCE GENES IN MASTITIS ISOLATES

5.1 Phenotyping

Phenotyping or susceptibility testing is performed using broth or agar dilution,

gradient diffusion, or disk diffusion methods. Genotyping enables the detection and

characterization of specific antimicrobial genes by nucleic acid hybridisation or

amplification techniques (Sundsfjord et al. 2004). One disadvantage of susceptibility

testing is the time involved, since at least 24-48 hours are required before a result can be

achieved, whereas genotyping is able to provide a yes or no answer in a shorter time

period enabling more rapid therapeutic predictions (Sundsfjord et al. 2004). However, the

presence of a gene does not always indicate its expression or clinically relevant resistance

levels and genotyping may lead to misguided therapy choices. In addition, genotyping

can only detect known resistance determinants and is useless for the detection of new

uncharacterized resistance mechanisms. Conversely, low-level resistance mechanisms are

35

often not detected using susceptibility testing because isolates with such mechanisms

may be misclassified as susceptible based on clinical breakpoints only (Constable and

Morin2003).

5.2 Antimicrobial Susceptibility Testing

Susceptibility testing is based essentially on minimum inhibitory concentration

(MIC), and break point testing (Clinical and Laboratory Standards Institute 2011). The

MIC is the lowest concentration of antimicrobial which inhibits the growth of bacteria

(Clinical and Laboratory Standards Institute 2011). There are currently databases

available for the MICs of subclinical mastitis pathogens, but fewer databases are

available for clinical isolates (Constable and Morin 2003). A breakpoint is an agreed

definite MIC value which characterizes bacteria as susceptible, intermediate or resistant

(Clinical and Laboratory Standards Institute 2011). Standard testing procedures and

breakpoints have been developed by a number of national and supranational committees

and institutes. Globally, those defined by the Clinical Laboratory Standard Institute

(CLSI, formerly NCCLS) are the most commonly used.

The broth microdilution method uses 96-well plates to accurately determine the

MIC (Constable and Morin 2003). Agar dilution is another MIC method where

antimicrobials are added to agar media containing various antimicrobial concentrations

(Constable and Morin 2003). Because of the medium used, some discrepancies in MIC

estimates between the two approaches are not infrequent. Another method, gradient

diffusion, uses a strip with a gradient of concentrations of an antibiotic (Smaill 2000).

The disc diffusion method frequently referred to as the Kirby-Bauer method determines

the size of the zone of clearing around an antimicrobial disk. Although disk diffusion

36

results correlate with the other methods, this method does not provide an actual MIC. It

describes an organism as susceptible, resistant or intermediate based upon previously

known zone diameter distributions (Smaill 2000). Disc diffusion is not the most accurate

testing method but is the least expensive and is therefore still the most frequently used for

testing mastitis isolates in clinical settings.

5.3 Polymerase Chain Reaction

PCR is a technique which amplifies specific nucleic acid sequences in vitro

(Mullis and Faloona 1987). PCR is often used for screening purposes and to confirm the

presence of a specific gene found on a microarray and has been revolutionary in the

diagnosis of microbial and other diseases. Multiplex PCR amplifies two or more target

sequences in the same reaction by adding more than one pair of primers (Markoulatos et

al. 2002).

One of the major advantages of using PCR is the rapidity of the technique; results

are normally acquired in less than 24 hours. As well, PCR is often highly sensitive and

inexpensive, especially multiplex PCR which reduces the amount of reagents required.

Microarrays on the other hand are generally more expensive and are thus better suited for

a small number of isolates (Markoulatos et al. 2002). A major disadvantage of PCR is

that it can produce false positives due to the carry-over of PCR products and

contamination. As well, in regards to multiplex PCR there is often target bias, the largest

target will use up large quantities of the PCR reagents leading to reduced sensitivity for

the other targets (Gunson et al. 2008).

37

5.4 Microarrays

DNA microarrays consist of a variety of gene-specific probes deposited on a solid

surface. The DNA to be tested for the presence of specific sequences is extracted,

labelled and hybridised to the array (Sundsfjord et al. 2004). Microarrays can be PCR

product-based or oligonucleotide-based (Dorrell et al. 2005). PCR product-based

microarrays make use of amplified gene fragments as probes. This method is

inexpensive, flexible, and easy to produce; however, there is a significant potential for

cross-hybridisation due to the large size of probes and potential gene overlap with non-

target genes. The more expensive oligonucleotide-based microarrays use synthesized

oligonucleotide probes ranging from 20 to 120 bp, thus reducing the potential for cross-

hybridisation. The latter type of probe is able to differentiate between highly homologous

regions (Dorrell et al. 2005). A number of different micro-arrays have been applied to the

field of antimicrobial resistance. For instance, an array used to determine the

antimicrobial resistance genes present in E. coli from the Great Lakes. A classical

oligonucleotide array was utilized which showed 14% isolates contained antimicrobial

resistance genes (Hamelin et al. 2006). As well, another array detecting antimicrobial

resistance genes in E. coli was used to determine the presence of antimicrobial resistance

genes in broiler chickens (Bonnet et al. 2009), and another microarray has been produced

for the detection of 775 AMR genes identified by the National Center for Biotechnology

Information Database (Frye et al. 2009).

The microarrays used for the following study are the commercial AMR-ve and

MRSA ArrayTube oligonucleotide-based microarray from Identibac (Identibac, New

Haw, Addlestone, Surrey, KT15 3NB, UK) (Monecke et al. 2006, Batchelor et al. 2008).

38

Using the Array Tube, the test DNA is amplified using a random-priming method and

labelled with biotin-16-dUTP. This labelled DNA is hybridised to the probes on the

ArrayTube. Positive hybridization results are demonstrated by the addition of horse

radish peroxidase-streptavidin in the tube and its detection using a peroxidase substrate

(seramun green) producing a visible precipitate in the presence of peroxidase (Batchelor

etal. 2008).

The ArrayTube method presents multiple advantages, including its ease of use,

commercial availability and therefore external standardization, and the ability to get

results within less than 24 hours. However, in comparison to more comprehensive arrays,

the ArrayTube platform requires more template DNA and holds a smaller number of

probes than a classical glass slide-based array system. The classic array requires a smaller

amount of DNA because of the use of fluorescent instead of enzyme labelling. However,

the classical array is very labour intensive and requires significantly more expensive

hardware equipment. More specifically, the AMR-ve kit tests for 54 antimicrobial

resistance genes including the tetracyclines, trimethoprim, sulfonamides,

aminoglycosides, and pMactams (Table 23) (Batchelor et al. 2008). The MRSA kit also

used for this work follows the same protocol with the only difference being the use of

purified DNA not lysates as a template for the initial PCR/labelling reaction. The MRSA

kit contains 95 different probes testing for 72 virulence genes and 23 antimicrobial

resistance genes present in S. aureus from the following antimicrobial families:

aminoglycosides, (3-lactams, macrolides, lincosamides, streptogramins, and tetracyclines

(Table 24) (Monecke et al. 2006). Validation tests showed that there is a 1.2%

discrepancy between the AMR-ve ArrayTubes and PCR, where the ArrayTube was less

39

sensitive (Batchelor et al. 2008). Batchelor et al. suggested that these discrepancies were

due to sequence variability within the control strains; however, they did not sequence the

target genes in the negative isolates to confirm these claims. Cross-reactions also

occurred between the dfrAl gene and the dr£A17 probe as well as between WacMY-and

blcifox, due to strong similarity in these pairs of gene variants. The AMR-ve ArrayTube

has been used for multiple studies including the characterization of AMR in German E.

coli from cattle, swine and poultry and the identification of macrolide resistance in

isolates from Portuguese children (Guerra et al. 2003, Ojo et al. 2004). The MRSA array

has been successfully used to demonstrate the presence of virulence genes in bovine S.

aureus strains, including haemolysin beta (82% of isolates) and toxic shock syndrome

toxin-1 and enterotoxin N and it has been used for the characterisation of a Panton-

Valentine leukocidin positive community-acquired MRSA strain (Monecke et al. 2006,

Monecke et al. 2007).

6. THESIS PROPOSAL OVERVIEW

This project intends to characterize the antimicrobial resistance genes present in a

subset of Escherichia coli, Klebsiella spp., and Staphylococcus aureus bovine mastitis

isolates collected by the Canadian Bovine Mastitis Research Network (CBMRN).

Rationale. Because antimicrobial agents are the major treatment for bovine mastitis,

antimicrobial resistance can lead to therapy failure. Assessing AMR genotype is

important in order to identify the genes causing resistance as well as to determine their

transmission and how AMR in agents of mastitis relates to AMR in bacteria from other

body compartments in dairy cattle and in other host species. Currently, there has been

40

minimal research in regards to the AMR determinants causing resistance in E. coli,

Klebsiella spp. and S. aureus isolates from bovine mastitis in Canada. Only a few studies,

from Finland, Italy, Switzerland and the United States, have identified resistance genes in

bovine mastitis isolates, but only one has investigated the P-lactamase genes (Lanz et al.

2003, Haveri et al. 2007, Srinivasan et al. 2007, Locatelli et al. 2009). The p-lactam

antimicrobials are important because they are regularly used for the treatment of mastitis

and are linked to public health issues. Therefore, more information is needed on this

topic, both at the local and at the global level.

Objectives

The Specific Objectives of the work described here were:

- To identify the major antimicrobial resistance genes present in E. coli, Klebsiella

spp., and S. aureus bovine mastitis isolates with emphasis on the P-lactamase

genes.

To examine the genes and genetic environment surrounding an extended-

spectrum P-lactamase gene or P-lactamase gene of epidemiological importance in

E. coli or Klebsiella spp. isolates.

~ X V VlXC4J.C4-VLVi.XZjV IXXV I V O I O U U I V V VXVtVXXXXXXltlXXLO VSX kJ. VIM t M J XDOic t lVa UIOLJXCIjr XXXg

methicillin-resistance and to type the strain(s) in order to understand their

relationship with MRSA from other sources.

- To assess the sequence diversity of the blaz genes in S. aureus isolates from

mastitis.

41

http://VlXC4J.C4-VLVi.XZjV

Approach. Mastitic milk samples were collected systematically and bacteria were

isolated by the CBMRN from 89 farms in Alberta, Ontario, Quebec and the Maritime

provinces. Antimicrobial susceptibility testing of a subset of these E. coli (n=482),

Klebsiella spp. (n=149) and S. aureus (n=600) isolates was performed by Dr. J. Trenton

McClure at the University of Prince Edward Island using broth microdilution. A further

1030 S. aureus isolates were screened for resistance using penicillin-infused agar, and all

resistant isolates were tested to see if they were MRSA. Ampicillin-resistant E. coli

isolates (n=42) along with second generation cephalosporin resistant Klebsiella spp. (19),

any MRSA (n=l), and a subset of ampicillin and/or penicillin-resistant S. aureus isolates

(n=79) were chosen for further assessment of AMR genes. Screened isolates of interest

were further characterized using various genetic techniques.

Materials and Methods. Microarray analysis using the Identibac AMR-ve and

MRSA Array Tube technology along with polymerase chain reaction (PCR) was used to

determine the presence of antimicrobial resistance genes. The AMR-ve ArrayTube was

used for the E. coli and as an alternative a classical microarray was utilized for the

Klebsiella spp. isolates (Hamelin et al. 2006). The AMR-ve array allowed for the

detection of a variety of antimicrobial resistance genes conferring resistance towards

tetracyclines, trimethoprim, sulfonamides, aminoglycosides, and the ^-lactam family of

antimicrobials. The presence of any P-lactamase genes of interest were verified using

PCR and the product were sequenced to determine its variant, and its presence on a

plasmid or on the chromosome was assessed using hybridization and transformation

experiments. The genetic environment of some |3-lactamase genes of particular interest

(i.e.; ESBL genes and genes of particular public health relevance) were determined by

42

DNA sequencing using a combination of approaches based on primer walking, PCR-

product sequencing, and cloning.

The MRSA ArrayTube kit was used for S. aureus isolates. This array includes a

variety of virulence factors along with antimicrobial resistance gene probes. The

methicillin resistance cassette type of any S. aureus isolates presenting methicillin-

resistance were determined using PCR and DNA sequencing. As well, a sample of

isolates containing the blaz gene were further characterized by DNA sequencing to

determine the genetic diversity of this resistance determinant. Additionally,

representative 6/az-positive isolates as well as any MRSA were characterized by

multilocus sequence typing to assess their genetic relatedness within this study

population, and to place them in the global frame of S. aureus worldwide.

Expected Outcome. Antimicrobial resistance genes, especially the ^-lactamases,

have not been extensively studied in E. coli, Klebsiella spp. and S. aureus from bovine

mastitis. The results from this project will be the first of this kind in Canada. The further

characterization of specific isolates and of the genetic environment of their P-lactam

resistance genes will provide a better understanding of the transmission of resistance

determinants in the context of dairy cattle and mastitis. The results acquired from this

study will also help to increase our knowledge in regards to the epidemiology of

antimicrobial resistance in bovine mastitis and in the broader context of farm animals and

human beings.

43

CHAPTER TWO; ANTIMICROBIAL RESISTANCE IN ESCHERICHIA COLI

AND KLEBSIELLA SPP. FROM CANADIAN BOVINE MASTITIS ISOLATES

ABSTRACT

Objectives: The objective of this study was to identify the major P-lactam resistance

determinants present in E. coli and Klebsiella spp. bovine mastitis isolates and to

examine their genetic environment and associated resistance genes.

Methods: Microarray analysis using the Identibac AMR-ve ArrayTube technology

was used to determine the presence of antimicrobial resistance genes for tetracyclines,

trimethoprim, sulfonamides, aminoglycosides, and the P-lactam families. The genetic

environment of some P-lactamase genes of particular interest were determined by DNA

sequencing using a combination of approaches based on primer walking, PCR-product

sequencing, and cloning.

Results: An unexpected diversity of P-lactamase genes were detected in E. coli and

Klebsiella spp. isolates. The most common P-lactamase genes found in ampicillin-

resistant E. coli were blarsM, blaoxA-u and blacuY-2- For Klebsiella spp. isolates, MOTEM

and blaoxA-2b were the major P-lactamase genes besides the intrinsic blasuv- The blacuY-2

gene was located on multi-resistance plasmids of the repA/C type in two isolates, but was

chromosomal in a third isolate. Characterization of a blapsE-i plasmid showed that this

gene was part of an integron along with three other resistance genes in one of our bovine

mastitis isolate. We found a similar structure in isolates from chicken and beef cattle.

Conclusions: This study provides new information regarding the P-lactam resistance

determinants in E. coli and Klebsiella spp. isolates from bovine mastitis. A surprising

diversity of P-lactamase genes were detected considering the small sample size. The

45

presence of the now ubiquitous W«CMY-2 gene on multi-resistance plasmids indicates the

potential for both co-selection and treatment problems. Although AMR is infrequent in

bovine mastitis multi-resistance does occur.

INTRODUCTION

Bovine mastitis is a source of major economic losses in the dairy industry (Philpot

and Nickerson 2000). Escherichia coli and Klebsiella spp. are a major cause of

environmental mastitis and account for approximately one third of all clinical mastitis

cases in Canada, most of which are acute in nature (Canadian Bovine Mastitis Research

Network 2009). The main use of antimicrobials on dairy farms is for the treatment and

prevention of mastitis (Bradley 2002), with, as a consequence, potential selection of

bacteria resistant to antimicrobial agents. The P-lactams are the major antimicrobials used

for the control of bovine mastitis, but they are also important because they are significant

antimicrobials employed for treatment of human infections.

Antimicrobial resistance surveillance in agents of mastitis is essential in order to

ensure productivity, appropriate treatment and disease control. As well, it is important

from a public health perspective because resistant bacteria could potentially be

transmitted from animals to humans through direct contact or through the food chain via

raw milk and raw milk products. To date, few studies have looked at AMR in E. coli

from bovine mastitis in North America and most of these have only studied AMR at the

phenotypic level (Erskine et al. 2002, Leslie et al. 2003, Makovec and Ruegg 2003,

Srinivasan et al. 2007). The most frequent resistances in E. coli from bovine mastitis

isolates include resistance to ampicillin, streptomycin, sulfisoxazole, and tetracycline

46

(Srinivasan et al. 2007). For Klebsiella spp., which are intrinsically resistant to

ampicillin, resistance to tetracycline, ceftiofur and trimethoprim-sulfonamide

combinations are some of the most frequently found (Erskine et al. 2002). There are no

studies which detail the prevalence of P-lactamases in Klebsiella spp. from animal origin,

and, in humans, only information regarding extended-spectrum P-lactamases is readily

available.

Susceptibility testing is generally used for AMR monitoring and surveillance but

it does not provide as much information about the epidemiology of resistance as

genotypic detection. Genotyping enables the tracking of a gene and gene associations. As

a result of gene linkages on mobile elements, co-selection may occur, supporting the

spread of multiple resistances by the usage of single antimicrobials. Few studies have

looked at AMR genes in bovine mastitis agents in North America. Most investigations

only examined resistance genes for tetracycline, sulfonamides and streptomycin, and did

not attempt to detect P-lactam resistance genes (Makovec and Ruegg 2003, Srinivasan et

al. 2007).

The objective of this study was to identify the major AMR genes present in E.

coli resistant to P-lactams and multi-resistant Klebsiella spp. isolates from bovine mastitis

in Canada with emphasis on the P-lactamase genes. This study also aimed to characterize

the genetic environment of P-lactamase genes of particular epidemiological relevance in

E. coli isolates. The frequency, distribution and linkages between genes were analyzed.

MATERIALS AND METHODS

Bacterial Isolates. The collection of isolates was carried out over a two year period by

the Canadian Bovine Mastitis Research Network (CBMRN) from 2007 to 2008 and is

47

described by Reyher et al. (Reyher et al. 2011). For this study, 482 E. coli isolates and

149 Klebsiella spp. isolates from Alberta, Ontario, and Quebec and the Maritime region

(New Brunswick, Nova Scotia, and Prince Edward Island) were used. Of these isolates,

32 ampicillin-resistant E. coli from Alberta (n=9), Ontario (n=5), Quebec (n=5) and the

Maritime region (New Brunswick (n=2), Prince Edward Island (n=ll)) were detected

(Saini et al. 2010). Also, ten multi-resistant and nine ampicillin-resistant Klebsiella spp.

isolates from Alberta (n=9), Ontario (n=l), Quebec (n=2) and the Maritime region (New

Brunswick (n=l), Nova Scotia (n=0) and Prince Edward Island (n=6)) were selected. All

multi-resistant Klebsiella spp. isolates were used and ampicillin-resistant isolates were

selected randomly. The E. coli isolates came from 22 different farms and the Klebsiella

spp. isolates came from 15 different farms. Milk samples were first collected from

individual animals with a clinical mastitis infection. E. coli and Klebsiella spp. were

isolated from the milk samples and only one isolate per animal was used. An additional

five E. coli isolates from the Ministere de rAgriculture, des Pecheries et de l'Alimentation

du Quebec (MAPAQ) isolated between May and August 2010 and five E. coli from the

Animal Health Laboratory isolated between February and June 2010 were also used.

Eighteen E. coli isolates from chicken and one from beef cattle from the Public Health

Agency of Canada isolated between 2003 and 2005 were also used for characterization of

the blapsE-i genetic environment.

Animicrobial Susceptibility Testing. Susceptibility testing was performed using the

broth microdilution method according to CLSI standards (Clinical and Laboratory

Standards Institute 2011) using the Sensititre Automated Microbiology System (Trek

48

Diagnostic Systems Ltd., Cleveland, OH) and the NARMS/ CIPARS Gram-negative

panels.

