ANTIMICROBIAL RESISTANCE IN ESCHERICHIA COLI, …
Transcript of 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.
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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
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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
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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
bovine mastitis milk isolates 131
Table 12. Frequency of tetracycline resistance and resistance genes in E. coli from bovine
mastitis milk isolates 132
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Table 13. Frequency of tetracycline resistance and resistance genes in S. aureus from
bovine mastitis milk isolates 134
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
bovine mastitis milk isolates 136
Table 16. Frequency of aminoglycoside resistance and resistance genes in S. aureus from
bovine mastitis milk isolates 138
Table 17. Frequency of aminoglycoside resistance and resistance genes in Klebsiella spp.
from bovine mastitis milk isolates 140
Table 18. Frequency of sulfonamide resistance and resistance genes in E. coli from
bovine mastitis milk isolates 141
Table 19. Frequency of sulfonamide resistance and resistance genes in S. aureus from
bovine mastitis milk isolates 142
Table 20. Frequency of sulfonamide resistance and resistance genes in Klebsiella spp.
from bovine mastitis milk isolates 143
Table 21. Frequency of macrolide and lincosamide resistance in Staphylococcus aureus
from bovine mastitis milk isolates 144
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
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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
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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
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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
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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.
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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.
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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.
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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.
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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
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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).
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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
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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.
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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).
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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).
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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.
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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
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
44
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
Chloramphenicol (CHL)
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
Chloramphenicol (CHL)
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
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
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
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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
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
(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
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
(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
1994-2001 United States
1939 21.9%
100.0%
27.9%
ND
99.4%
ND
ND
ND
No Genes Detected
(Srinivasan et al. 2007)
E. coli
Both
1999-2000 United States
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.
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
(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
(Makovec and Ruegg 2003)
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
Reference Microorganism
Clinical or Subclinical Mastitis Year
Country
Number of Isolates Ampicillin Resistance Penicillin Resistance
Cephalothin Resistance Ceftiofur Resistance
Cloxacillin Resistance
AmoxiciUin/clavulanic acid Resistance
Oxacillin Resistance
Methicillin 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
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
(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
(Hendriksen et al. 2008)
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
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
(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
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
(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.
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
(Makovec and Ruegg 2003)
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.
Reference Microorganism
Clinical or Subclinical Mastitis Year
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
(Hendriksen et al. 2008)
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
(Lehtolainen et al. 2003)
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
Reference Microorganism
Clinical or Subclinical Mastitis Year
Country Number of Isolates
Tetracycline Resistance
Frequency tet(A)
Frequency tet(B)
Frequency tet(C)
Frequency tet(A) + tet(B)
Frequency tet(A) + te/(C)
Frequency tet(A) + tet(B) + tet(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
(Makovec and Ruegg 2003)
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
Number of Isolates Tetracycline Resistance
Frequency tet(A)
Frequency tet(B)
Frequency tet(C)
Frequency tet(A) + tetQi)
Frequency tet(A) + tet(C)
Frequency tet(A) + tet(B) + tet(C)
No Genes Detected
(Hendriksen et al. 2008)
S. aureus
Both
2002-2004 Denmark, England,
France, Italy, Latvia, The Netherlands, Portugal,
Norway, Spain, Sweden, Switzerland 691-1321
0-9.2%
NA
NA
NA
NA
NA
NA
No Genes Detected
(Makovec and Ruegg 2003)
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.
Reference Microorganism
Clinical or Subclinical Mastitis Year
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
(Makovec and Ruegg 2003)
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
(Lehtolainen et al. 2003)
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
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 + strA/strB
Additional Genes Detected
No Genes Detected
(Hendriksen et al. 2008)
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.
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
(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
(Hendriksen et al. 2008)
S. aureus
Both
2002-2004 Denmark, England,
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
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
(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.
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
(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.
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)
S. aureus
Clinical
2002-2003
Sweden
211
ND
ND
0%
NA
NA
NA
NA
NA NA
No Genes Detected
(Hendriksen et al. 2008)
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
(Makovec and Ruegg 2003)
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.
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 + sull
Frequency sull + sul3
Frequency sull + sul3 No Genes Detected
(Bengtsson et al. 2009)
Klebsiella spp.
clinical
2002-2003
Sweden
211
ND
ND
7.10%
NA
NA
NA
NA
NA
NA
No Genes Detected
(Makovec and Ruegg 2003)
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
Reference Microorganism
Clinical or Subclinical Mastitis Year
Country Number of 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
1994-2000 United States
832
6.9%
2.5%
ND
(Makovec and Ruegg 2003)
S. aureus
Both
1994-2001 United States
2132
6.7%
ND
ND
(Pitkala et al. 2004)
S. aureus
Both
2001 Finland
431
1.5%
4.8%
3.2%
144
Reference Microorganism
Clinical or Subclinical Mastitis
Year
Country
Number of Isolates Erythromycin Resistance
Pirlimycin Resistance Lincomycin Resistance
(Hendriksen et al. 2008)
S. aureus
Both
2002-2004 Denmark, England,
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
(Bengtsson et al. 2009)
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
agrD-I 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
Superantigenic toxins
Superantigenic toxins
leukocidin toxin protein
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
leukocidin toxin protein
leukocidin toxin protein
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
Epidermal cell differentiation inhibitor genes
Epidermal cell differentiation inhibitor genes
5e?C(SACOL0442)
ssll
sstt
ssl3
ssl4
ssl5
ssl6
ssl7
ssl8
ssl9
ssllO
sslll
staphylococcal exotoxin-like proteins
staphylococcal exotoxin-like proteins
staphylococcal exotoxin-like proteins
staphylococcal exotoxin-like proteins
staphylococcal exotoxin-like proteins
staphylococcal exotoxin-like proteins
staphylococcal exotoxin-like proteins
aphylococcal exotoxin-like proteins
staphylococcal exotoxin-like proteins
staphylococcal exotoxin-like proteins
staphylococcal exotoxin-like proteins
staphylococcal 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.
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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.
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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
164