Microarrays. The AMR-ve Array Tube (Identibac, New Haw, Addlestone, Surrey, UK)

was used for AMR gene detection of the 42 ampicillin-resistant E. coli. Bacterial lysates,

biotin-labelling and hybridizations were performed following the manufacturer's

instructions. The Array Tube was read using the Clondiag ArrayTube reader (Clondiag

GmbH, Jena, Germany) and analyzed with the IconoClust software (AT-Version;

Clondiag GmbH) using the *7*/normalization probe for data analysis. A classical slide

microarray was used for detection of resistance genes in Klebsiella spp. isolates (Hamelin

et al., 2006). The lysate preparation, labeling, hybridization, and washing were performed

as previously described (Hamelin et al. 2006). Fluorescence signal detection was

completed with a ScanArray operator and ScanArray Express software (PerkinElmer,

Waltham, MA).

Transformations. Plasmids from blapsE-i- and WacMY-2-positive isolates were isolated

using the Qiagen plasmid midi kit (Qiagen, Germantown, MD) following manufacturer's

instructions. Plasmid DNA was transferred into electrocompetent E. coli DH10B cells

(Invitrogen, Carlsbad, CA) following standard procedures (Sambrook and Russell

2001b). Selection of transformants was performed on Luria-Bertani agar (Becton

Dickinson, Franklin Lakes, NJ) containing 50 ug/ml ampicillin (Roche, Indianapolis, IN)

for the WflpsE-i plasmids and 8 ug/ml ceftiofur (Sigma-Aldrich, Saint Louis, MO) for the

blacMY-2 plasmids.

Southern Blotting. Plasmid DNA was isolated using the Qiagen plasmid midi kit

(Qiagen) and total DNA was isolated using the Agencourt Genfind V2 Blood and Serum

49

Genomic Isolation Kit (Agencourt Bioscience Corporation, Beverly, MA) following

manufacturer's instructions. Plasmid DNA and total genomic DNA was digested with

BgUl (New England Biolabs, Ipswich, MA) and EcoRI (New England Biolabs),

respectively, and used for Southern blotting (Sambrook and Russell 2001a).

Detection and Identification of ^-lactamase Genes and ampC Promoter Mutations.

Bacterial lysates for PCR were prepared as previously described (Miserez et al. 1998).

Polymerase chain reaction for the blajEM, blacwiY, blapsE-i, and the ampC promoter region

of E. coli isolates was carried out with these bacterial lysates using previously described

protocols (Caroff et al. 1999, Chen et al. 2004, Kozak et al. 2009). Replicon typing of the

blacMY-2 plasmids was carried out using an already published protocol (Carattoli et al.

2005). PCR products were purified using the Qiagen PCR Purification Kit (Qiagen) and

sequenced at the Guelph Molecular Supercentre, Laboratory Services, University of

Guelph. Sequence identification and detection of mutations were done using sequences

alignment with Clustal W (Chenna et al. 2003) and comparison with GenBank sequences

using BLASTn (Altschul et al. 1990).

Overlapping Polymerase Chain Reaction for blapsE-u 19 E. coli isolates (18 from

chicken and one from beef cattle) from the Canadian Integrated Program for

Antimicrobial Resistance Surveillance (CIPARS), Public Health Agency of Canada

isolated between 2006 and 2007 and known to include bla?sE-i were used for the

detection of the blapsE-i integron. Bacterial lysates for PCR were prepared as previously

described (Miserez et al. 1998). Polymerase chain reactions for the integron of the E. coli

isolates were carried out with these bacterial lysates using primers and annealing

temperatures described in Table 26.

50

Serotyping. The 19 E. coli isolates from CIPARS, and any blacuY-2 positive ampicillin-

resistant isolates were serotyped by using O- and H-specific antisera (Statens Serum

Institut, DK) at the Laboratory for Foodborne Zoonoses, Public Health Agency of Canada

Guelph, Canada (Edwards and Ewing, 1986).

Restriction fragment length polymorphism (RFLP) analysis of plasmids. Plasmid

DNA was isolated from transformants as described above and digested with the

endonuclease Bgtll (New England Biolabs). The DNA fragments were analyzed by

electrophoresis in 0.7% agarose gels. The restriction profiles of the two blacuY-2 plasmids

mentioned above along with six other MCICMY-2 plasmids from E. coli collected between

2003 to 2005 from chicken, swine and beef cattle across Canada were compared to one

another using Bionumerics (Version 3.5; Applied Maths, Austin, TX). Similarities

between restriction profiles were estimated using Dice coefficients after band matching

using band tolerance and optimization factors of 0.87% and 0.44%, respectively. A tree

was built based on the matrix of Dice coefficients using Unweighted Pair Group Method

with Arithmetic Mean (UPGMA).

Plasmid sequencing. Sequencing of the blapsE-i plasmid was performed using a

combination of pyro-sequencing (National Microbiology Laboratory, Winnipeg, Canada)

arirl aar> rlndncr Ticina nrim^r walkina nti the nricnnal nlasmid and on lone range PCR

products. Sequence data was analyzed and assembled using Sequencher Software

Version 4.5 (Gene Codes Corporation, Ann Arbor, MI).

Statistical Analysis. All statistical analyses were performed using STATA 9.0 and 11.0

(StataCorp LP, College Station, TX). Associations between genes and agreement

between susceptibility testing data and genotypic data were reported using the same

51

software. Statistically significant associations were investigated using a Kappa statistic

and statistical significance of p < 0.05. Statistically significant agreement between

phenotypic susceptibility and genotypes were investigated using exact logistic regression

and odds ratios.

RESULTS

Distribution of antimicrobial resistance genes in ampicillin-resistant E. coli. A (3-

lactamase gene was detected with the Identibac ArrayTube in 34 (88.1%) of the 42

ampicillin-resistant E. coli isolates investigated (Table 2). The P-lactamase genes

identified were blajBu (n=26; 61.9%), MaoxA (n=4; 9.5%), MOCMY-Z (n=3; 7.1%) and

WapsE-i (n=l; 2.4%). Sequencing of the blajEu isolates showed they were blajEM-i-

Mutations were detected in the ampC promoter of four isolates resistant to cefoxitin

(three with no detectable acquired P-lactamase gene and one with a MCITEM gene; Figure

1). No specific P-lactamase gene was detected for the five (11.9%) ampicillin-resistant

isolates which did not exhibit a cephamycinase phenotype. Other resistance-associated

genes for non-P-lactam antimicrobials included: aminoglycosides (strA, strB, aadAl),

tetracyclines (tet(A), tet(B)), sulfonamides and trimethoprim (sull, su!2, dfrA5, dfrAl,),

phenicols (catAl,floR), and integrons (intll and intll) (Table 2).

Distribution of antimicrobial resistance genes in Klebsiella spp. All of the isolates (6

Klebsiella oxytoca and 13 Klebsiella pneumoniae) contained blasm- Other P-lactamase

genes detected were blajEu, blaoxxib, and blaoxy- The blasuv gene was detected alone in

six (31.6%) isolates (all K pneumoniae). Five isolates (26.3%) had a combination of

blasm and WATEM; four (21.1%) isolates contained blasHv, blaoxxib, and bla-rEM, three

(15.8%) isolates contained W^SHV and blaoxA2b, and one isolate (5.3%) possessed W#SHV,

52

blaoxA2b> and blaaxY- AMR genes detected among isolates resistant to antimicrobial

agents other than P-lactams included tet(A), tet(B), tet(C), aphA, aacC4, aadA, dfrA,

sull, sul2,floR, and catAl (Table 3).

Agreement between E. coli genotypic and phenotypic data. Exact logistic regression

analysis showed good to excellent agreement between the susceptibility testing data and

genotypic microarray data (Table 4). However, a discrepancy was detected for the

presence of sull and resistance to sulfonamide. For streptomycin resistance the presence

of the aadAl gene also did not always predict in vitro phenotypic resistance to

streptomycin.

E. coli gene associations. Associations between blajEM and other non-pMactamase genes

were investigated using the Kappa statistic and significant associations are reported in

Table 5. Associations were detected between blajEM and strA, strB, tet(A), intll, and

dfrAS (Kappa = 0.34, 0.38, 0.28, 0.41 and 0.47 respectively). Two of these associations

[blajEM + tet(A) and 6/ATEM + dfrAS] revealed a significant McNemar's test which

suggests that the Kappa results have to be interpreted with caution in these two cases.

blacMY-2 a n d WapsE-i characterization. Southern blotting and transformation

experiments with plasmid preparations of the three E. coli isolates positive for blacuY-2

(two from the CBMRN, one from MAPAQ) showed that two WACMY-2 were plasmid-

borne and one chromosomal. The two transformants were multi-resistant, and both

plasmids conferred resistance to chloramphenicol, sulfisoxazole, and tetracycline in

addition to the amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur resistances and

reduced susceptibility to ceftriaxone encoded by MCICMY-2- RFLP analysis of the plasmids

showed that both plasmids were similar to multiple blacuY-2 multi-resistance plasmids

53

found in E. coli from beef cattle in Canada (Figure 2). Replicon-typing of the plasmids

indicated that they were both rep type A/C.

Southern blotting showed that the blctpsE-\ gene is located on a plasmid. Replicon-

typing of this plasmid indicated it was rep type Incll. Initial primer walking on this

plasmid around WapsE-i revealed that this gene was part of an array of gene cassettes also

including ereA, dfrA16, and aadA2 in the variable region of a class 1 integron (Figure 3)

(GenBank: DQ157752.1). Pyrosequencing of the entire bla^sE-i plasmid (pEC_PSE-l)

showed strong similarities to an already sequenced plasmid from Salmonella enterica

subsp. enterica serovar Heidelberg; however, some differences included the presence of

the WapsE-i gene and its associated class 1 integron (GenBank: CP001118.1). The

pEC_PSE-l plasmid also harbours a number of genes of interest including a shufflon and

a porcine ETEC pilus gene absent from the Salmonella Heidelberg plasmid (Table 25).

Nineteen E. coli isolates positive for bla^sE-i were analyzed by overlapping PCR

and primer walking for the presence of a similar WapsE^-containing integron cassette

(data not shown). Despite the diversity of the isolates investigated (each isolate was from

a different serotype), the W^PSE-I gene was present in the same class 1 integron in all of

them (Figure 3).

DISCUSSION

This study is the first to identify pMactamase genes in E. coli and Klebsiella spp.

from bovine mastitis. To our knowledge no other investigations have looked at these

genes in isolates from bovine mastitis. However, a study done on fecal E. coli isolates

from dairy cattle by Walk and collaborators in the United States found that 92.2%

(n=129) of their ampicillin-resistant E. coli isolates were positive for blajEM and no other

54

genes were detected, even though WasHV and blaoxA-i were both investigated (Walk et al.

2007). A group from Denmark found that 94.3% of ampicillin-resistant E. coli isolates

(n=35) from food animals contained blajEu, and they also found that 2.9% harboured

blaoxA and 2.9% contained ampC promoter mutations (Olesen et al. 2004). These results

are in agreement with the high frequency of blajEu and sporadic presence of WaoxA-i in

our E. coli isolates. However, neither blacuY-i nor bla^sE-i have apparently been

described previously in E. coli isolates from bovine mastitis although blacuY-i plasmids

have been detected in fecal isolates from dairy cattle in the United States (Call et al.

2010). In comparison to fecal isolates, E. coli from bovine mastitis in Canada seem to

carry an even larger diversity of P-lactamase determinants (Call et al. 2010). Since E. coli

from mastitic milk are thought to be essentially the same as those from fecal matter

(Nemeth et al. 1994), this large diversity of (̂ -lactamases is interesting. Although the

small number of isolates investigated may not allow for reliable frequency estimates,

another likely explanation may be a true difference in AMR determinants and

antimicrobial use in comparison to other countries. It should also be noted that ten of the

E. coli isolates were not from the CBMRN. These isolates may bias the results and are

less likely to reflect Canadian dairies.

A seemingly high number of ampC promoter mutations (9,5?/o) were detected in

the E. coli isolates. These promoter mutations have been shown to lead to the

overproduction of an intrinsic E. coli cephamycinase (Caroff et al. 1999). It has been

postulated that because mastitis is usually a mono-infection and the udder is well-

separated from the rest of the animal the likelihood of acquiring resistance genes in this

body compartment is rather low (Mattel et al. 1995). Thus, most p-lactam resistances

55

emerging under selective pressure in the udder may be the result of mutation rather than

horizontal gene transfer. Therefore an increased proportion of ampC promoter mutants

may not be surprising among agents of mastitis resistant to P-lactams. Further prevalence

studies with a larger number of isolates are nevertheless necessary to confirm this

hypothesis. An alternative explanation is that resistant bacteria may be more virulent, as

has been noted in some medical studies (Martinez and Baquero 2002). We also detected a

number of resistant isolates for which none of the investigated P-lactamase genes were

detected. Since the AMR-ve ArrayTube does not contain probes for all known P-

lactamase genes but only for 6/asHv, blavsM, blaoxA, bla^ox, blacuY, W«LEN, MCIACC,

blauox, blapsE, and blacrx-M, further investigation may reveal the presence of other rare

P-lactamases in these isolates.

All Klebsiella spp. isolates contained the blasw gene. This was not surprising

because the blasm gene has been thought to be ubiquitous within K. pneumoniae (Babini

and Livermore 2000). Thus, our results provide further evidence that both K. pneumoniae

and K. oxytoca contain an intrinsic blasm/ gene. Despite this, 67% of Klebsiella spp.

isolates harboured one or two additional p-lactamase genes. The reason most isolates

contained more than one P-lactamase is unknown. The P-lactamase gene may be linked to

other resistance genes on a mobile genetic element and co-selected by the usage of a non-

P-lactam antimicrobial. However, no obvious associations between non-P-lactam and P-

lactam genes were evident in our isolates. The presence of more than one P-lactamase

gene may also increase the minimum inhibitory concentrations for the P-lactam and

provide a selective advantage in the presence of high concentrations of these

antimicrobials. Interestingly, besides the ubiquitous WajEM, the other P-lactamase genes

56

were not the same as those from E. coli. This suggests that the epidemiology of

antimicrobial resistance in these two related organisms may be different and further

comparisons will be needed to investigate this point more precisely.

AMR gene associations may be due to a physical linkage on the same genetic

element (Travis et al. 2006) or the clonal spread of a single strain carrying two or more

resistance determinants (Maidhof et al. 2002). We detected significant associations

between: blajEM and strA/strB, an association that has been detected before in fecal E.

coli from grow-finish pigs (Rosengren et al. 2009) but not from bovine mastitis isolates.

Further molecular characterization of these isolates is required to determine if they are

present on the same genetic element. An association between WOTEM and intll was also

detected but seems unlikely because blajEM has not been found as part of a gene cassette

of an integron although it may reside beside a transposon containing an integron. Two

other associations were found between blajEu + tet(A) and blaj^u + dfrA5. However, a

significant McNemar's test was detected for both of these associations indicating the

presence of a bias. The associations detected provide evidence to support the idea that the

use of non-pMactam antimicrobials may be selecting for P-lactamase resistance in E. coli

and potentially Klebsiella spp.. Serotyping of the isolates containing associations would

help to understand if these genes are physically linked or clonally spread.

We detected discrepancies between genotype and phenotypic susceptibility testing

data for aadAl and sull conferring streptomycin resistance and sulfonamide resistance

respectively. The classification of aadA 1 -positive isolates as susceptible for streptomycin

had already been noticed before in dairy cattle (Srinivasan et al. 2007) as well as swine

(Boerlin et al. 2005) and various animal sources (Lanz et al. 2003). It has been

57

hypothesized that E. coli carrying the aadAl gene may not be expressing this gene

(Srinivasan et al. 2007), or that the breakpoint for resistance has been set too high.

Further investigations using a large variety of isolates may support the above hypotheses.

The sull gene was also found in a number of sulfonamide and trimethoprim-sulfonamide

susceptible isolates. To our knowledge this finding has not been described before and

may need some further clarification. One explanation may be that the Array Tube is not

specific for the sull gene.

E. coli isolates harbouring either blacMY-2 or WQPSE-I were further characterized

because of their public health importance. Specifically, blacMY-2 confers resistance to

most p-lactams including third-generation cephalosporins which have been identified as

critically important for the treatment of serious human infections (Food and Agriculture

Organization of the United Nations 2008). The two WacMY-2 plasmids investigated here

encoded multiple resistances besides P-lactam resistance and were similar to those from

E. coli from beef cattle (Martin and Boerlin, manuscript in preparation). blacMY-2

plasmids from bovine and human Salmonella isolates with the same resistance profile as

our plasmids have been detected before (Winokur et al. 2000, Carattoli et al. 2002). The

presence of these multi-resistance plasmids is a concern because they could lead to the

co-selection of extended-spectrum cephalosporin resistance by non-p-lactam

antimicrobials commonly used in dairy cattle. These plasmids could also cause a decrease

in treatment options. The similarity of the bovine mastitis blacuv-2 plasmid to those froni

beef cattle may suggest that E. coli isolates harbouring these repA/C plasmids are better

adapted to survival in cattle. Another hypothesis for this similarity, is that these blacMY-2-

plasmids, are ubiquitous or nearly so, they have been detected in Salmonella enterica

58

serovar Newport from cattle in the United States and have now been found in several

bacterial species from a broad range of domestic animal species in North America (Call

etal. 2010).

An E. coli isolate carrying blapsE-i was also further characterized. This isolate is of

interest because the WapsE-i gene is normally detected in the Salmonella genomic island 1

(SGI1) first found in Salmonella enterica subsp. enterica serovar Typhimurium DTI 04

(Boyd et al. 2002). The W<ZPSE-I gene has been only rarely detected in E. coli. It has been

found in blood, bile and urine isolates from human patients in China (Ling et al. 1994)

and in 1.3% (n=371) of human isolates from six different countries (Medeiros et al.

1982). However, the WapsE-i genetic elements have only rarely been characterized but

were detected on transposons in E. coli, Salmonella Typhimurium and Pseudomonas

aeruginosa from humans (Medeiros et al. 1982, Levesque and Jacoby 1988). Our results

show that in E. coli, blapsE-i is part of a class 1 integron but not of an SGI-like structure.

Nevertheless, one {aadAT) of the three gene cassettes encoding resistance to

trimethoprim, erythromycin, and streptomycin found on the same integron is identical to

one found close to 6/tfpsE-i on SGI1. Some of the major antimicrobials used to treat

bovine mastitis include streptomycin and the pMactams, suggesting that the dissemination

of this plasmid could potentially lead to a decrease in the therapeutic options available for

bovine mastitis treatment.

Nineteen blapsE-i positive E. coli isolates from other animals and from a large

diversity of serotypes also contained the same integron described above. This shows that

the mobile element carrying the WapsE-i gene is able to move by horizontal transfer across

a broad diversity of unrelated strains. Further conjugation experiments may help to

59

provide more information on the transfer mechanisms and potential host spectrum of the

mobile element carrying the blapsE-i integron.

Besides numerous genes associated with replication, partition and transfer,

sequencing identified afaeG operon, a Type IV pilus operon and a shuffion on the

blapsE-i plasmid. The shuffion, located next to the Pil transfer protein genes, is

responsible for switching on and off the type IV pilus thus increasing under some

circumstances the conjugation and transfer efficiency of the plasmid which could

potentially lead to the transfer and preservation of this plasmid in a number of animal

species (Komano 1999). Also of interest, the K88 fimbrial adhesin is normally associated

with adhesion of ETEC in swine indicating this plasmid may have passed through

bacteria of several animal hosts and carries a great potential for spread through bacterial

populations (Jin and Zhao 2000).

In conclusion, this study provides new information regarding the pMactam

resistance determinants in E. coli and Klebsiella spp. isolates from bovine mastitis. An

unexpectedly large diversity of p-lactamase genes were detected for both E. coli and

Klebsiella spp. in comparison to beef cattle, despite the small number of isolates

investigated. Some resistance gene associations which have been described before in

bacteria from other animal species were also detected in bovine mastitis agents. The

ubiquitous blacMY-2 gene is present in bacteria from the mastitic milk samples of

Canadian dairy cattle. Its presence on the same multi-resistance plasmid as in beef cattle

brings with it the potential for co-selection and treatment problems. Characterization of

the uncommon blapsE-i and of its genetic environment showed that it was part of an

integron along with three other resistance genes in our bovine mastitis isolate, as well as

60

in those from chicken and beef cattle. Its association with a type IV pilus, thus increasing

conjugation efficiency, and with an adhesin from porcine ETEC suggest a vast potential

for spread and maintenance in bacterial populations between animals. Although AMR in

bovine mastitis normally occurs at low levels the presence of multi-resistant isolates and

the potential for increased dissemination of these variants is a concern.

ACKNOWLEDGEMENTS

We would like to thank Matt Saab for providing the isolates and performing the

antimicrobial susceptibility testing. We also wish to thank Dr. Marie Nadeau and Dr.

Durda Slavic for providing isolates. As well, thanks to Laura Martin for providing the

chicken and bovine WapsE-i isolates and for carrying out the RFLP on the blacuY-2

isolates. Special thanks to Philippe Garneau and Dr. Josee Harel for help in running the

Klebsiella microarrays. Thanks to Dr. Mike Mulvey for sequencing the bla^sE-i plasmid.

Also, thanks to Kim Ziebell and collaborators for serotyping the E. coli isolates and

Gabhan Chalmers for Rep-typing the blacMY-2 plasmids. Finally, thanks to Fiona

Coutinho for performing primer-walking on the WapsE-i isolates. Thanks to the

Veterinary Laboratories Agency for aid in running the ArrayTubes. This research was

financed by the Natural Science and Engineering Research Council, Alberta Milk, Dairy

Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait

Inc., Dairy Farmers of Canada, Canadian Dairy Network, Agriculture and Agri-Food

Canada, Public Health Agency of Canada, Technology PEI Inc., Universite de Montreal

and University of Prince Edward Island, through the Canadian Bovine Mastitis Research

Network.

61

Table 1. Antimicrobial susceptibility testing results for ampicillin-resistant E. coli (n=42) and Klebsiella spp. (n^lf?) from

bovine mastitis in Canada.

Antimicrobial Agent Testing Range Breakpoint

(ug/mL) (ug/mL)a Resistant E. coli Resistant Klebsiella spp.

Amikacin (AMK)

Amoxicillin/Clavulanic Acid (AMC)

Ampicillin (AMP)C

Cefoxitin (FOX)

Ceftiofur (TIO)

Ceftriaxone (CRO)

Chloramphenicol (CHL)

Ciprofloxacin (CIP)

Gentamicin (GEN)

Kanamycin (KAN)

Nalidixic Acid (NAL)

0.5-64 >64 0 (0.0%) 0 (0.0%)

1-32

1-32

0.5-32

0.12-8

0.25-64

2-32

0.015-4

0.25-16

8-64

0.5-32

>32

>32

>32

>8

>64

>32

>4

>16

>64

>32

8 (19.0%)

42(100.0%)

6 (14.3%)

5(11.9%)

2 (4.8%)

11(26.2%)

0 (0.0%)

1 (2.4%)

15 (35.7%)

0 (0.0%)

0 (0.0%)

20 (100.0%)

0 (0.0%)

0 (0.0%)

0 (0.0%)

2 (10.0%)

0 (0.0%)

2 (10.0%)

5 (25.0%)

0 (0.0%)

62

Streptomycin (STR)

Sulfisoxazole (SOX)

Tetracycline (TET)

Trimethoprim/Sulphamethoxazole (SXT)

32-64

16-256

4-32

0.12-4

>64

>512

>16

>4

30(71.4%)

33 (78.6%)

31 (73.8%)

24(57.1%)

4 (20.0%)

6 (30.0%)

6 (30.0%)

3 (15.0%)

Antimicrobial susceptibility testing was performed following CLSI standards for broth microdilution and the Sensititre

Automated Microbiology System (Trek Diagnostic Systems Ltd). Ten isolates resistant to one or more antimicrobials on the

testing panel and nine susceptible isolates were selected.c Resistance to ampicillin was the selection criterion for inclusion of

E. coli isolates in the study.

63

Table 2. Frequency of AMR and integrase genes among 42 ampicillin-resistant E. coli from bovine mastitis in Canada.

Antimicrobial Agent(s) Gene(s) Resistant Isolates3 (n=42)

(3-lactams


Gentamicin (GEN)

Streptomycin (STR)

Sulfisoxazole (SOX)

Tetracycline (TET)

Trimethoprim/ Sulfamethoxazole (SXT)

Integrons

blapsE-i', blacMY-2', blaoxA-V, W#TEM-I

catAl;floR

aac3Iva

strAlstrB; aadAl; aadA2; aadA4

sull; sul2

tet(A); tet(B); tet(C)

dfrAl; dfrA5; dfrA7; dfrA12; dfrAll

intll; intI2

2.4%; 7.1%; 9.5%; 61.9%

14.3%; 16.7%

4.8%

66.7%; 26.2%; 2.4%; 2.4%

23.8%; 42.9%

38.1%; 31.0%; 2.4%

14.3%; 33.3%; 2.4%; 2.4%; 4.8%

57.1%; 7.1%

a Number of isolates positive for this specific gene. Numbers represent the percentage of isolates positive for the specific gene.

The following (^-lactamase genes were not detected: blasm, blauox, blaixn, MCIACC, blauox, and blacvx-M-

64

Table 3. Frequency of AMR and intll genes among multi-resistant and susceptible Klebsiella spp. isolates from bovine

mastitis in Canada.

Antimicrobial Agent(s) Gene(s) Resistant Isolates" „ x.,, T , . b , ™

, _.«> Susceptible Isolates (n=9)

pMactams


Gentamicin (GEN)

blasuv', blaoxA2b', blaoxy', blajEM-i

catAl

aac31Va; aadB

10; 5; 1; 6

2

9;1

9; 3; 0; 3

0

8;0

Kanamycin (KAN)

Streptomycin (STR)

aphAl; aph; aadB

strAlstrB; aadAl; aadAl; aph6

4;3;1

4;4;1;4

3;0;0

0; 0; 0; 0

Sulfisoxazole (SUL) sull; sul2 2:4 0;1

Tetracycline (TET)

Tetracycline (TET)

tet(A); tet(B); tet(C)

tet(D); tet(M); tet(R); tet(Y)

2; 4; 2

1;2;1;1

1;0;1

0;0;1;0

Trimethoprim/ Sulfamethoxazole (SXT)

Integrons

dfrA7

intll

Number of resistant isolates positive for this specific gene. Number of susceptible isolates positive for this specific gene.

65

Table 4. Associations between susceptibility testing results and genotypes for E. coli

antimicrobial resistance genes tested.

Antimicrobial „ Odds . Genes „ .. p-value

Ratio r 95% Confidence

Interval

Streptomycin

Sulfisoxazole

Tetracycline

Chloramphenicol

Trimethoprim/ Sulphamethoxazole

strAlstrB 110.57a <0.00001

aadAl

sull

2.61

5.24'

sul2 14.1

0.8923

0.1413

0.0066

tet(A) 29.64 0.0009

tet(B) 31.28a 0.0004

floR 92.23 0.0002

catAl 74.58 0.0008

dfrV 51.69a O.00001

dfrAl 19.21a 0.005

13.36-oo

0.11-188.36

0.61 -oo

1.9-00

2.88 -1622.69

3.98-oo

5.63 - 7064.09

4.16-5885.53

7.05 - oo

2.25-00

a Median unbiased estimates (MUE).

66

Table 5. Associations between J3-lactamases and other resistance determinants.

Gene v a , 95% Confidence , , , T , „ . , . . .. Kappa p-value T ^ , McNemars Test p-value

Association rv r Intervals

6/aiEM + strA 034 oTol 0.04-0.63 O08 0.78

MajEu + strB 0.38 0.01 0.09-0.67 0.33 0.56

b!aTEM + tet(A) 0.28 0.02 0.03-0.53 6.25 0.01b

blajEu + intll 0.41 0.00 0.13-0.69 0.33 0.56

blaxEu + dfrV 0.47 0.00 0.26-0.69 12.00 0.0005b

a A Kappa statistic <0.2 indicates a slight agreement, 0.2-0.4 describes a fair agreement,

0.4-0.6 is a moderate agreement, 0.6-0.8 is a substantial agreement and a Kappa > 0.8 is

an almost perfect agreement; b A significant McNemar's test indicates discordance

between the results.

67

EC229 EC346 EC26 EC44 ATCC25922 (REF)

EC229 EC346 EC26 EC44 ATCC25922 (REF)

GCTATC|TGACAGTTGTCACGCTGATTGGTITCGTTACAATCTAACGIATCGCCAATGTA 60 GCTATCITGACAGTTGTCACGCTGATTGGTITCGTTACAATCTAACGIATCGCCAATGTA 60 GCTATCITGACAGTTGTCACGCTGATTGGTITCGTTACAATCTAACGIATCGCCAATGTA 60 GCTATC|TGACAGTTGTCACGCTGATTGGTITCGTTACAATCTAACG|ATCGCCAATGTA 60 GCTATC|TGACAGTTGTCACGCTGATTGGT|TCGTTACAATCTAACG|ATCGCCAATGTA 6 0 ****** *********************** **************** ************

AATCCGGCCCGCCTATGGCGGGCCGTTTTGTATGGAAACCAGACCITATG 110 AATCCGGCCCGCCTATGGCGGGCCGTTTTGTATGGAAACCAGACCITATG 110 AATCCGGCCCGCCTATGGCGGGCCGTTTTGTATGGAAACCAGACCITATG 110 AATCCGGCCCGCCTATGGCGGGCCGTTTTGTATGGAAACCAGACCITATG 110 AATCCGGCCCGCCTATGGCGGGCCGTTTTGTATGGAAACCAGACC|TATG 110 ********************************************* ****

Figure 1. Promoter mutations of the ampC gene. Red are mutated and green are non-

mutated nucleotides.

68

BD11TF

B341TF

B3B0TF

ED44TF

B323TF

B218

E2415

B346TF

CHcten

Fbdne

Chdcn

Btxine

Bxine

Bcxine

Bains

Oicten

AB

CN

NB

QC

PB

CN

CC

CC

PtJCMPKXTiOCFD

PUCPtJPKXHCKFD

PNCfiM>KX1\OCFD

/My^FCKiiocRD(H.axrcy9cr

/M>^FO(T10CRDCH.3CXTCy9Cr

/M^^FD(TlCKroCH.9CKTCy

/MV^FOOKXKXH-SXTCY

/M^PFCKTICXRD

2004

2003

2005

20C4

2003

2007

2010

2003

K

-

FfepB

PLC

PLC

PLC

PLC

11

Figure 2. blacuY-2 dendrogram representing the similarity between restriction profiles of

blacuY-i plasmids from bovine mastitis E. coli (EC218 (from the CBMRN), EC2415

(from MAPAQ)), plasmids from beef cattle, and representative blacuY-2 plasmids from

each of four other replicon types. For abbreviations see Table 1.

o o , l ° , , , ,r

\

69

dfr_pse_F dfr_pse_R pse_aadA2_F pse_aadA2_R aadA2_ereA_F aadA2_ereA_R

5400 bp

Figure 3. WapsE-i class 1 integron structure and PCR primer positions.

70

CHAPTER THREE: B-LACTAM RESISTANCE IN STAPHYLOCOCCUS

AUREUS FROM CANADIAN BOVINE MASTITIS ISOLATES

ABSTRACT

Objectives: The primary objective of this study was to identify the major antimicrobial

resistance (AMR) and virulence genes present in S. aureus bovine mastitis isolates with

emphasis on the P-lactamase genes. Secondary objectives included the characterization of

any methicillin (oxacillin)-resistant S. aureus (MRS A), and assessment of the sequence

diversity of the blaz gene.

Methods: Microarray analysis using the Identibac MRS A ArrayTube technology

was used to determine the presence of virulence genes and antimicrobial resistance genes

for the following antimicrobial families: P-lactams, aminoglycosides, tetracyclines,

macrolides, lincosamides, and streptogramins. The Staphylococcal cassette chromosome

mec (SCCmec) type of the MRSA isolate was determined using PCR and DNA

sequencing. Isolates containing the blaz gene were further characterized by DNA

sequencing. The MRSA as well as subsets of penicillin-resistant and susceptible isolates

were characterized by multilocus sequence typing (MLST).

Results: All of the resistant S. aureus isolates contained the blaz gene and resistant

isolates were significantly more likely to harbour virulence genes. The first MRSA from

bovine mastitis in Canada was identified and found to be ST8 SCCmec IYc. The blaz

gene presented a relatively high sequence diversity and variants clustered per farm of

origin. Most MLST types detected had been previously associated with bovine mastitis.

Conclusions: Overall, the prevalance of resistance to P-lactams was low in S. aureus

and, as expected, blaz was detected in all penicillin-resistant S. aureus isolates. The

71

characterization of the first MRSA from bovine mastitis in Canada showed that it was a

community-acquired ST normally associated with humans. Resistant isolates were more

likely to harbour virulence genes including specific enterotoxins.

INTRODUCTION

Mastitis has a major economic impact on dairy farming (Philpot and Nickerson

2000). One of the main causes of contagious mastitis is Staphylococcus aureus which

accounts for approximately 9% of all mastitis cases, most of which are subclinical

(Wilson et al. 1997). Infection with S. aureus is often chronic in nature and difficult to

treat. The major antimicrobials used for treatment of S. aureus mastitis are the P-lactams.

As with other antimicrobials, overuse of P-lactams can potentially lead to antimicrobial

resistance (AMR). P-lactams are also the major antimicrobials employed for treatment of

human infections.

Surveillance of AMR in agents of mastitis is essential in order to ensure productivity,

appropriate treatment and disease control. AMR in S. aureus mastitis cases has not been

thoroughly studied in North America especially in Canada. However, the major

resistances found include resistance to the P-lactams (Werckenthin et al. 2001). In the

United States, penicillin-resistance frequency in S. aureus isolates from bovine mastitis

ranges from 35 to 50% (Erskine et al. 2002, Makovec and Ruegg 2003). Tetracycline,

macrolide, chloramphenicol, and aminoglycoside resistances are less frequent. The major

resistance gene responsible for resistance to P-lactams in S. aureus is blaz (Haveri et al.

2005). This gene has not been thoroughly investigated, especially with respect to its

sequence diversity in bovine mastitis. One publication by Olsen and collaborators

72

sequenced 105 blaz genes from humans and cattle and found broad diversity (Olsen et al.

2006).

MRSA is a significant pathogen in humans and is frequently resistant to a number of

non-P-lactam antimicrobials, including tetracyclines, aminoglycosides, macrolides, and

lincosamides. Its ability to cause serious infections in both humans and animals is well

known (Voss and Doebbeling 1995). MRSA has only occasionally been found in bovine

mastitis cases, but its frequency seems to have increased in the past few years (Lee 2003,

Kwon et al. 2005, Moon et al. 2007, Juhasz-Kaszanyitzky et al. 2007). It has not yet been

found in Canadian dairy cattle. Typing and characterization of MRSA when occurring in

relation with mastitis is essential in order to understand their origin and because these

isolates do not respond well to treatment and could potentially enter the food chain.

The major objectives of this study were: 1) to identify the antimicrobial resistance

genes present in a subset of penicillin-resistant S. aureus bovine mastitis isolates with

emphasis on the P-lactamases; 2) to assess the sequence diversity of the blaz genes

encoding penicillin-resistance; 3) to characterize the SCCmec cassette and molecular type

of the first MRSA from bovine mastitis found in Canada.

MATERIALS AND METHODS

Bacterial Isolates. The collection of bovine mammary gland derived bacterial isolates

was carried out over a two year period by the Canadian Bovine Mastitis Research

Network (CBMRN) from 2007-2008 and has been described (Reyher et al. 2011). For

this study, a collection of 1630 S. aureus isolates from 89 farms located in Alberta,

Ontario, Quebec and the Maritime region (New Brunswick, Nova Scotia and Prince

Edward Island) were used. The 57 penicillin-resistant S. aureus isolates selected for this

73

study originated from Alberta (n=3), Ontario (n=32) and Quebec (n=21) (Saini et al,

2010). The 22 penicillin-susceptible S. aureus isolates were randomly selected from the

same farms as the penicillin-resistant isolates and originated from Alberta (n=4), Ontario

(n=5), Quebec (n=8) and the Maritime region (New Brunswick (n=3) and Prince Edward

Island (n=2)). Milk samples were collected from individual dairy cattle with either

subclinical or clinical mastitis. Only one isolate per cow was used for this study. On the

farm where MRSA was detected, all S. aureus isolates were tested, including multiple

samples from the same cow.

Susceptibility Testing. Susceptibility testing was performed by plating on selective agar.

Briefly, 25ul of a pure culture suspension (0.5 McFarland) was pipetted onto Mueller-

Hinton agar containing 0.25 ug/mL of penicillin G for the detection of penicillin-resistant

isolates, and on Denim Blue Agar (Oxoid, Basingstoke, United Kingdom) for the

detection of MRSA. Both plates were incubated at 37°C for 18-24 hours.

Microarray. The MRSA Array Tube (Identibac, New Haw, Addlestone, Surrey, UK) was

used for AMR and virulence gene detection. The array consists of 168 probes in duplicate

representing 95 resistance and virulence genes. Genomic isolation, biotin-labelling and

hybridizations were performed following the manufacturer's instructions. The ArrayTube

was read using the Clondiag ArrayTube reader (Clondiag GmbH, Jena, Germany) and

analyzed with the use of IconoClust software (AT-Version; Clondiag GmbH).

Multi-locus Sequence Typing (MLST) and spa typing. MLST analysis was performed

as described previously (Enright et al. 2000). Sequence types were analysed and an

eBurst analysis was performed using the S. aureus MLST database

(http://saureus.mlst.net/). Spa typing was carried out as previously described (Harmsen et

74

http://saureus.mlst.net/

al. 2003). Spa repeats and spa type codes were determined using the Ridom SpaServer

website (www.spaserver.ridom.de).

Detection of the bla% Gene. Bacterial lysates were prepared as previously described

(Miserez et al. 1998). Polymerase chain reaction for the blaz gene of S. aureus isolates

was carried out for the bacterial lysates using the blazF and blazR primers as described

by Nannini and collaborators (Nannini et al. 2003).

SCCmec Cassette characterization. Genomic DNA was isolated using the QIAamp

DNA Mini Kit (Qiagen, Germantown, MD). For the determination of the Staphylococcal

Chromosome Cassette mec (SCCmec) type a polymerase chain reaction protocol was

utilized to test for types I to IV (Oliveira and de Lencastre 2002). Then primers were

designed using Primer 3 Software (Rozen and Skaletsky 2000) based upon the SCCmec

Type IVc backbone (GenBank: AB096217.1).

DNA Sequencing. PCR products from MLST, spa Typing, blaz, and SCCmec

characterization were purified using the Qiagen PCR Purification Kit (Qiagen) and

sequenced at the Guelph Molecular Supercentre, Laboratory Services, University of

Guelph. Sequence data was analyzed and assembled using Sequencher Software Version

4.5 (Gene Codes Corporation, Ann Arbor, MI). Sequence identification was performed

using GenBank sequences with BLASTn (Altschul et al. 1990). Sequences were aligned

using Clustal X version 1.83 (Chenna et al. 2003) and trees were created using the

Neighbour-Joining method with NJplot software (Perriere and Gouy 1996).

Statistical Analysis. All statistical analyses were performed using STATA 9.0 and 11.0

(StataCorp LP, College Station, TX). Poisson regression analysis was used to compare

the number of virulence genes detected between susceptible and penicillin-resistant

75

http://www.spaserver.ridom.de

isolates and was reported using an incidence risk ratio. The comparison of individual

virulence gene frequencies in susceptible and resistant isolates was investigated using

exact logistic regression analysis. A p-value < 0.05 was considered significant.

RESULTS

Distribution of resistance genes. A total of 118 out of 1630 S. aureus isolates tested

were penicillin-resistant (Saini et al. 2010). 57 of these resistant isolates as well as 22

randomly selected penicillin-susceptible isolates were run on the MRSA ArrayTube. The

resistant isolates were from 11 farms and the susceptible isolates were from 17 farms.

The blaz gene was detected in all 57 ampicillin-resistant S. aureus isolates and in none of

the susceptible isolates. No resistance determinants other than blaz were detected in these

79 isolates, with the exception of one multi-resistant MRSA isolate. This MRSA isolate

was identified and found to harbour four resistance genes (mecA, dfrV, tet(K) and aacA).

Three additional isolates collected from the same cow harbouring MRSA were also

identified as MRSA.

Distribution of virulence genes. The frequency of virulence genes detected in

susceptible and resistant isolates is reported in Table 27. The major virulence genes

identified were staphylococcal superantigen-like proteins, protein A, leukocidins,

accessory gene regulator genes, hemolysins, and enterotoxins G, I, O, X, and Y.

Poisson regression analysis showed that the resistant isolates contained a higher

number of virulence genes in comparison to the susceptible isolates and is reported using

an incidence risk ratio (IRR) (IRR=1.22, p O.0001, 95% confidence interval 1.12-1.33).

The following virulence genes were shown by exact logistic regression analysis to be

76

significantly more frequent in resistant isolates: agrBII, agrCII, seg, sei, seo, sey, and a

specific variant of protein A (Table 6). Conversely, both agrBI and agrDI were more

frequent in susceptible isolates (Table 6).

bla-L sequence diversity. 55 of the 57 blaz genes were sequenced to assess sequence

diversity in bovine mastitis. Sequences were compared to a database composed of blaz

gene sequences from Genbank. Eight different sequence types were identified among the

55 isolates investigated (Figure 5). Four of these variants had not been reported before.

The number of each blaz variant per farm is shown in Table 7. In most cases the isolates

from the same farm clustered together with a few exceptions (farms 205, 220 and 319).

These farms were distributed across Canada and were located in Alberta (n=3 farms),

Ontario (n=3 farms) and Quebec (n=5 farms).

Characterization of the Staphylococcal Chromosome Cassette in MRSA. The

MRSA isolate was shown to harbour a Type IV cassette. Further detailed characterization

of this cassette performed using sequencing revealed it was similar to a Type IVc cassette

with some new variations (Figure 7).

MLST analysis and spa Typing. Fourteen penicillin-resistant S. aureus strains,

including the four MRSA isolates, from nine different farms, were selected and matched

to nine susceptible isolates from the same farms. The four MRSA isolates came from one

cow. All 23 isolates were typed using MLST (Table 8). The main sequence types (ST's)

found were ST151 (n=7, 4 farms), ST352 (n=5, 5 farms), and ST705 (n=3, 1 farm). These

three ST's have been previously found in bovine mastitis isolates from The Netherlands,

Japan and Spain. One penicillin-resistant isolate was ST45, a sequence type previously

identified in humans. Four MRSA isolates were recovered from a single infected quarter

77

of a cow over a period of 33 days and were all ST8 (USA3 00 / CMRSA-10) and spa type

?451, which is normally associated with humans and is often community-associated

MRSA (caMRSA) (GenBank: CP000255). Non-MRSA isolates (n=3) from the same

farm as the MRSA isolate were ST350. A Neighbour-Joining tree based on concatenated

sequences of each MLST ST shows a high diversity with few STs closely related (Figure

6) and an eBurst analysis showed only singletons with the exception of a close

relationship between ST151 and ST705.

DISCUSSION

The results of this study on P-lactam resistance in S. aureus from mastitis are in

full agreement with previous investigations, showing that blaz and mecA are the two

major resistance determinants for this class of antimicrobials (Haveri et al. 2005,

Deurenberg et al. 2007).

The MRSA isolate characterized in this study also contained a number of

resistance genes for other antimicrobial agent classes. The low frequency of resistance

genes other than blaz in bovine mastitis compares to what has been found before and

highlights the importance of MRSA isolates which often harbour multiple resistance

genes (Monecke et al. 2007, Piccinini et al. 2010).

The major enterotoxins we found were genes encoding seg, set, seo, sex, and sey.

There have been a number of studies which have looked at the enterotoxins and other

virulence genes present in bovine mastitis isolates however there are few from North

America. Srinivasan et al. found that 93.6% of all bovine mastitis S. aureus isolates from

the United States harboured enterotoxin genes (Srinivasan et al. 2006). The seg, sei and

78

seo toxin genes have also been found by several other researchers (Akineden et al. 2001,

Srinivasan et al. 2006, Piccinini et al. 2010). We did not frequently detect sed, sej, sen,

and sent, but others have identified them regularly (Akineden et al. 2001, Srinivasan et al.

2006, Piccinini et al. 2010). No global common picture seems to emerge from these

multiple studies. However, the differences in enterotoxin gene patterns and frequencies

may be the result of the over-representation of penicillin-resistant isolates in our study or

it may be because of the small sample size or a difference in geographical locations.

Overall, enterotoxins normally associated with food poisoning {sea, seb, sec, sed, see)

were detected only rarely or not at all in our S. aureus isolates. This is consistent with the

low prevalance of food poisoning outbreaks caused by S. aureus compared to other

foodborne pathogens due to drinking raw milk (Oliver et al. 2009).

Other major virulence genes of interest which were frequently detected in our

isolates included the leukocidins, agr genes and the hemolysins. The major leukocidins

detected were lukE, lukD, and lukF. These have been detected before in S. aureus isolates

from mastitis and other sources (Haveri et al. 2005). Similar to other studies (Haveri et

al. 2005, Piccinini et al. 2010), toxic shock syndrome toxin (tsst-1) was absent from our

isolates.

A large diversity of virulence genes were detected in both susceptible and

resistant isolates; however, these genes were not evenly distributed and penicillin-

resistant isolates harboured more virulence genes than susceptible isolates. The reason for

the apparent association between resistance and virulence may be due to an increased rate

of horizontal gene transfer in comparison to susceptible isolates increasing their ability to

take up virulence genes frequently located on mobile genetic elements (Martinez and

79

Baquero 2002). Alternatively, more virulent strains may also undergo antimicrobial

therapy more frequently because of the severity of infection. A study by Haveri et al.

detected a similar association between the presence of blaz and pyrogenic toxin

superantigens (Haveri et al. 2007). They also found that those isolates harbouring blaz

caused persistent cases of mastitis. Similarly, MRSA is also more virulent than MSSA

and causes an increased rate of mortality in bacteraemic infections (Melzer et al. 2003).

A relationship between virulence and resistance has been postulated before in a number

of microorganisms; however, the reasons behind this may be multiple and remain largely

hypothetical (Martinez and Baquero 2002).

Univariable analysis demonstrated that specific virulence genes were more

frequent in penicillin-resistant isolates than in susceptible ones. Various enterotoxins

(seg, sei, seo, and sey) were significantly associated with resistant isolates. There is little

information regarding the significance of these novel enterotoxins in bovine mastitis or

any animal infection and requires further investigation. This higher prevalance of specific

enterotoxins in the resistant isolates may be a real association between resistance and

virulence but it could also be the result of the spread of a single clonal lineage which has

accumulated both types of genes. However, MLST analysis of these resistant isolates

showed the presence of a large diversity of unrelated sequence types among penicillin-

resistant isolates.

Penicillin-resistant isolates were more likely to harbour a different variant of the

accessory gene regulator B and C (agrBII, agrCII compared to agrBI, agrCI) than

susceptible isolates. Agr is a two-component regulatory system responsible for up-

regulating exo-proteins and down-regulating cell-wall associated proteins (Chien and

80

Cheung 1998). Recently, it was shown that MRSA isolates from infections were more

likely to harbour agr type 1 compared to colonisation isolates (Thomsen et al. 2011). A

study conducted in Italy using the same Array Tube found that high prevalance isolates

were normally associated with agr type II (Piccinini et al. 2010). Therefore, the presence

of a specific agr type in the resistant isolates may be associated with particular types of

infection/colomzation caused by resistant isolates. Further investigation using a larger

sample size and infection model studies may shed more light on this theory.

Little is known regarding the diversity of the blaz gene. A previous study by

Olsen et al. also found a high diversity among 105 human and bovine isolates, detecting

69 different sequence types (Olsen et al. 2006). Although 69 sequence types were

detected they translated into a limited number of protein types. Our study only detected

eight variants out of a total of 55 isolates. The small sample size and inclusion of several

isolates per farm may have decreased the potential number of types detected. This later

hypothesis is supported by a apparent clustering of blaz type at the farm level (only three

farms had more than one blaz type although samples were taken from multiple cows in

eleven farms). Given this clustering, it appears that once a blaz positive isolate is

introduced on a farm it may be able to persist and become or remain the sole blaz type.

Further molecular invest!aation of these nersistent isolates mav heln to identify whether a

single resistant strain is responsible for the majority of penicillin-resistant blaz infections

in each farm, or if the blaz gene is spread horizontally through plasmid transfer (Voladri

and Kernodle 1998) across a diversity of strains present on each premise.

Characterization of the MRSA isolate from this study showed that it was ST8

caMRSA SCCmec type IVc similar to USA-300 / CMRSA-10, the human strain capable

81

of causing severe infections in North America (Tenover and Goering 2009). European

studies identified ST398, first detected in factory farmed pigs, as the major MRSA ST

from bovine mastitis (Fessler et al. 2010, Huber et al. 2010, Vanderhaeghen et al. 2010).

Other ST's from bovine mastitis identified worldwide include: ST1 and 72 from Korea

and ST5 from France and Japan (Hata et al. 2010, Haenni et al. 2011, Nam et al. 2011).

To our knowledge caMRSA ST8 has only been detected in bovine mastitis in Turkey

(Turkyilmaz et al. 2010). ST8 has also been found in companion animals, and horses (Lin

et al. 2010, van Duijkeren et al. 2010). Due to the association of this strain with humans,

it is likely that the cow was originally infected by a human during routine handling or

milking procedures. The SCCmec cassette was found to be type IVc (with some

additional new variations), which fits with the MRSA isolate being a community-

acquired strain of human origin (Yamamoto et al. 2010). The MRSA strain subsequently

persisted in the udder of this cow for a period of at least a month and a half. Thus, even

though this strain is similar to a human strain it may be able to persist for a significant

time in the bovine udder. Although only MSSA of different STs were recovered from

other cows from the same farm, further investigations are needed in order to assess the

potential of this strain to survive and spread further in cows and establish itself in dairy

cattle populations.

Most of the MSSA isolates from this study belonged to three ST's normally

associated with bovine mastitis. One penicillin-resistant isolate was ST45, a sequence

type often associated with human infections and colonization which has not yet been

described in relation with animals to date (Cuny et al. 2010). The ability of a human-

specific strain (ST8) to persist and cause intramammary infections in dairy cattle may be

82

a sign of S. aureus evolving toward an increased zoonotic potential. Increased evidence is

showing that MRSA is an emerging zoonotic agent that could potentially have reservoirs

in livestock (Springer et al. 2009).

In conclusion, this study provides a better understanding of pMactam resistance in

S. aureus. All of the penicillin-resistant S. aureus isolates contained the blaz gene. The

blaz variant clustered per farm, suggesting either a clonal spread of resistant strains

within farms or the horizontal transfer of blaz between strains witin a farm. A caMRS A

ST8 SCCmec IVc isolate was also identified, highlighting the potential that human

sequence types may be able to infect dairy cows and persist for several weeks in the

udder. Penicillin-resistance was shown to be associated with increased virulence potential

in S. aureus from mastitis. Specific enterotoxins and agr variants are found with resistant

isolates. MLST analysis suggests a true association between virulence and pencillin

resistance rather than an association related to the clonal spread of a particular clone. The

overall resistance in S. aureus from bovine mastitis is low, but the presence of a multi-

resistant MRSA isolate and penicillin-resistant strains with an increased number of

virulence genes is a possible cause for concern.

ACKNOWLEDGEMENTS

We would like to thank Matt Saab from the University of Prince Edward Island for his

technical assistance. This research was financed by the Natural Science and Engineering

Research Council, Alberta Milk, Dairy Farmers of New Brunswick, Nova Scotia, Ontario

and Prince Edward Island, Novalait Inc., Dairy Farmers of Canada, Canadian Dairy

Network, Agriculture and Agri-Food Canada, Public Health Agency of Canada,

83

Technology PEI Inc., Universite de Montreal and University of Prince Edward Island,

through the Canadian Bovine Mastitis Research Network.

84

Table 6. Associations of virulence genes compared between penicillin-resistant S. aureus and susceptible S. aureus isolates from bovine mastitis.

/- n i r\AA T> 4.- 95% Confidence Gene P-value Odds Ratio T . ,

Interval

femA O02

katA 0.042

agrB-I 0.022

agrB-II <0.001

agrC-II 0.007

agrD-I 0.001

entG 0.011

entl 0.014

en«9 0.028

entY 0.036

Mm 0.021

ProteinA 0.032

set2-var2 0.004

setf-varl 0.023

set6-\ar2 0.007

5^5 0.001

^e/P-varl 0.05

13.37

7.84

0.09

33.93

23.05

0.05

10.89

44.67

26.62

7.08

0.06

7.71

16.99

0.12

38.7

37.29

0.14

1.50-119.55

1.08-57.12

0.01-0.71

5.36-214.59

2.35-226.30

0.01-0.27

1.72-68.98

2.16-923.28

1.41-501.61

1.13-44.14

0.005-0.65

1.19-50.01

2.44-118.23

0.02-0.75

2.69-557.51

4.86-286.25

0.02-0.90

85

ttrfP-varl-2 0.01 0.05 0.005-0.48

Only statistically significant associations (p < 0.05) calculated using exact logistic

regression using gene as the x variable and susceptible/resistant as the y variable while

correcting for clustering of the farms, are listed.

86

Table 7. Number and identity ofblaz variants found within farms across Canada.

Province

Farm

AB

108

AB

110

AB

111

ON

205

ON

220

ON

221

QC

308

QC

310

QC

318

QC

319

QC

321

Vl a 3(2)

V2 l(l)b 1(1) 1(1)

V3 1(1)

V4 17(12) 1(1) 11(9)

V5 1(1) 2(1) 9(7) 3(3)

V6 1(1)

V7 2(1)

V8 1(1)

a VI-V8, abbreviations for variants 1 to 8; b The first number represents the number of

isolates and the numbers in brackets represent the number of cows. AB = Alberta, ON =

Ontario, QC = Quebec.

87

Table 8. MLST analysis of eleven penicillin-resistant and nine susceptible S. aureus

isolates.

T , Resistant/Susceptible Herd(s) Sequence Type

6 R 205,220,308 151

1 S 318 151

352

352

705

1

4

3

1

3

1

1

Not available.

R

S

R

R

S

R

S

111

220,223,308,319

319

108

108

110

108

350

45

NAa

88

0.005

-SA5 ISA1029 1SA822 'SA815

SA37 SA391 SA428 SA575 SA978 SA909 SA906 SA905 SA903 SA902 SA1323 SA1322 SA1321 SA1319 SA1212 SA1210 SA1209 SA1204 SA1201 SA486 SA382

SA1326 SA1 SA911 SA908 SA904 SA267 SA435 SA424 SA394 SA175 SA1171 SA1152 SA1150 SA1131 SA1129 SA1058 SA1024 SA966 SA965 SA581 SA536 SA454 SA288 SA207

-SA1155 - 1 J S A 9 5 9

"SA258

ISA1107 1—--SA1153 'SA577

Figure 4. Neighbour-Joining plot depicting the diversity of the blaz gene in bovine

mastitis isolates.

89

I SA5_ST45_blaZ_C_l 10

I SA2057_STNA_108

I SA815_ST352_111 1 SA379_ST352_220

SA287_ST352_223

' SA136_ST352_308

SA132_ST352_319

SA2053_ST8_108 1 SA2058_ST8_108

SA2052_ST8_108

SA822_ST8_blaZ_B_l 08

SA909_ST151_blaZ_D_205

SA1129_ST15 l_blaZ_E_220

SAl_ST151_blaZ_D_205

SA16_ST151_318

SA1153_ST705_blaZ_H_319

SA1107_ST705_blaZ_G_319

I SA577_ST705_blaZ_G_319

SA1326_ST151_blaZ_F_205

SA1155_ST15 l_blaZ_A_308

SA394_ST151_blaZJD_205

SA2056_ST350_108

SA2064_ST350_108

SA2054_ST350_108

Figure 5. Neighbour-Joining plot depicting the diversity of the MLST sequence types in

bovine mastitis. Isolate numbers are followed by sequence types, blaz types and herd

numbers.

90

Jl-IVc IS256 IS256

SCCmec IVc

IS1272 IS431 Tn4001 24,106

IS2S6 IS2S6

r 0

Jl-IVK Jl-IVc

IS1272 IS431 Tn4001 24,106

SCCmec IVc MRSASA822

Figure 6. SCCmec type IVc with some recombinations from the bovine mastitis MRS A

isolate SA822. Light grey coloured line indicates an unsequenced section of the SCCmec.

91

DISCUSSION AND CONCLUSIONS

The work presented in this thesis provides a better understanding of AMR in

major agents of bovine mastitis across Canada. To date, there have been few studies

worldwide which have investigated AMR determinants in bovine mastitis, and even

fewer which have looked at the p-lactamase genes. This is the first cross-Canada study

able to provide a genotypic AMR baseline for E. coli, Klebsiella spp. and S. aureus from

bovine mastitis. We detected a large diversity of P-lactamase genes in E. coli and

Klebsiella spp. isolates. All of the penicillin-resistant S. aureus isolates from this study

harboured the blaz gene and the first MRSA isolate from bovine mastitis in Canada was

characterized.

Klebsiella spp. isolates were chosen as part of this project for a number of

reasons. Klebsiella spp. are responsible for most peracute cases of mastitis, they are

frequently multi-resistant and are the second most common cause of coliform mastitis

(Hogan and Smith 2003; Colodner 2005).

Among the diverse P-lactamase resistance determinants detected in E. coli isolates

ampC promoter mutations were found repeatedly for those isolates not harbouring a

horizontally acquired P-lactamase gene. This may not be surprising among agents of

mastitis because of the decreased likelihood of resistance gene acquisition in the udder as

previously postulated (Martel et al. 1995). However, it is an interesting finding and fits

with the observations that mastitis is the major cause of antimicrobial use in dairy cattle

and that resistance genes are relatively rarely detected in mastitis isolates. Associations

between blajEu and other resistance genes were also found. Although these associations

had been detected before in swine E. coli (Rosengren et al. 2009), they provide evidence

92

to support the idea that the use of non-P-lactam antimicrobials may be selecting for |3-

lactamase resistance.

Discrepancies between genotype and phenotypic susceptibility testing data for

aadAl and sull were found. This stresses the need for further investigation and

correlation between the presence of AMR genes and resistance levels as well as on the

significance of clinical and epidemiological breakpoints for surveillance of antimicrobial

resistance.

Multi-resistance plasmids harbouring the ubiquitous blacuY-2 gene were detected

among the E. coli isolates indicating that this gene is present in mastitis isolates from

dairy cattle. Its presence on a multi-resistance plasmid accords the potential for co-

selection and treatment failures. Co-selection for extended-spectrum cephalosporin

resistance can occur by simply using non-P-lactam antimicrobials which are sometimes

used to treat mastitis. These plasmids could also result in a decrease in treatment options.

They are similar to plasmids detected in beef cattle because they may encode factors

important to the survival of E. coli in cattle or are just belong to one of the two most

widespread blacMY-2 plasmids type detected in many animal species across North

America (Call et al. 2010). Characterization of the plasmid carrying the WOPSE-I gene and

screening of further plasmids carrying this gene showed that its genetic environment is

different in E. coli and S. Typhimurium. In E. coli, it is part of an integron along with

three other resistance genes encoding resistance towards antimicrobials often used to treat

bovine mastitis.

Although a low diversity of resistance determinants was found for S. aureus from

mastitis, a high diversity of virulence genes was observed and associations between

93

penicillin-resistance and virulence were identified. Of the major virulence genes found, a

number of enterotoxins were significantly more prevalent in resistant isolates than

susceptible isolates. As well, specific variants of agr were associated with resistant or

susceptible isolates. The increased prevalance of specific enterotoxins or agr variants in

the resistant isolates may increase the virulence of these isolates by increasing their

ability to survive within the host.

Further characterization of the MRSA isolate showed it was ST8 caMRSA

SCCmecIVc similar to USA-300 / CMRSA-10. Due to its close relatedness to human

MRSA we hypothesized that the microorganism may have been transferred during

handling by a human. Of interest, the ability of this human-specific strain to persist

within the bovine udder provides evidence towards the hypothesis that many MRSA

strains are spreading to animal populations.

A comparison of the occurence of resistance between E. coli, Klebsiella spp.

isolates and S. aureus highlights the differences between these microorganisms. S. aureus

is a contagious organism which is able to reside on the bovine udder and because of this

is able to transfer between farms. Therefore, specific strains of S. aureus are thought to be

responsible for causing bovine mastitis. E. coli and Klebsiella spp. isolates are

environmental and isolates responsible for causing bovine mastitis are not known to

contain specific characteristics (Suojala et al, 2011). The emergence of resistance in both

E. coli and Klebsiella spp. isolates is often due to the presence of mobilizable elements

rather than the transfer of specific strains. Overall, although these organisms are

strikingly different they are both able to use their own mechanisms for the selection and

emergence of resistance.

94

Although microarrays provide a plethora of data, they are currently only feasible

for use with a limited number of samples. Even though we generated a large amount of

data, this study may need to be repeated using a larger sample size in order to obtain

increased statistical power. As well, although the ArrayTube was able to detect most

resistance genes, there were instances where an isolate was phenotypically resistant but

no specific gene was detected. Using a microarray technique guarantees the detection of

most gene variants but it does not identify rare genes. For some of the ampicillin-resistant

E. coli isolates we could not detect a specific P-lactamase gene even though they were

phenotypically resistant. In the future, as the cost of microarray technology decreases, the

use of a larger microarray with an increased number of probes may be plausible and

would ensure this problem does not occur again.

Due to the low AMR frequency in bovine mastitis we had difficulty acquiring

enough isolates for analysis. Ampicillin-resistant E. coli isolates were obtained from

other sources which may produce a bias in the results.

The previously described research brings up a number of future directions.

Overall, the reproduction of this work using an increased number of isolates and a larger

microarray may provide a better baseline of resistance for bovine mastitis in Canada. In

terms of reproducibility, this project could be easily reproduced because the sampling

platform has been set-up and there is little sampling bias. Also, this data may supply a

starting point for a surveillance system to monitor resistance genes in Canadian dairy

cattle. Infection models using the penicillin-resistant S. aureus isolates may provide

insight into the hypothesis that resistant isolates are often more virulent, with special

95

regard to the MRSA isolate. Conjugation experiments using the blapsE-i and blacuY-2

plasmids may provide knowledge of their transfer mechanisms.

A number of questions are raised by this work including: Why are different 0-

lactamases detected between E. coli and Klebsiella spp.? Are the blacMY-2 and bla^sE-i

plasmids emerging in mastitis isolates from dairy cattle or have they been there for

awhile? Are resistant S. aureus isolates from bovine mastitis also more virulent? Are

specific strains of S. aureus better able to survive in the udder? Is MRSA from humans

able to persist and cause disease in dairy cattle?

Overall, the research presented in this thesis enhances our understanding of

antimicrobial resistance in E. coli, Klebsiella spp., and S. aureus from bovine mastitis in

Canada and provides much needed information required to ensure prudent usage of

antimicrobials for treatment of dairy cattle by providing data on the prevalance of

specific AMR determinants. The study of the characterization of resistance in E. coli and

Klebsiella spp. isolates detected a surprisingly high diversity of pMactamase genes and

perhaps the presence of multi-resistance plasmids and genes not known to occur

previously in bacteria from mastitis samples from dairy cattle. The first MRSA mastitis

isolate from dairy cattle in Canada was characterized in detail and resistant isolates were

significantly more likely to harbour virulence genes including different variants of agr

and specific enterotoxins. Although the prevalance of P-lactam resistance in E. coli,

Klebsiella spp. and S. aureus isolates from bovine mastitis is low the potential for multi-

resistance and increased virulence of these strains requires monitoring.

96

REFERENCES

Aarestrup, F. (2006) Antimicrobial Resistance in Bacteria of Animal Origin. Washington,

D.C.: ASM Press.

Akineden, O., Annemiiller, C , Hassan, A.A., Lammler, C , Wolter, W. and Zschock, M.

(2001) Toxin genes and other characteristics of Staphylococcus aureus isolates from milk

of cows with mastitis. Clin Diagn Lab Immunol 8, 959-964.

Allen, K.J. and Poppe, C. (2002) Occurrence and characterization of resistance to

extended-spectrum cephalosporins mediated by beta-lactamase CMY-2 in Salmonella

isolated from food-producing animals in Canada. Can J Vet Res 66,137-144.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local

alignment search tool. JMol Biol 215,403-410.

Ambler, R.P. (1980) The structure of beta-lactamases. Philos Trans R Soc Lond B Biol

Sci 289,321-331.

Babini, G.S. and Livermore, D.M. (2000) Are SHV beta-lactamases universal in

Klebsiella pneumoniae! Antimicrob Agents Chemother 44, 2230.

Batchelor, M., Hopkins, K.L., Liebana, E., Slickers, P., Ehricht, R., Mafura, M.,

Aarestrup, F., Mevius, D., Clifton-Hadley, F.A., Woodward, M.J., Davies, R.H.,

Threlfall, E.J. and Anjum, M.F. (2008) Development of a miniaturised microarray-based

assay for the rapid identification of antimicrobial resistance genes in Gram-negative

bacteria. Int J Antimicrob Agents 31, 440-451.

97

Bengtsson, B., Unnerstad, H.E., Ekman, T., Artursson, K., Nilsson-Ost, M. and Waller,

K.P. (2009) Antimicrobial susceptibility of udder pathogens from cases of acute clinical

mastitis in dairy cows. Vet Microbiol 136,142-149.

Bennett, P.M. (2008) Plasmid encoded antibiotic resistance: acquisition and transfer of

antibiotic resistance genes in bacteria. Br J Pharmacol 153 Suppl 1, S347-57.

Boerlin, P., Travis, R., Gyles, C.L., Reid-Smith, R., Janecko, N., Lim, H., Nicholson, V.,

McEwen, S.A., Friendship, R. and Archambault, M. (2005) Antimicrobial resistance and

virulence genes of Escherichia coli isolates from swine in Ontario. Appl Environ

Microbiol 71, 6753-6761.

Bonnet, C , Diarrassouba, F., Brousseau, R., Masson, L., Topp, E. and Diarra, M.S.

(2009) Pathotype and antibiotic resistance gene distributions of Escherichia coli isolates

from broiler chickens raised on antimicrobial-supplemented diets. Appl Environ

Microbiol 75, 6955-6962.

Botrel, M.A., Haenni, M., Morignat, E., Sulpice, P., Madec, J.Y. and Calavas, D. (2010)

Distribution and antimicrobial resistance of clinical and subclinical mastitis pathogens in

dairy cows in Rhone-Alpes, France. Foodborne Pathog Dis 7,479-487.

Boyd, D., Cloeckaert, A., Chaslus-Dancla, E. and Mulvey, M.R. (2002) Characterization

of variant Salmonella genomic island 1 multidrug resistance regions from serovars

Typhimurium DT104 and Agona. Antimicrob Agents Chemother 46,1714-1722.

98

Bradford, P.A. (2001) Extended-spectrum beta-lactamases in the 21st century:

characterization, epidemiology, and detection of this important resistance threat. Clin

Microbiol Rev 14, 933-51.

Bradley, A.J. (2002) Bovine Mastitis: An Evolving Disease. The Veterinary Journal 164,

116-128.

Brenner, D.J., Staley, J.T. and Krieg, N.R. (2001) Classification of Procaryotic

Organisms and the Concept of Bacterial Speciation. In Bergey's Manual of Systematic

Bacteriology ed. Garrity, G.M. pp. 29-31. New York, United States: Bergey's Manual

Trust.

Bryskier, A. (2005) Antibiotics and Antibacterial Agents: Classifications and Structure-

Activity Relationship. Washington, United States: ASM Press.

Bush, K., Jacoby, G.A. and Medeiros, A.A. (1995) A functional classification scheme for

beta-lactamases and its correlation with molecular structure. Antimicrob Agents

Chemother 39,1211-1233.

Call, D.R., Singer, R.S., Meng, D., Broschat, S.L., Orfe, L.H., Anderson, J.M., Herndon,

D.R., Kappmeyer, L.S., Daniels, J.B. and Besser, T.E. (2010) blaCMY-2-positive IncA/C

plasmids from Escherichia coli and Salmonella enterica are a distinct component of a

larger lineage of plasmids. Antimicrob Agents Chemother 54, 590-596.

Canadian Animal Health Institute (2009) Compendium of Veterinary Products 2009.

Michigan, United States: North American Compendiums Inc.

99

Canadian Bovine Mastitis Research Network (2009) Canadian Bovine Mastitis Research

Network Annual Report 2008-2009. 2, 8.

Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K.L. and Threlfall, E.J. (2005)

Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 63, 219-

228.

Carattoli, A., Tosini, F., Giles, W.P., Rupp, M.E., Hinrichs, S.H., Angulo, F.J., Barrett,

T.J. and Fey, P.D. (2002) Characterization of plasmids carrying CMY-2 from expanded-

spectrum cephalosporin-resistant Salmonella strains isolated in the United States between

1996 and 1998. Antimicrob Agents Chemother 46, 1269-1272.

Caroff, N., Espaze, E., Berard, I., Richet, H. and Reynaud, A. (1999) Mutations in the

ampC promoter of Escherichia coli isolates resistant to oxyiminocephalosporins without

extended spectrum beta-lactamase production. FEMS Microbiol Lett 173,459-465.

Catry, B., Laevens, H., Devriese, L.A., Opsomer, G. and De Kruif, A. (2003)

Antimicrobial resistance in livestock. J Vet Pharmacol Ther 26, 81-93.

Chebel, R.C. (2007) Mastitis Effects on Reproduction. NMC Regional Meeting

Proceedings, 43-44-55.

Chen, S., Zhao, S., White, D.G., Schroeder, CM., Lu, R., Yang, H., McDermott, P.F.,

Ayers, S. and Meng, J. (2004) Characterization of multiple-antimicrobial-resistant

salmonella serovars isolated from retail meats. Appl Environ Microbiol 70,1-7.

100

Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Higgins, D.G. and

Thompson, J.D. (2003) Multiple sequence alignment with the Clustal series of programs.

Nucleic Acids Res 31, 3497-3500.

Chien, Y. and Cheung, A.L. (1998) Molecular interactions between two global

regulators, sar and agr, in Staphylococcus aureus. J Biol Chem 273,2645-2652.

Clinical and Laboratory Standards Institute (2011) Performance Standards for

Antimicrobial Susceptibility Testing. M100-S21.

Colodner, R. (2005) Extended-spectrum beta-lactamases: a challenge for clinical

microbiologists and infection control specialists. Am J Infect Control 33,104-107.

Constable, P.D. and Morin, D.E. (2003) Treatment of clinical mastitis. Using

antimicrobial susceptibility profiles for treatment decisions. Vet Clin North Am Food

AnimPract 19,139-155.

Cuny, C , Friedrich, A., Kozytska, S., Layer, F., Nubel, U., Ohlsen, K., Strommenger, B.,

Walther, B., Wieler, L. and Witte, W. (2010) Emergence of methicillin-resistant

Staphylococcus aureus (MRSA) in different animal species. Int J Med Microbiol 300,

109-117.

Deurenberg, R.H., Vink, C , Kalenic, S., Friedrich, A.W., Bruggeman, C.A. and

Stobberingh, E.E. (2007) The molecular evolution of methicillin-resistant Staphylococcus

aureus. Clin Microbiol Infect 13, 222-235.

101

Dingwell, R.T., Kelton, D.F. and Leslie, K.E. (2003) Management of the dry cow in

control of peripartum disease and mastitis. Vet Clin North Am FoodAnim Pract 19, 235-

265.

Dorrell, N., Hinchliffe, S.J. and Wren, B.W. (2005) Comparative phylogenomics of

pathogenic bacteria by microarray analysis. Curr Opin Microbiol 8, 620-626.

Edwards, P.R. and Ewing, W.H. (1986) Edwards and Ewing's Identification of

Enterobacteriaceae. New York: Elsevier.

Enright, M.C., Day, N.P., Davies, C.E., Peacock, S.J. and Spratt, B.G. (2000) Multilocus

sequence typing for characterization of methicillin-resistant and methicillin-susceptible

clones of Staphylococcus aureus. J Clin Microbiol 38,1008-1015.

Erskine, R.J., Wagner, S. and DeGraves, F.J. (2003) Mastitis therapy and pharmacology.

Vet Clin North Am FoodAnim Pract 19,109-38, vi.

Erskine, R.J., Walker, R.D., Bolin, C.A., Bartlett, P.C. and White, D.G. (2002) Trends in

antibacterial susceptibility of mastitis pathogens during a seven-year period. J Dairy Sci

85,1111-1118.

European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the

European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (2000)

EUCAST Definitive Document E.DEF 2.1, August 2000: Determination of antimicrobial

susceptibility test breakpoints. Clin Microbiol Infect 6, 570-572.

102

Fessler, A., Scott, C , Kadlec, K., Ehricht, R., Monecke, S. and Schwarz, S. (2010)

Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of

bovine mastitis. JAntimicrob Chemother 65, 619-625.

Fluit, A.C. and Schmitz, F.J. (1999) Class 1 integrons, gene cassettes, mobility, and

epidemiology. Eur J Clin Microbiol Infect Dis 18, 761-770.

Food and Agriculture Organization of the United Nations (2008), World Health

Organization, and Office International des Epizooties. Joint FAO/WHO/OIE expert

meeting on critically important antimicrobials. Report of a meeting held in FAO, Rome,

Italy, 26-30 November 2007. FAO, Rome, Italy and WHO, Geneva, Switzerland.

Foster, P.L. (2007) Stress-induced mutagenesis in bacteria. Crit Rev Biochem Mol Biol

42, 373-397.

Frye, J.G., Lindsey, R.L., Rondeau, G., Porwollik, S., Long, F., McClelland, M., Jackson,

C.R., Englen, M.D., Meinersmann, R.J., Berrang, M.E., Davis, J.A., Barrett, J.B., Turpin,

J.B., Thitaram, S.N. and Fedorka-Cray, P.J. (2009) Development of a DNA Microarray to

Detect Antimicrobial Resistance Genes Identified in the National Center for

Biotechnology Information Database. Microb Drug Resist 16, 9-19.

Gentilini, E., Denamiel, G., Llorente, P., Godaly, S., Rebuelto, M. and DeGregorio, O.

(2000) Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine

mastitis in Argentina. J Dairy Sci 83,1224-1227.

103

Giguere, S., Prescott, J.F., Baggot, J.D., Walker, R.D. and Dowling, P.M. (2006)

Antimicrobial Therapy in Veterinary Medicine. Ames, Iowa: Blackwell Pub.

Guerra, B., Junker, E., Schroeter, A., Malorny, B., Lehmann, S. and Helmuth, R. (2003)

Phenotypic and genotypic characterization of antimicrobial resistance in German

Escherichia coli isolates from cattle, swine and poultry. J Antimicrob Chemother 52,

489-492.

Gundogan, N. and Yakar, U.A. (2007) Siderophore production, serum resistance,

hemolytic activity and extended-spectrum beta-lactamase-producing Klebsiella species

isolated from milk and milk products. Journal of Food Safety, 251-251-264.

Gunson, R.N., Bennett, S., Maclean, A. and Carman, W.F. (2008) Using multiplex real

time PCR in order to streamline a routine diagnostic service. J Clin Virol 43, 372-375.

Gyles, C.L., Prescott, J.F., Songer, J.G. and Thoen, CO. (2004) Pathogenesis of

Bacterial Infections in Animals. Ames, Iowa: Blackwell Publishing.

Haenni, M., Galofaro, L., Ponsin, C, Bes, M., Laurent, F. and Madec, J.Y. (2011)

Staphylococcal bovine mastitis in France: enterotoxins, resistance and the human

Geraldine methicillin-resistant Staphylococcus aureus clone. J Antimicrob Chemother 66,

216-218.

Hamelin, K., Bruant, G., El-Shaarawi, A., Hill, S., Edge, T.A., Bekal, S., Fairbrother,

J.M., Harel, J., Maynard, C, Masson, L. and Brousseau, R. (2006) A virulence and

antimicrobial resistance DNA microarray detects a high frequency of virulence genes in

104

Escherichia coli isolates from Great Lakes recreational waters. Appl Environ Microbiol

72,4200-4206.

Harbottle, H., Thakur, S., Zhao, S. and White, D.G. (2006) Genetics of antimicrobial

resistance. Anim Biotechnol 17,111-124.

Harmon, R.J. (1994) Physiology of mastitis and factors affecting somatic cell counts. J

Dairy Sci 77,2103-2112.

Harmsen, D., Claus, H., Witte, W., Rothganger, J., Claus, H., Turnwald, D. and Vogel,

U. (2003) Typing of methicillin-resistant Staphylococcus aureus in a university hospital

setting by using novel software for spa repeat determination and database management. J

Clin Microbiol 41, 5442-5448.

Hata, E., Katsuda, K., Kobayashi, H., Uchida, I., Tanaka, K. and Eguchi, M. (2010)

Genetic variation among Staphylococcus aureus strains from bovine milk and their

relevance to methicillin-resistant isolates from humans. J Clin Microbiol 48, 2130-2139.

Haveri, M., Roslof, A., Rantala, L. and Pyorala, S. (2007) Virulence genes of bovine

Staphylococcus aureus from persistent and nonpersistent intramammary infections with

different clinical characteristics. J Appl Microbiol 103,993-1000.

Haveri, M., Suominen, S., Rantala, L., Honkanen-Buzalski, T. and Pyorala, S. (2005)

Comparison of phenotypic and genotypic detection of penicillin G resistance of

Staphylococcus aureus isolated from bovine intramammary infection. Vet Microbiol 106,

97-102.

105

Hendriksen, R.S., Mevius, D.J., Schroeter, A., Teale, C, Meunier, D., Butaye, P., Franco,

A., Utinane, A., Amado, A., Moreno, M., Greko, C , Stark, K., Berghold, C , Myllyniemi,

A.L., Wasyl, D., Sunde, M. and Aarestrup, F.M. (2008) Prevalence of antimicrobial

resistance among bacterial pathogens isolated from cattle in different European countries:

2002-2004. Acta Vet Scand 50.

Hogan, J. and Smith, K.L. (2003) Coliform Mastitis. Veterinary Research 34, 507-519.

Hornish, R.E. and Kotarski, S.F. (2002) Cephalosporins in veterinary medicine - ceftiofur

use in food animals. Curr Top Med Chem 2, 717-731.

Huber, H., Koller, S., Giezendanner, N., Stephan, R. and Zweifel, C. (2010) Prevalence

and characteristics of meticillin-resistant Staphylococcus aureus in humans in contact

with farm animals, in livestock, and in food of animal origin, Switzerland, 2009. Euro

Surveil! 15, 19542.

Jacoby, G.A. and Bush, K. (2009) Amino Acid Sequences for TEM, SHV and OXA

extended-spectrum and inhibitor resistant fi-lactamases. 2009.

Jin, L.Z. and Zhao, X. (2000) Intestinal receptors for adhesive fimbriae of

enterotoxigenic Escherichia coli (ETEC) K88 in swine—a review. Appl Microbiol

Biotechnol 54, 311-318.

Juhas, M., van der Meer, J.R., Gaillard, M., Harding, R.M., Hood, D.W. and Crook, D.W.

(2009) Genomic islands: tools of bacterial horizontal gene transfer and evolution. FEMS


106

Juhasz-Kaszanyitzky, E., Janosi, S., Somogyi, P., Dan, A., van der Graaf-van Bloois, L.,

van Duijkeren, E. and Wagenaar, J.A. (2007) MRSA transmission between cows and

humans. Emerg Infect Dis 13, 630-632.

Kim, T. and Heald, C.W. (1999) Inducing inference rules for the classification of bovine

mastitis. Computers and Electronics in Agriculture 23, 27-28-42.

Kloos, W.E. (1980) Natural populations of the genus staphylococcus. Annual Review of

Microbiology 34, 559-559-592.

Komano, T. (1999) Shufflons: multiple inversion systems and integrons. Annu Rev Genet

33,171-191.

Kozak, G.K., Boerlin, P., Janecko, N., Reid-Smith, RJ. and Jardine, C. (2009)

Antimicrobial resistance in Escherichia coli isolates from swine and wild small mammals

in the proximity of swine farms and in natural environments in Ontario, Canada. Appl

Environ Microbiol 75, 559-566.

Kwon, N.H., Park, K.T., Moon, J.S., Jung, W.K., Kim, S.H., Kim, J.M., Hong, S.K.,

Koo, H.C., Joo, Y.S. and Park, Y.H. (2005) Staphylococcal cassette chromosome mec

(SCCmec) characterization and molecular analysis for methicillin-resistant

Staphylococcus aureus and novel SCCmec subtype IVg isolated from bovine milk in

Korea. JAntimicrob Chemother 56, 624-632.

107

Lanz, R., Kuhnert, P. and Boerlin, P. (2003) Antimicrobial resistance and resistance gene

determinants in clinical Escherichia coli from different animal species in Switzerland.

Vet Microbiol 91, 73-84.

Lee, J.H. (2003) Methicillin (Oxacillin)-resistant Staphylococcus aureus strains isolated

from major food animals and their potential transmission to humans. Appl Environ

Microbiol 69, 6489-6494.

Lehtolainen, T., Shwimmer, A., Shpigel, N.Y., Honkanen-Buzalski, T. and Pyorala, S.

(2003) In vitro antimicrobial susceptibility of Escherichia coli isolates from clinical

bovine mastitis in Finland and Israel. J Dairy Sci 86,3927-3932.

Leslie, K.E., Schukken, Y.H., Archambault, M., Dingwell, R.T., Bashiri, A. and Leslie,

C.F. (2003) Antimicrobial resistance patterns of Staphylococcus aureus isolated from

intramammary infections before and after the dry period of dairy cattle in Canada. NMC

Council Annual Meeting Proceedings, 324-325.

Levesque, R.C. and Jacoby, G.A. (1988) Molecular structure and interrelationships of

multiresistance beta-lactamase transposons. Plasmid 19, 21-29.

Lewis, k., Salyers, A.A., Taber, H.W. and Wax, R.G. (2002) Bacterial Resistance to

Antimicrobials. New York, United States: Marcel Dekker.

Li, J.P., Zhou, H.J., Yuan, L., He, T. and Hu, S.H. (2009) Prevalence, genetic diversity,

and antimicrobial susceptibility profiles of Staphylococcus aureus isolated from bovine

mastitis in Zhejiang Province, China. JZhejiang Univ Sci B 10, 753-760.

108

Li, X.Z., Mehrotra, M., Ghimire, S. and Adewoye, L. (2007) beta-lactam resistance and

beta-lactamases in bacteria of animal origin. Vet Microbiol 121,197-214.

Lin, Y., Barker, E., Kislow, J., Kaldhone, P., Stemper, M.E., Pantrangi, M., Moore, F.M.,

Hall, M., Fritsche, T.R., Novicki, T., Foley, S.L. and Shukla, S.K. (2010) Evidence of

multiple virulence subtypes in nosocomial and community-associated MRSA genotypes

in companion animals from the upper Midwestern and Northeastern United States. Clin

Med Res.

Ling, T.K., Lyon, D.J., Cheng, A.F. and French, G.L. (1994) In-vitro antimicrobial

susceptibility and beta-lactamases of ampicillin-resistant Escherichia coli in Hong Kong.

JAntimicrob Chemother 34, 65-71.

Lipman, L.J., de Nijs, A. and Gaastra, W. (1995) Isolation and identification of fimbriae

and toxin production by Escherichia coli strains from cows with clinical mastitis. Vet

Microbiol 47', 1-7.

Livermore, D.M. (1995) beta-Lactamases in laboratory and clinical resistance. Clin


Locatelli, C , Caronte, I., Scaccabarozzi, L., Migliavacca, R., Pagani, L. and Moroni, P.

(2009) Extended-spectrum beta-lactamase production in E. coli strains isolated from

clinical bovine mastitis. Vet Res Commun 33, 141-144.

109

Locatelli, C , Scaccabarozzi, L., Pisoni, G. and Moroni, P. (2010) CTX-M1 ESBL-

producing Klebsiella pneumoniae subsp. pneumoniae isolated from cases of bovine

mastitis. J Clin Microbiol 48, 3822-3823.

Maidhof, H., Guerra, B., Abbas, S., Elsheikha, H.M., Whittam, T.S. and Beutin, L.

(2002) A multiresistant clone of Shiga toxin-producing Escherichia coli 0118:[HI6] is

spread in cattle and humans over different European countries. Appl Environ Microbiol

68, 5834-5842.

Makovec, J.A. and Ruegg, P.L. (2003) Antimicrobial resistance of bacteria isolated from

dairy cow milk samples submitted for bacterial culture: 8,905 samples (1994-2001). J Am

Vet Med Assoc 222, 1582-1589.

Markoulatos, P., Siafakas, N. and Moncany, M. (2002) Multiplex polymerase chain

reaction: a practical approach. J Clin Lab Anal 16,47-51.

Mattel, J.L., Chaslus-Dancla, E., Coudert, M., Poumarat, F. and Lafont, J.P. (1995)

Survey of antimicrobial resistance in bacterial isolates from diseased cattle in France.

Microb Drug Resist 1, 273-283.

Martinez, J.L. and Baquero, F. (2002) Interactions among strategies associated with

bacterial infection: pathogenicity, epidemicity, and antibiotic resistance. Clin Microbiol

Rev 15, 647-679.

Medeiros, A.A., Hedges, R.W. and Jacoby, G.A. (1982) Spread of a "Pseudomonas-

specific" beta-lactamase to plasmids of enterobacteria. JBacteriol 149, 700-707.

110

Melzer, M., Eykyn, S.J., Gransden, W.R. and Chinn, S. (2003) Is methicillin-resistant

Staphylococcus aureus more virulent than methicillin-susceptible S. aureus? A

comparative cohort study of British patients with nosocomial infection and bacteremia.

Clin Infect Dis 37,1453-1460.

Miserez, R., Frey, J., Buogo, C , Capaul, S., Tontis, A., Burnens, A. and Nicolet, J.

(1998) Detection of alpha- and epsilon-toxigenic Clostridium perfringens type D in sheep

and goats using a DNA amplification technique (PCR). LettAppl Microbiol 26, 382-386.

Monecke, S., Kuhnert, P., Hotzel, H., Slickers, P. and Ehricht, R. (2007) Microarray

based study on virulence-associated genes and resistance determinants of Staphylococcus

aureus isolates from cattle. Vet Microbiol 125,128-140.

Monecke, S., Slickers, P., Hotzel, H., Richter-Huhn, G., Pohle, M., Weber, S., Witte, W.

and Ehricht, R. (2006) Microarray-based characterisation of a Panton-Valentine

leukocidin-positive community-acquired strain of methicillin-resistant Staphylococcus

aureus. Clin Microbiol Infect 12, 718-728.

Moon, J.S., Lee, A.R., Kang, H.M., Lee, E.S., Kim, M.N., Paik, Y.H., Park, Y.H., Joo,

Y.S. and Koo, H.C (2007) Phenotypic and genetic antibiogram of methicillin-resistant

staphylococci isolated from bovine mastitis in Korea. J Dairy Sci 90,1176-1185.

Moroni, P., Pisoni, G., Antonini, M., Villa, R., Boettcher, P. and Carli, S. (2006) Short

Communication: Antimicrobial Drug Susceptibility of Staphylococcus aureus from

Subclinical Bovine Mastitis in Italy. Journal of Dairy Science 89,2973-2973-2976.

I l l

Mullis, K.B. and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a

polymerase-catalyzed chain reaction. Methods Enzymol 155, 335-350.

Munoz, M.A., Welcome, F.L., Schukken, Y.H. and Zadoks, R.N. (2007) Molecular

epidemiology of two Klebsiella pneumoniae mastitis outbreaks on a dairy farm in New

York State. J Clin Microbiol 45, 3964-3971.

Nam, H.M., Lee, A.L., Jung, S.C., Kim, M.N., Jang, G.C., Wee, S.H. and Lim, S.K.

(2011) Antimicrobial susceptibility of Staphylococcus aureus and characterization of

methicillin-resistant Staphylococcus aureus isolated from bovine mastitis in Korea.

Foodborne PathogDis 8,231-238.

Nam, H.M., Lim, S.K., Kang, H.M., Kim, J.M., Moon, J.S., Jang, K.C., Kim, J.M., Joo,

Y.S. and Jung, S.C. (2009) Prevalence and antimicrobial susceptibility of gram-negative

bacteria isolated from bovine mastitis between 2003 and 2008 in Korea. J Dairy Sci 92,

2020-2026.

Nannini, E.C., Singh, K.V. and Murray, B.E. (2003) Relapse of type A beta-lactamase-

producing Staphylococcus aureus native valve endocarditis during cefazolin therapy:

revisiting the issue. Clin Infect Dis 37, 1194-1198.

Nemeth, J., Muckle, C.A. and Gyles, C.L. (1994) In vitro comparison of bovine mastitis

and fecal Escherichia coli isolates. Vet Microbiol 40,231-238.

112

Nunes, S.F., Bexiga, R., Cavaco, L.M. and Vilela, C.L. (2007) Technical note:

Antimicrobial susceptibility of Portuguese isolates of Staphylococcus aureus and

Staphylococcus epidermidis in subclinical bovine mastitis. J Dairy Sci 90, 3242-3246.

Ojo, K.K., Ulep, C , Van Kirk, N., Luis, H., Bernardo, M., Leitao, J. and Roberts, M.C.

(2004) The mef(A) gene predominates among seven macrolide resistance genes identified

in gram-negative strains representing 13 genera, isolated from healthy Portuguese

children. Antimicrob Agents Chemother 48, 3451-3456.

Olde Riekerink, R.G., Barkema, H.W., Kelton, D.F. and Scholl, D.T. (2008) Incidence

rate of clinical mastitis on Canadian dairy farms. J Dairy Sci 91,1366-1377.

Olesen, I., Hasman, H. and Aarestrup, F.M. (2004) Prevalence of beta-lactamases among

ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in

Denmark. Microb Drug Resist 10, 334-340.

Oliveira, D.C. and de Lencastre, H. (2002) Multiplex PCR strategy for rapid

identification of structural types and variants of the mec element in methicillin-resistant

Staphylococcus aureus. Antimicrob Agents Chemother 46,2155-2161.

Oliver, S.P., Boor, K.J., Murphy, S.C. and Murinda, S.E. (2009) Food safety hazards

associated with consumption of raw milk. Foodborne Pathog Dis 6, 793-806.

Olsen, J.E., Christensen, H. and Aarestrup, F.M. (2006) Diversity and evolution of blaZ

from Staphylococcus aureus and coagulase-negative staphylococci. J Antimicrob

Chemother 57, 450-460.

113

Onile, B.A. (1985) Review of group B streptococci and their infections. Afr J Med Med

Sci 14,131-143.

Paulin-Curlee, G.G., Sreevatsan, S., Singer, R.S., Isaacson, R., Reneau, J., Bey, R. and

Foster, D. (2008) Molecular subtyping of mastitis-associated Klebsiella pneumoniae

isolates shows high levels of diversity within and between dairy herds. J Dairy Sci 91,

554-563.

Perriere, G. and Gouy, M. (1996) WWW-query: an on-line retrieval system for biological

sequence banks. Biochimie 78,364-369.

Philpot, W.N. and Nickerson, S.C. (2000) Winning the Fight Against Mastitis.

Naperville, IL: Westfalia Surge, Inc.

Piccinini, R., Borromeo, V. and Zecconi, A. (2010) Relationship between S. aureus gene

pattern and dairy herd mastitis prevalence. Vet Microbiol 145,100-105.

Piccinini, R., Cesaris, L., Dapra, V., Borromeo, V., Picozzi, C, Secchi, C. and Zecconi,

A. (2009) The role of teat skin contamination in the epidemiology of Staphylococcus

aureus intramammary infections. J Dairy Res 76, 36-41.

Pitkala, A., Haveri, M., Pyorala, S., Myllys, V. and Honkanen-Buzalski, T. (2004)

Bovine mastitis in Finland 2001 - prevalence, distribution of bacteria, and antimicrobial

resistance. J Dairy Sci 87,2433-2441.

Poole, K. (2004) Resistance to beta-lactam antibiotics. Cell Mol Life Sci 61,2200-2223.

114

Rajala-Schultz, P., Smith, K.L., Hogan, J.S. and Love, B.C. (2004) Antimicrobial

susceptibility of mastitis pathogens from first lactation and older cows. Vet Microbiol

102,33-42.

Reyher, K.K., Dufour, S., Barkema, H.W., Des Coteaux, L., Devries, T.J., Dohoo, I.R.,

Keefe, G.P., Roy, J.P. and Scholl, D.T. (2011) The National Cohort of Dairy Farms-A

data collection platform for mastitis research in Canada. J Dairy Sci 94,1616-1626.

Roberson, J.R. (2003) Establishing treatment protocols for clinical mastitis. Vet Clin

North Am FoodAnim Pract 19, 223-34, viii.

Roberts, M.C. (2008) Update on macrolide-lincosamide-streptogramin, ketolide, and

oxazolidinone resistance genes. FEMS Microbiol Lett 282,147-159.

Roberts, M.C. (2002) Resistance to tetracycline, macrolide-lincosamide-streptogramin,

trimethoprim, and sulfonamide drug classes. Mol Biotechnol 20,261-283.

Rosengren, L.B., Waldner, C.L. and Reid-Smith, RJ. (2009) Associations between

antimicrobial resistance phenotypes, antimicrobial resistance genes, and virulence genes

of fecal Escherichia coli isolates from healthy grow-finish pigs. Appl Environ Microbiol

75, 1373-1380.

Rozen, S. and Skaletsky, H. (2000) Primer3 on the WWW for general users and for

biologist programmers. Methods Mol Biol 132, 365-386.

115

Sabour, P.M., Gill, J.J., Lepp, D., Paean, J.C., Ahmed, R., Dingwell, R. and Leslie, K.

(2004) Molecular typing and distribution of Staphylococcus aureus isolates in Eastern

Canadian dairy herds. J Clin Microbiol 42, 3449-3455.

Saini, V., McClure, J.T., Leger, D. and Barkema, H.W. (2010) "Quantifying on-farm

antimicrobial use on Canadian dairy farms." Canadian Bovine Mastitis Research Network

2010 Annual Scientific Meeting, Toronto, ON.

Sambrook, J. and Russell, D.W. (2001a) Southern Blotting. In ed. Argentine, J. pp. 6.45-

6.46-6.49. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.

Sambrook, J. and Russell, D.W. (2001b) Transformation of E. coli by Electroporation. In

Molecular Cloning A Laboratory Manual ed. Argentine, J. pp. 1.119-1.120-1.122. Cold

Spring Harbour, NY, USA: Cold Spring Harbour Laboratory Press.

Sandholm, M., Honkanen-Buzalski, T., Kaartinen, L. and Pyorala, S. (1995) The Bovine

Udder and Mastitis. Helsinki, Finland: University of Helsinki.

Sanford, C.J., Keefe, G.P., Dohoo, I.R., Leslie, K.E., Dingwell, R.T., DesCoteaux, L. and

Barkema, H.W. (2006) Efficacy of using an internal teat sealer to prevent new

intramammary infections in nonlactating dairy cattle. J Am Vet Med Assoc 228, 1565-

1573.

Saudagar, P.S., Survase, S.A. and Singhal, R.S. (2008) Clavulanic acid: a review.

Biotechnol Adv 26, 335-351.

116

Schnappinger, D. and Hillen, W. (1996) Tetracyclines: antibiotic action, uptake, and

resistance mechanisms. Arch Microbiol 165, 359-369.

Schwarz, S. and Chaslus-Dancla, E. (2001) Use of antimicrobials in veterinary medicine

and mechanisms of resistance. Vet Res 32, 201-225.

Shi, D., Hao, Y., Zhang, A., Wulan, B. and Fan, X. (2010) Antimicrobial resistance of

Staphylococcus aureus isolated from bovine mastitis in China. Transbound Emerg Dis

57,221-224.

Shpigel, N.Y., Elazar, S. and Rosenshine, I. (2008) Mammary pathogenic Escherichia

coli. Curr Opin Microbiol 11, 60-65.

Singh, S.V., Jaiswal, S., Singh, D.D. and Singh, H.N. (2006) Bovine Mastitis and Udder

Affection. New Delhi, India: International Book Distribution Company.

Singh, G.S. (2004) Beta-lactams in the new millennium. Part-II: cephems, oxacephems,

penams and sulbactam. Mini Rev Med Chem 4, 93-109.

Skold, O. (2001) Resistance to trimethoprim and sulfonamides. Vet Res 32,261-273.

Smaill, F. (2000) Antibiotic susceptibility and resistance testing: an overview. Can J

Gastroenterol 14, 871-875.

Songer, G.L. and Post, K.W. (2005) Veterinary Microbiology Bacterial and Fungal

Agents of Animal Disease. St. Louis, Missouri: Elsevier Saunders.

117

Springer, B., Orendi, U., Much, P., Hoger, G., Ruppitsch, W., Krziwanek, K., Metz-

Gercek, S. and Mittermayer, H. (2009) Methicillin-resistant Staphylococcus aureus: a

new zoonotic agent? Wien Klin Wochenschr 121, 86-90.

Srinivasan, V., Gillespie, B.E., Lewis, M.J., Nguyen, L.T., Headrick, S.I., Schukken,

Y.H. and Oliver, S.P. (2007) Phenotypic and genotypic antimicrobial resistance patterns

of Escherichia coli isolated from dairy cows with mastitis. Vet Microbiol 124,319-328.

Srinivasan, V., Sawant, A.A., Gillespie, B.E., Headrick, S.J., Ceasaris, L. and Oliver, S.P.

(2006) Prevalence of enterotoxin and toxic shock syndrome toxin genes in

Staphylococcus aureus isolated from milk of cows with mastitis. Foodborne Pathog Dis

3, 274-283.

Sundsfjord, A., Simonsen, G.S., Haldorsen, B.C., Haaheim, H., Hjelmevoll, S.O.,

Littauer, P. and Dahl, K.H. (2004) Genetic methods for detection of antimicrobial

resistance. APMIS112, 815-837.

Suojala, L., Pohjanvirta, T., Simojoki, H., Myllyniemi, A.L., Pitkala, A., Pelkonen, S. and

Pyorala, S. (2011) Phylogeny, virulence factors and antimicrobial susceptibility of

Escherichia coli isolated in clinical bovine mastitis. Vet Microbiol 147, 383-388.

Tenover, F.C. and Goering, R.V. (2009) Methicillin-resistant Staphylococcus aureus

strain USA300: origin and epidemiology. JAntimicrob Chemother 64,441-446.

Thomsen, I., McKenna, B.D., Saye, E.J., Jimenez, N., Edwards, K.M. and Creech, C.B.

(2011) Molecular Distinctions Exist Between Community-associated Methicillin-resistant

118

Staphylococcus aureus Colonization and Disease-associated Isolates in Children. Pediatr

Infect Dis J.

Travis, R.M., Gyles, C.L., Reid-Smith, R., Poppe, C , McEwen, S.A., Friendship, R.,

Janecko, N. and Boerlin, P. (2006) Chloramphenicol and kanamycin resistance among

porcine Escherichia coli in Ontario. JAntimicrob Chemother 58, 173-177.

Turkyilmaz, S., Tekbiyik, S., Oryasin, E. and Bozdogan, B. (2010) Molecular

epidemiology and antimicrobial resistance mechanisms of methicillin-resistant

Staphylococcus aureus isolated from bovine milk. Zoonoses Public Health 57,197-203.

Turutoglu, H., Hasoksuz, M., Ozturk, D., Yildirim, M. and Sagnak, S. (2009) Methicillin

and aminoglycoside resistance in Staphylococcus aureus isolates from bovine mastitis

and sequence analysis of their mecA genes. Vet Res Commun.

Vakulenko, S.B. and Mobashery, S. (2003) Versatility of aminoglycosides and prospects

for their future. Clin Microbiol Rev 16,430-450.

van den Borne, B.H., Nielen, M , van Schaik, G., Melchior, M.B., Lam, TJ. and Zadoks,

R.N. (2010) Host adaptation of bovine Staphylococcus aureus seems associated with

bacteriological cure after lactational antimicrobial treatment. J Dairy Sci 93,2550-2558.

van Duijkeren, E., Moleman, M., Sloet van Oldruitenborgh-Oosterbaan, M.M., Multem,

J., Troelstra, A., Fluit, A.C., van Wamel, W.J., Houwers, D.J., de Neeling, A.J. and

Wagenaar, J.A. (2010) Methicillin-resistant Staphylococcus aureus in horses and horse

personnel: an investigation of several outbreaks. Vet Microbiol 141,96-102.

119

Vanderhaeghen, W., Cerpentier, T., Adriaensen, C , Vicca, J., Hermans, K. and Butaye,

P. (2010) Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with

clinical and subclinical mastitis in Belgian cows. Vet Microbiol 144,166-171.

Voladri, R.K. and Kernodle, D.S. (1998) Characterization of a chromosomal gene

encoding type B beta-lactamase in phage group II isolates of Staphylococcus aureus.

Antimicrob Agents Chemother 42, 3163-3168.

Voss, A. and Doebbeling, B.N. (1995) The worldwide prevalence of methicillin-resistant

Staphylococcus aureus. Int J Antimicrob Agents 5,101-106.

Walk, S.T., Mladonicky, J.M., Middleton, J.A., Heidt, A.J., Cunningham, J.R., Bartlett,

P., Sato, K. and Whittam, T.S. (2007) Influence of antibiotic selection on genetic

composition of Escherichia coli populations from conventional and organic dairy farms.

Appl Environ Microbiol 73, 5982-5989.

Walsh, C. (2000) Molecular mechanisms that confer antibacterial drug resistance. Nature

406,775-781.

Wang, Y., Wu, CM., Lu, L.M., Ren, G.W., Cao, X.Y. and Shen, J.Z. (2008) Macrolide-

lincosamide-resistant phenotypes and genotypes of Staphylococcus aureus isolated from

bovine clinical mastitis. Vet Microbiol 130,118-125.

Wax, R.G., Lewis, K., Salyers, A.A. and Taber, H. (2008) Bacterial Resistance to

Antimicrobials. Boca Raton, Florida, United States: CRC Press.

120

Wenz, J.R., Barrington, G.M., Garry, F.B., Ellis, R.P. and Magnuson, R.J. (2006)

Escherichia coli isolates' serotypes, genotypes, and virulence genes and clinical coliform

mastitis severity. J Dairy Sci 89, 3408-3412.

Werckenthin, C, Cardoso, M., Martel, J.L. and Schwarz, S. (2001) Antimicrobial

resistance in staphylococci from animals with particular reference to bovine

Staphylococcus aureus, porcine Staphylococcus hyicus, and canine Staphylococcus

intermedius. Vet Res 32, 341-362.

White, D.G. and McDermott, P.F. (2001) Emergence and transfer of antibacterial

resistance; 2000 Joint ADSA/ASAS Annual Meeting. J Dairy Sci 84, E151-E155.

Wilson, D.J., Gonzalez, R.N., Case, K.L., Garrison, L.L. and Grohn, Y.T. (1999)

Comparison of seven antibiotic treatments with no treatment for bacteriological efficacy

against bovine mastitis pathogens. J Dairy Sci 82,1664-1670.

Wilson, D.J., Gonzalez, R.N. and Das, H.H. (1997) Bovine mastitis pathogens in New

York and Pennsylvania: prevalence and effects on somatic cell count and milk

production. J Dairy Sci 80,2592-2598.

Winokur, P.L., Brueggemann, A., DeSalvo, D.L., Hoffmann, L., Apley, M.D.,

Uhlenhopp, E.K., Pfaller, M.A. and Doern, G.V. (2000) Animal and human multidrug-

resistant, cephalosporin-resistant Salmonella isolates expressing a plasmid-mediated

CMY-2 AmpC beta-lactamase. Antimicrob Agents Chemother 44,2777-2783.

121

Yamamoto, T., Nishiyama, A., Takano, T., Yabe, S., Higuchi, W., Razvina, O. and Shi,

D. (2010) Community-acquired methicillin-resistant Staphylococcus aureus: community

transmission, pathogenesis, and drug resistance. J Infect Chemother 16,225-254.

122

APPENDIX 1; FREQUENCY OF ANTIMICROBIAL RESISTANCE AND ANTIMICROBIAL RESISTANCE

GENES IN ESCHERICHIA COLL KLEBSIELLA SPP. AND STAPHYLOCOCCUS AUREUS ISOLATES FROM

MASTITIS

Table 9. Frequency of P-lactam resistance and resistance genes in Escherichia coli from bovine mastitis milk isolates.

Reference Microorganism

Clinical or Subclinical Mastitis

Year

Country

Number of Isolates

Ampicillin Resistance

Penicillin Resistance Cephalothin Resistance

Ceftiofur Resistance Cloxacillin Resistance

Amoxicillin/clavulanic acid Resistance

Oxacillin Resistance Methicillin Resistance

Genes Detected

(Lanz et al. 2003)

E. coli

Clinical

2000-2001

Switzerland

211

21.0%

ND

ND

ND

ND

ND

ND

ND

No Genes Detected

(Lehtolainen et al. 2003)

E. coli

Clinical

NA

Finland and Israel

100 Finnish, 100 Israeli 10.0% Israeli; 7.0%

Finnish ND

ND

ND

ND

ND

ND

ND

No Genes Detected

(Hendriksen et al. 2008)

E. coli

both

2002-2004 Sweden, Switzerland,

The Netherlands unknown

0-33.5%

ND

ND

0.0%

ND

0.0-1.0%

ND

ND

No Genes Detected

(Name/al. 2009)

E. coli

Unknown

2003-2008

Korea

127

67.8%

ND

15.7% ND

ND

81.0%

ND

ND

No Genes Detected

ND: Not determined

123


Clinical or Subclinical Mastitis Year

Country

Number of Isolates Ampicillin Resistance Penicillin Resistance

Cephalothin Resistance Ceftiofur Resistance

Cloxacillin Resistance


Oxacillin Resistance

Methicillin Resistance

Genes Detected

(Suojala etal. 2011)

E. coli

Clinical

Unknown

Finland

154

18.7%

ND

ND

ND

ND

ND

ND

ND

No Genes Detected

(Botreletal. 2010)

E. coli

Both

2007-2008

France

1770

ND

ND

0.7%

ND

ND

9.7%

ND

ND

No Genes Detected

124



Country Number of Isolates

Ampicillin Resistance Penicillin Resistance





Genes Detected

(Erskine et al. 2002)

E. coli

Both

1994-2000 United States

638 15.7%

ND

25.5%

4.6%

ND

ND

ND

ND

No Genes Detected

(Makovec and Ruegg 2003)

E. coli

Both


1939 21.9%

100.0%

27.9%

ND

99.4%

ND

ND

ND

No Genes Detected

(Srinivasan et al. 2007)

E. coli

Both


135

98.4%

ND

ND

ND

ND

ND

ND

ND

97% ampC

125

Table 10. Frequency of p-lactam resistance and resistance genes in S. aureus from bovine mastitis milk isolates.



Country



Cloxacillin Resistance Amoxicillin/clavulanic acid Resistance


Genes Detected

(Erskim etal. 2002)

& aureus

Both

1994-2000

United States

832

49.6%

49.6%

0.2%

0.2%

ND ND

0.6%

ND

No Genes Detected


S. aureus

Both

1994-2001

United States

2132

34.9%

35.4%

0.1%

ND

1.8% ND ND

ND

No Genes Detected

(Gentiliniefa/. 2000)

S. aureus

Both

1996-1998

Argentina

206

ND

40.0%

0.0%

ND

ND ND

0.0%

ND

No Genes Detected

126



Country




AmoxiciUin/clavulanic acid Resistance



Genes Detected

(Lee 2003)

S. aureus

Unknown

2001-2003 Korea

265

ND

ND

ND

ND

ND

ND

ND

4.5%

4.5% mecA

(Pitkula etal. 2004)

S. aureus

both

2001 Finland

431

ND

52.0%

ND

ND

ND

ND

4.1%

No Genes Detected

(Kwon etal. 2005)

S. aureus

unknown

1999,2000,2003 Korea

75335

ND

ND

ND

ND

ND

ND

ND

0.01%

0.007% mecA

(Moronietal. 2006)

S. aureus

Subclinical

2004 Italy

68

98.5%

69.0%

ND

ND

0.0%

20.0%

ND

ND

No Genes Detected

127



Year

Country







Genes Detected

(Nunes etal. 2007)

S. aureus

Subclinical

2006

Portugal

30

66.7%

ND

ND

ND

ND

ND

ND

ND

No Genes Detected

(Moon et al. 2007)

S. aureus

Unknown

Unknown

Korea

835 ND

ND

ND

ND

ND

ND

ND

2.5%

62% mecA


S. aureus

Both

2002-2004 Denmark, England,

France, Italy, Latvia, The Netherlands,

Portugal, Norway, Spain, Sweden,

Switzerland

691-1321

ND

3-49%

ND

0-1%

ND

ND

0-8.3%

ND

No Genes Detected

(Li et al. 2009)

S. aureus

Subclinical

2007-2008

China

3178

77.3%

77.3%

ND

ND

ND

ND

ND

ND No Genes Detected

128










Genes Detected

(Turutoglue/a/. 2009)

S. aureus

Unknown

2002-2004 Turkey

18

ND

ND

ND

ND

ND

ND

ND

16.7%

16.7% mecA

(Fessler ef a/. 2010)

S. aureus

unknown

2008-2009 Germany

25

ND

ND

ND

ND

ND

ND

ND

100.0%

100% mecA

(Huber etal. 2010)

S. aureus

unknown

2009 Switzerland

142

ND

ND

ND

ND

ND

ND

ND

1.4%

mecA

(Turkyilmaz et al 2010)

S. aureus

Unknown

2002-2006 Turkey

93

ND

ND

ND

ND

ND

ND

ND

17.2%

17.2% mecA

129



Year Country







Genes Detected

(Uaemxietal. 2011)

& aureus

Unknown

2007-2008 France

139

ND

41.0%

ND

ND

ND

ND

ND

0.7%

0.7% mecA

(Name/al. 2011)

S. aureus

Unknown

2003-2009 Korea

402

66%

ND

ND

ND

ND

ND

ND

7.7%

7.7% mecA

130

Table 11. Frequency of P-lactam resistance and resistance genes in Klebsiella spp. from bovine mastitis milk isolates.






Cloxacillin Resistance Amoxicillin/clavulanic acid Resistance



Genes Detected


Klebsiella spp.

Both

1994-2001

United States

607

89.1%

100.0%

12.1%

ND

100.0% ND ND

ND

No Genes Detected

(Name/al 2009)

Klebsiella spp.

Unknown

2003-2008

Korea

54

8.1%

ND

59.2%

ND

ND 100.0%

ND

ND No Genes Detected

131

Table 12. Frequency of tetracycline resistance and resistance genes in E. coli from bovine mastitis milk isolates.



Country

Number of Isolates Tetracycline Resistance

Frequency tet(A)

Frequency tet(B)

Frequency tet(C)

Frequency tet(A) + tet(B)

Frequency tet(A) + tet{C)

Frequency tet(A) + tet(B) + tet(C)

No Genes Detected


E. coli

Both

2002-2004

Sweden, Switzerland, The Netherlands

Unknown

5-84.5%

NA

NA

NA

NA

NA

NA

No Genes Detected

(Lanz et al. 2003)

E. coli

Clinical

2000-2001

Switzerland

211

20%

57%

38%

ND

5%

ND

ND


E. coli

Clinical

Finland and Israel

100 Finnish, 100 Israeli

15% Israeli

14% Finnish

NA

NA

NA

NA

NA

NA

No Genes Detected

(Nam et al. 2009)

E. coli

Both

2003-2008

Korea

127

47.30%

NA

NA

NA

NA

NA

NA

No Genes Detected

NA: Not Available.

132




Tetracycline Resistance

Frequency tet(A)

Frequency tet(B)

Frequency tet(C)


Frequency tet(A) + te/(C)


No Genes Detected

(Botrelefa/. 2010)

E. coli

Both

2007-2008

France

1770

10.40%

NA

NA

NA

NA

NA

NA

No Genes Detected

(Suojala ef a/. 2011)

E. coli

clinical

unknown

Finland

154

16.70%

NA

NA

NA

NA

NA

NA

No Genes Detected


E. coli

both

1994-2001

United States

1939

37.4

NA

NA

NA

NA

NA

NA

No Genes Detected

(Srinivasanefa/. 2007)

E. coli

Both

1999-2000

United States

135

24.80%

ND

ND

43.80%

ND

43.80%

ND

133

Table 13. Frequency of tetracycline resistance and resistance genes in S. aureus from bovine mastitis milk isolates.

Reference

Microorganism Clinical or Subclinical Mastitis

Year

Country


Frequency tet(A)

Frequency tet(B)

Frequency tet(C)

Frequency tet(A) + tetQi)

Frequency tet(A) + tet(C)


No Genes Detected


S. aureus

Both


France, Italy, Latvia, The Netherlands, Portugal,

Norway, Spain, Sweden, Switzerland 691-1321

0-9.2%

NA

NA

NA

NA

NA

NA

No Genes Detected


S, aureus

both

1994-2001

United States

2132

8.60%

NA

NA

NA

NA

NA

NA

No Genes Detected

(Pitkala et al. 2004)

S. aureus

Both

2001

Finland

431

5%

NA

NA

NA

NA

NA

NA

No Genes Detected

(Name*al. 2011)

S. aureus

Unknown

2003-2009

Korea

402

4%

NA

NA

NA

NA

NA

NA

No Genes Detected

134

Table 14. Frequency of tetracycline resistance and resistance genes in Klebsiella spp. from bovine mastitis milk isolates.



Country


Frequency tet{A)

Frequency tet(B)

Frequency tet(C)


Frequency tet(A) + tet(C)

Frequency tet{A) + tet(B) + tet(C)

No Genes Detected


Klebsiella spp.

Both

1994-2001

United States

607

30%

NA

NA

NA

NA

NA

NA

No Genes Detected

(Name?al. 2009)

Klebsiella spp.

Both

2003-2008

Korea

54

42.60%

NA

NA

NA

NA

NA

NA

No Genes Detected

135

Table 15. Frequency of aminoglycoside resistance and resistance genes in E. coli from bovine mastitis milk isolates.

Reference

Microorganism Clinical or Subclinical Mastitis

Year Country

Number of Isolates Streptomycin Resistance Gentamicin Resistance Kanamycin Resistance Neomycin Resistance

Other Resistances

Frequency aadAl3

Frequency strA/strB

Frequency strB/aadAl

Frequency aac(3)IV

Frequency aadAl + strAlstrB

Additional Genes Detected

No Genes Detected

(Erskine et al. 2002)

E. coli

Both

1994-2000

United States 638

2.0%

ND

ND

ND

NA

NA

NA

NA

NA

NA

No Genes Detected

(Srinivasan et al. 2007) E. coli

both

1999-2000

United States 135

40.3% ND

ND

ND

ND

55.8%

NA

NA

NA

21.0%

NA

(Lanz et al. 2003)

E. coli

clinical

Switzerland

211 22.0%

1.0% 16.0%

ND 4.0%

spectinomycin

48.0%

15.0%

1.0%

NA

36.0%

NA


E. coli

Clinical

Unknown

Finland and Israel 100 Finnish, 100 Israeli

13.0% Israeli; 9.0% Finnish ND

ND

ND

ND

NA

NA

NA

NA

NA

NA

No Genes Detected

136



Year

Country

Number of Isolates

Streptomycin Resistance

Gentamicin Resistance Kanamycin Resistance

Neomycin Resistance

Other Resistances

Frequency aadAl3

Frequency strA/strB


Frequency aac(3)IV

Frequency aadAl + strA/strB

Additional Genes Detected

No Genes Detected


E. coli

Both

2002-2004

Sweden, Switzerland, The Netherlands

Unknown

15.2% The Netherlands, 24.0% Sweden

6.3% Switzerland ND

7.0% The Netherlands, 7.0% Sweden, 18.0% Switzerland

ND

NA

NA

NA

NA

NA

NA

No Genes Detected

(Nam et al. 2009)

E. coli

both

2003-2008

Korea

127

52.8%

10.3%

30.0%

ND

ND

NA

NA

NA

NA

NA

NA

No Genes Detected

(Botrelefa/. 2010)

E. coli

Both

2007-2008

France

1770

13.4%

0.7%

6.0%

ND

ND

NA

NA

NA

NA

NA

NA

No Genes Detected

(Suojala ef a/. 2011)

E. coli

clinical

Unknown

Finland

154

18.1%

0.0%

6.3%

ND

ND

NA

NA

NA

NA

NA

NA

No Genes Detected

137

Table 16. Frequency of aminoglycoside resistance and resistance genes in & aureus from bovine mastitis milk isolates.



Country

Number of Isolates Streptomycin Resistance Gentamicin Resistance Kanamycin Resistance Neomycin Resistance

Other Resistances

Frequency aadAl3

Frequency strA/strB


Frequency aac(3)IV

Frequency aadAl + strAlstrB

Additional Genes Detected No Genes Detected

(Erskinee/a/. 2002)

S. aureus

Both

1994-2000

United States

832 ND 1.1%

ND

ND

ND

NA

NA

NA

NA

NA

NA

No Genes Detected

(Gentiliniefa/. 2000)

S. aureus

both

1996-1998

Argentina

206 ND 3.4%

ND

ND

ND

NA

NA

NA

NA

NA

NA

No Genes Detected


S. aureus

Both


France, Italy, Latvia, The Netherlands, Portugal,

Norway, Spain, Sweden, Switzerland 691-1321 0.0-8%

0.0-3.1%

ND

0.0-5.3%

ND

NA

NA

NA

NA

NA

NA

No Genes Detected

(Moroni et al 2006)

S. aureus

Subclinical

2004

Italy

68 ND ND

16.0%

ND

ND

NA

NA

NA

NA

NA

NA

No Genes Detected

138




Streptomycin Resistance Gentamicin Resistance Kanamycin Resistance Neomycin Resistance

Other Resistances

Frequency aadAl3

Frequency strA/strB


Frequency aac(3)IV Frequency aadAl + strAlstrB Additional Genes Detected

No Genes Detected

(Pitkalaefa/. 2004)

S. aureus

Both

2001

Finland 431

4.0% ND

ND

ND

ND

NA

NA

NA

NA NA NA

No Genes Detected

(Nam et al. 2011)

S. aureus

unknown

2003-2009

Korea 402 ND

11.9%

ND

ND

ND

NA

NA

NA

NA NA NA

No Genes Detected

(Shi et al. 2010)

S. aureus

unknown

2005-2006

China 835 ND

17.4%

17.4%

ND

ND

NA

NA

NA

NA NA NA

No Genes Detected

(Liet al. 2009)

S. aureus

Subclinical

2007-2008

China 3178 ND

28.0%

ND

ND

ND

NA

NA

NA

NA NA NA

No Genes Detected

139

Table 17. Frequency of aminoglycoside resistance and resistance genes in Klebsiella spp. from bovine mastitis milk isolates.



Country

Number of Isolates

Streptomycin Resistance Gentamicin Resistance Kanamycin Resistance Neomycin Resistance

Other Resistances

Frequency aadAl3

Frequency strA/strB


Frequency aac(3)IV Frequency aadAl + strAlstrB Additional Genes Detected

No Genes Detected

(Nam et al. 2009) Klebsiella spp.

Both 2003-2008

Korea

54

59.3% 18.6% 46.3%

ND

ND

NA

NA

NA

NA NA NA

No Genes Detected

140

Table 18. Frequency of sulfonamide resistance and resistance genes in E. coli from bovine mastitis milk isolates.

Reference

Microorganism Clinical or Subclinical

Mastitis Year

Country

Number of Isolates Sulfonamide Resistance

Sulfametoxazole Resistance Sulfisoxazole Resistance

Frequency sull Frequency sul2 Frequency sul3

Frequency sull + sul2 Frequency sull + sul3 Frequency sul2 + sul3

No Genes Detected

(Bengtsson et al. 2009) E. coli

Clinical

2002-2003

Sweden

163

ND

8.50%

ND

NA

NA

NA

NA NA

NA No Genes Detected

(Hendriksen et al. 2008) E. coli

Both

2002-2004

Sweden, Switzerland, The

Netherlands

Unknown

8-41% sulfonamides

ND

ND

NA

NA

NA

NA NA

NA

No Genes Detected

(Lanz et al. 2003) E. coli

Clinical

2000-2001

Switzerland

211

ND

ND

ND

13%

57%

NA

30% NA

NA

(Makovec and Ruegg 2003) E. coli

both

1994-2001

United States

1939

ND

ND

16.30%

NA

NA

NA

NA NA

NA

No Genes Detected

(Srinivasan et al. 2007) E. coli

Both

1999-2000

United States

135

ND

ND

34.10%

27%

22.70%

NA

2.30% NA

NA

141

Table 19. Frequency of sulfonamide resistance and resistance genes in S. aureus from bovine mastitis milk isolates.



Year

Country

Number of Isolates Sulfonamide Resistance

Sulfametoxazole Resistance

Sulfisoxazole Resistance Frequency sull Frequency sul2 Frequency sul3

Frequency sull + sul2 Frequency sull + sul3 Frequency sul2 + sul3

No Genes Detected

(Bengtsson et al. 2009)

S. aureus

Clinical

2002-2003

Sweden

211

ND

ND

0%

NA

NA

NA

NA

NA NA

No Genes Detected


S. aureus

both

2002-2004

Denmark, England, France, Italy, Latvia,

The Netherlands, Portugal, Norway,

Spain, Sweden, Switzerland

691-1321

0% sulfonamides

ND

ND

NA

NA

NA

NA

NA NA

No Genes Detected


S. aureus

Both

1994-2001

United States

2132

ND

ND

4.50%

NA

NA

NA NA

NA NA

No Genes Detected

142

Table 20. Frequency of sulfonamide resistance and resistance genes in Klebsiella spp. from bovine mastitis milk isolates.




Sulfonamide Resistance Sulfametoxazole Resistance

Sulfisoxazole Resistance Frequency sull Frequency sul2 Frequency sul3

Frequency sull + sull

Frequency sull + sul3

Frequency sull + sul3 No Genes Detected


Klebsiella spp.

clinical

2002-2003

Sweden

211

ND

ND

7.10%

NA

NA

NA

NA

NA

NA

No Genes Detected


Klebsiella spp.

Both

1994-2001

United States

607

ND

ND

11.70%

NA

NA

NA

NA

NA

NA

No Genes Detected

143

Table 21. Frequency of raacrolide and lincosamide resistance in Staphylococcus aureus from bovine mastitis milk isolates




Erythromycin Resistance

Pirlimycin Resistance Lincomycin Resistance

(Gentilinie/a/. 2000)

S. aureus

Both

1996-1998 Argentina

206

11.6%

3.4%

ND

(Erskineefa/. 2002)

S. aureus

both


832

6.9%

2.5%

ND


S. aureus

Both


2132

6.7%

ND

ND

(Pitkala et al. 2004)

S. aureus

Both

2001 Finland

431

1.5%

4.8%

3.2%

144



Year

Country

Number of Isolates Erythromycin Resistance

Pirlimycin Resistance Lincomycin Resistance


S. aureus

Both


France, Italy, Latvia, The

Netherlands, Portugal, Norway,

Spain, Sweden, Switzerland 691-1321

0-11.4%

ND

ND

(Wang et al. 2008)

S. aureus

clinical

2006

China

72

93.10%

ND

ND


S. aureus

Clinical

2002-2003

Sweden

211

1.9%

ND

ND

(Lie/al. 2009)

S. aureus

Subclinical

2007-2008

China

3178

48.0%

ND

ND

145

Table 22. Distribution of macrolide-lincosamide-streptogramin (MLS) resistance determinants in Escherichia coli, Klebsiella

spp., and Staphylococcus aureus (Roberts 2008).

Type rRNA Methylase

Efflux

Major Facilitators Inactivating Genes

Transferases

Phosphorylases

Resistance MLS

lincomycin, erythromycin, olenadomycin, spiramycin, tylosin streptogramin A, or

lincomycin and streptogramin A

Erythromycin

streptogramin A

Macrolides

Gene erm(F)

erm(G)

erm(Q)

erm(33)

msr(D)

Isa(B)

mefiA)

ere(A)

ere(B)

Inu(B)

Inu(F)

mph(A)

mph(D)

E. coli

X

X

X

X

Klebsiella spp.

X

X

X X

S. aureus X X X X

X X

X X X

146

APPENDIX 2: GENE LIST FROM THE AMR-VE AND MRSA IDENTIBAC

ARRAYTUBES

Table 23. Genes present on the AMR-ve ArrayTube.

Gene Antimicrobial Class

aadAl Aminoglycoside

aadAl Aminoglycoside

aadA4 Aminoglycoside

aac(3)-Ia Aminoglycoside

aac(3)-Iva Aminoglycoside

aac(6')-Ib Aminoglycoside

ant(2")-Ia Aminoglycoside

strA

strB

6/apsE-i

WtfDHA

blaAcc-i

blaxcc-i

Aminoglycoside

Aminoglycoside

P-lactam

P -lactam

P -lactam

P -lactam

147

blauox

blacMY

blauox

blasm

bhuEN-l

bldTEM-l

blaoxA-i

blaoxA-2

bldoxA-7

blaoxk-9

blacTx-M-i

blacrx-u-2

blacrx-M-9

blacrx-ui

blacrx-yae

blauox-cuY

cmlAl

catAl

B -lactam

B -lactam

P -lactam

B -lactam

P -lactam

P -lactam

P -lactam

P -lactam

p -lactam

P -lactam

P -lactam

P -lactam

P -lactam

P -lactam

P -lactam

P -lactam

Chloramphenicol

Chloramphenicol

148

catlll

catB3

floR

intll

intI2

qnrA

qnrB

qnrS

sull

sul2

sul3

tet(A)

tet(B)

tet(C)

tet(D)

tet(E)

tet(G)

dfrAl

dfrAl

Chloramphenicol

Chloramphenicol

Chloramphenicol

Integrase

Integrase

Quinolone

Quinolone

Quinolone

Sulfonamide

Sulfonamide

Sulfonamide

Tetracycline

Tetracycline

Tetracycline

Tetracycline

Tetracycline

Tetracycline

Trimethoprim

Trimethoprim

149

dfrll Trimethoprim

dfrA14 Trimethoprim

dfrA19 Trimethoprim

150

Table 24. Genes present on the MRSA ArrayTube.

Gene Gene Description

Coa Coagulase

femA Gene for a factor involved in peptidoglycan synthesis

gap A Glyceraldehyde 3-phosphate dehydrogenase

katA Catalase

Spa ProteinA

sarA S. aureus virulence factor regulator

Sbi IgG-binding protein

agrB-I accessory gene regulator

agrB-II accessory gene regulator

agrB-III accessory gene regulator

agrB-IV accessory gene regulator

agrC-I accessory gene regulator

agrC-II accessory gene regulator

agrC-III accessory gene regulator

agrC-TV accessory gene regulator

agrD-I accessory gene regulator



151

agrD-II accessory gene regulator

agrD-III accessory gene regulator

mecA Methicillin resistance

blaz Beta-lactamase

ermA Erythromycin resistance and inducible or constitutive

clindamycin resistance ermC Erythromycin resistance and inducible or constitutive

clindamycin resistance

HnA Clindamycin / Hncomycin resistance

msrA Macrolide resistance

vatA streptogramin resistance gene

vatB streptogramin resistance gene

Vga streptogramin resistance gene

vgaA streptogramin resistance gene

Vgb streptogramin resistance gene

aacA-aphD Gentamicin / tobramycin resistance

aadD Neomycin / tobramycin resistance

aphA-3 Neomycin resistance

Sat Streptothricin resistance

dfrA Trimethoprim resistance

fori Fusidic acid resistance

mupR Mupirocin resistance

152

tet(K) tetracycline resistance

tetM tetracycline resistance

vanA Enterococcal genes involved in glycopeptide resistance

vanB Enterococcal genes involved in glycopeptide resistance

vanZ Enterococcal genes involved in glycopeptide resistance

tstl Toxic shock syndrome toxin

seA Enterotoxin gene A

seB Enterotoxin gene B

seC Enterotoxin gene C

seD Superantigenic toxins

seE Superantigenic toxins

seG Superantigenic toxins

She Superantigenic toxins

sel Superantigenic toxins

seJ Superantigenic toxins

•JCiV K Superantigenic toxins

seL Superantigenic toxins

seM Superantigenic toxins

seN Superantigenic toxins

seO Superantigenic toxins

153

seQ

seR

seU/seY

lukF

lukS

hlgA

lukF-PV

lukS-PV

lukF-P83

lukM

lukD

lukE

Putative leucocidin F subunit

Putative leucocidin S subunit

HI

Hla

Mb

Hid

hl-III

etA

Superantigenic toxins



leukocidin toxin protein


Gamma-haemolysin components

PVL, F subunit

PVL, S subunit

Bovine bicomponent leucocidin, F subunit

Bovine bicomponent leucocidin, S subunit

LukD/E leucocidin

LukD/E leucocidin



Unnamed haemolysin

Haemolysin alpha

Haemolysin beta

Haemolysin delta

Unnamed haemolysin

Exfoliative toxin gene A

154

etB Exfoliative toxin gene B

etD

Sak

splA

splB

edinA

edinB

edinC

Exfoliative toxin gene D

Staphylokinase

Serine protease-like exoprotein A

Serine protease-like exoprotein B

Epidermal cell differentiation inhibitor genes



5e?C(SACOL0442)

ssll

sstt

ssl3

ssl4

ssl5

ssl6

ssl7

ssl8

ssl9

ssllO

sslll

staphylococcal exotoxin-like proteins







aphylococcal exotoxin-like proteins





155

setB3 staphylococcal exotoxin-like proteins

setB2 staphylococcal exotoxin-like proteins

setBl staphylococcal exotoxin-like proteins

156

APPENDIX 3; DETERMINATION OF ANTIMICROBIAL RESISTANCE IN

ESCHERICHIA COLIAND KLEBSIELLA SPP. FROM CANADIAN BOVINE

MASTITIS ISOLATES 6/apSF.i PLASMID GENES AND POLYMERASE CHAIN

REACTION CONDITIONS

Table 25. Genes of interest detected on the 6/apsE-i plasmid.

Gene Gene Description

faeG, H, I, J Fimbrial Proteins

faeF K88 Minor Fimbrial Protein

K88 Fimbrial Protein A

cshB Fimbrial Biogenesis Outer

Membrane Usher clpE Pili Assembly Chaperone

pilJ, pilK, pilL, pilM, pilN, pilO, pilP, pilQ, pilR, Conjugal Transfer Proteins pilS, pilT, pilU

traE, traF, traG, traH, tral, traJ Conjugal Transfer Proteins

traM, traN, traO, traP, traQ, traR, traT, trail, traV, Conjugal Transfer Proteins traW, traX, traY

trb, A, B, C Conjugal Transfer Proteins

jinQ Conjugal Transfer Proteins

incll Shufflon Shufflon Protein

Rci Shufflon specific DNA

Recombinase ibfA* Abortive Infection Protein

ibfC* Abortive Infection Protein

157

par A Homolog

parM

Putative nikB

psiB

psiA

resA

excA

Plasmid Segregation protein

Plasmid Segregation protein

Mobilization Protein

Plasmid SOS Inhibition Protein

Plasmid SOS Inhibition Protein

Resolvase

Surface Exclusion Protein

sogL DNA Primase

These genes are involved in aborting infection by phage.

158

Table 26. Polymerase chain reaction conditions for the detection of the cassette array

containing blapse-i in E. coli isolates from chicken and swine.

PCRa Gene(s) Primers Primer Sequence ~. „ .

1 aadA2-ereA aadA2_ereA_F CAGCCCGTCTTACTTGAAGC 903

aadA2_ereA_R CAAATCGCTGTTGACGTGTT

1 bla?SE-l-aadA2 pse_aadA2_F GCCCCAATTATTGTGAGCA 765

pse_aadA2_R GCTGCGAGTTCCATAGCTTC

1 dfrl6-bla?SE-l dfr_pse_F ATCGAGCGAGATGGAGACAT 864

dfr_pse_R GATAGCGCGGAACCAAATAA

a PCR 1 was carried out using the following thermal cycling conditions: one cycle

consisting of 15 min at 95°C, 30 cycles consisting of 1 min at 95°C, 1 min at 65°C, and 1

min at 72°C, and a final elongation of 10 min at 72°C. The concentration of each primer

was 0.2 uM.

159

APPENDIX 4: FREQUENCY OF VIRULENCE GENES IN STAPHYLOCOCCUS

AUREUS FROM CANADIAN BOVINE MASTITIS ISOLATES

Table 27. Frequency of virulence genes among penicillin-resistant and susceptible

Staphylococcus aureus from bovine mastitis in Canada.

Gene*

femA

katA

katAJl

5,2-entC

5,3-entC

agrB-I

agrB-II

agrB-III

agrC-I

agrC-II

agrD-I

agrD-II

agrD-III

entD

Resistant Isolates (n=57)c

42 (73.7%)

37 (64.9%)

52(91.2%)

3 (5.3%)

4 (7.0%)

2 (3.5%)

52(91.2%)

1 (1.8%)

1 (1.8%)

42 (73.7%)

3 (5.3%)

20(35.1%)

0 (0%)

0 (0%)

Susceptible Isolates (n=22)d

11(50%)

10(45.5%)

17 (77.3%)

1 (4.6%)

1 (4.6%)

8 (36.4%)

6 (27.3%)

0 (0%)

1 (4.6%)

3 (13.6%)

13 (59.1%)

0 (0%)

1 (4.6%)

3 (13.6%)

entG

entl

entL

entM

entN

entO

entX

entY

HI

Hla

hlbjl

hlb_12

Hid

MgA

hl-III

hp_entCM14_611

hp_entCM14_612

hp_entN_611

hp_entU_611

hp_tst_611

39 (68.4%)

31 (54.4%)

4 (7.0%)

6 (10.5%)

3 (5.3%)

24(42.1%)

43 (75.4%)

39 (68.4%)

54 (94.7%)

42 (73.7%)

41 (71.9%)

25 (43.9%)

44 (77.2%)

42 (73.7%)

10(17.5%)

39 (68.4%)

41 (71.9%)

35 (61.4%)

24(42.1%)

3 (5.3%)

6 (27.3%)

3 (13.6%)

1 (4.6%)

1 (4.6%)

1 (4.6%)

2(9.1%)

15 (68.2%)

8 (36.4%)

20 (90.9%)

12 (54.6%)

15 (68.2%)

12 (54.6%)

12 (54.6%)

12 (54.6%)

11(50%)

3 (13.6%)

5 (22.7%)

5 (22.7%)

2(9.1%)

1 (4.6%)

lukD

lukE

lukF

lukF-PV

lukM

lukS

lukX

lukY-varl J1

lukY-var2_ll

proteinA _11

proteinA_12

Sak

sarA

sbi-varl_ll

sbi-varl_12

SCtj. **

setl-varl

setl-var2

setl-var4

set21

51 (89.5%)

51 (89.5%)

54 (94.7%)

41 (71.9%)

42 (73.7%)

43 (75.4%)

3 (5.3%)

52(91.2%)

2 (3.5%)

45 (79.0%)

54 (94.7%)

1 (1.8%)

29 (50.9%)

50 (87.7%)

47 (82.5%)

36 (63.2%)

40 (70.2%)

9(15.8%)

53 (93.0%)

2 (3.5%)

16 (72.7%)

17 (77.3%)

18 (81.8%)

17 (77.3%)

17 (77.3%)

12 (54.6%)

1 (4.6%)

18(81.8%)

1 (4.6%)

12 (54.6%)

20 (90.9%)

0 (0%)

8 (36.4%)

17(77.3%)

16 (72.7%)

14 (63.6%)

14 (63.6%)

2(9.1%)

17(77.3%)

1 (4.6%)

162

set2-varl

set3

set4-varl

set4-var2

set5-varl

set5-var2

set6-varl

set6-var2

set6-var2

set6-var4

set7-varl

set7-var2

set8

set9-varl_ll

set9-varl_12

setB-SA117S

setB-SAR1139

setB-SAR1140

setB-SAR1141

setC

38 (66.7%)

13 (22.8%)

26 (45.6%)

26 (45.6%)

38 (66.7%)

1 (1.8%)

1 (1.8%)

3 (5.3%)

41 (71.9%)

13 (22.8%)

52 (91.2%)

15 (26.3%)

50 (87.7%)

10(17.5%)

12(21.1%)

52(91.2%)

1 (1.8%)

1 (1.8%)

26 (45.6%)

54 (94.7%)

163

4(18.2%)

12 (54.6%)

16 (72.7%)

15 (68.2%)

12 (54.6%)

0 (0%)

2(9.1%)

2 (9.1%)

5 (22.7%)

2(9.1%)

15 (68.2%)

3 (13.6%)

5 (22.7%)

10(45.5%)

13 (59.1%)

17(77.3%)

1 (4.6%)

1 (4.6%)

14 (63.6%)

19 (86.4%)

set-SAR0425_ll 0 (0%) 1 (4.6%)

set-SAR0425J2 3 (5.3%) 3 (13.6%)

splA 24(42.1%) 14(63.6%)

splB 52 (91.2%) 18 (81.8%)

a The following genes were not found in either resistant or susceptible isolates: agrC-II,

agrC-IV, entB, set-SAR0429-l1, and tet-1-16, 2; b Gene abbreviations are listed below; °

Number of resistant isolates positive for this specific gene. Numbers in brackets represent

the percentage of isolates positive for the specific gene out of a total number of 57

resistant isolates. d Number of susceptible isolates positive for this specific gene.

Numbers in brackets represent the percentage of isolates positive for the specific gene out

of a total number of 22 susceptible isolates.

Gene Abbreviations

agr accessory gene regulator ent enterotoxin femA factor essential for methicillin resistance hi hemolysin hp hypothetical hot A catalase A luk leukocidin toxin protein sak staphylokinase sarA staphylococcal accessory regulator A sbi surface protein set staphylococcal superantigen-like protein spl serine protease tst toxic shock toxin

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antimicrobial resistance in escherichia coli, …

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