An Investigation of Class 1 Integrons and Integrase Gene In

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AN INVESTIGATION OF CLASS 1 INTEGRONS AND INTEGRASE GENE IN

ESCHERICHIA COLI AND MANNHEIMIA HAEMOLYTICA FROM BEEF CATTLE IN

WESTERN CANADA

A Thesis

Presented to

The Faculty of Graduate Studies

The University of Guelph

MATTHEW LESLIE

In partial fulfillment of requirements

for the degree of

Master of Science

May, 2011

395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada

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1*1 Canada ABSTRACT

AN INVESTIGATION OF CLASS 1 INTEGRONS AND INTEGRASE GENE IN

ESCHERICHIA COII AND MANNHEIMIA HAEMOIYTICA ISOLATED FROM BEEF

CATTLE IN WESTERN CANADA

Matthew Leslie Advisor: University of Guelph, 2011 Dr. P. Boerlin

This study examined and characterized class 1 integrons and class 1 integrase-associated genetic elements in Escherichia coli and Mannheimia haemolytica from Canadian beef cattle. Both E. coli and M. haemolytica isolated from beef cattle on four feedlots in

Alberta Canada were screened for the presence of the class 1 integrase gene (intll). All isolates which possessed this gene were further characterized to determine if they contained a typical class 1 integron or an incomplete integron, dubbed 'lonely integrase'.

A variety of gene cassettes, gene arrays and variants were identified in E. coli. The genetic environment surrounding the 'lonely integrase' was characterized using cloning and PCR. The 'lonely integrase' structure was shown to contain a duplicated class 1 integrase gene and to be associated with both tet(M) and tet(A). This same structure was observed in the majority of the isolates possessing a 'lonely integrase'. ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. Patrick Boerlin, my mentor over the last two years, who has not only given me this opportunity but has also provided me with ongoing support, encouragement and guidance. His enthusiasm for science motivated me to improve my own knowledge and his support helped me gain confidence in my abilities. I would like to thank my committee members Dr. Lucy Mutharia, Dr. John Prescott and

Dr. Richard Reid-Smith for their wisdom and advice over the past two years. Thank you to Dr. David Pearl for his crash course and continued guidance in statistics. I thank our technicians Vivian Nicholson and Gabhan Chalmers for getting me acquainted with the laboratory and various techniques and for the tips and tricks that they've passed on.

Thank you to my lab mates (Stina Nilsson, Fiona Coutinho, Jennie Pouget, Shaun

Kernaghan, Walter Wang, Heidi Mascarenhas) who have helped me learn valuable lessons along the way and who have always had time to have a little fun (i.e., Lab

Olympics); I truly value the friendships I have made within the lab. A special shout out

Kelly Keithlin, my parallel life partner, who kept me motivated and with whom I competed with throughout our undergraduate and graduate studies. A very heartfelt thank you goes out to my incredibly supportive family and group of friends; you've helped me through a lot of the struggles that happen outside of the lab. TABLE OF CONTENTS

ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii LIST OF TABLES iv LIST OF FIGURES vi LIST OF ABBREVIATIONS vii DECLARATION OF WORK ix INTRODUCTION 2 CHAPTER ONE: LITERATURE REVIEW 5 1. Antimicrobial Use in Food Animal Production and Antimicrobial Resistance 5 2. Escherichia coli as a Commensal and Reservoir for AMR Genes 7 3. Mannheimia haemolytica: Opportunistic Pathogen of Ruminants 10 4. Integrons and Their Role in AMR 13 5. Predominant AMR Genes in Integrons and the Resistance Mechanisms They Confer 19 6. AMR in Bacteria Isolated from Canadian Beef Cattle 27 7. Ancestral Integrons, Modified Integrons and the 'Lonely Integrase' 29 8. The Dissemination of Class 1 Integrons 33 9. Proposal 34 CHAPTER TWO: AN INVESTIGATION AND CHARACTERIZATION OF CLASS ONE INTEGRONS IN ESCHERICHIA COLI AND MANNHEIMIA HAEMOLYTICA FROM FEEDLOT CATTLE IN WESTERN CANADA 39 ABSTRACT 39 INTRODUCTION 40

ii MATERIALS AND METHODS 42

RESULTS 45

DISCUSSION 49

ACKNOWLEDGEMENTS 54

CHAPTER 3: CHARACTERIZATION OF A CLASS 1INTEGRASE GENE ASSOCIATED WITH TETRACYLINE RESISTANCE IN ESCHERICHIA COLI ISOLATED FROM CANADIAN BEEF CATTLE 66

ABSTRACT 66

INTRODUCTION 67

MATERIALS AND METHODS 68

RESULTS 71

DISCUSSION 73

ACKNOWLEDGEMENTS 76

DISCUSSION AND CONCLUSIONS 86

REFERENCES 91

APPENDIX 1: FORMULAE USED TO DETERMINE THE ADJUSTED PREVALENCE FOR STRATIFIED RANDOM SAMPLES 110

APPENDIX 2: STATISTICAL ANALYSES OF INTEGRASE GENE, CLASS 1 INTEGRONS AND 'LONELY INTEGRASE' 111

APPENDIX 3: NUCLEOTIDE AND AMINO ACID ALIGNMENTS FOR VARIANTS OF THE AADA1 AND AADB-AADA2 GENE CASSETTES 114

APPENDIX 4 PLASMID SEQUENCING RESULTS OF TWO LARGE PLASMIDS CONTAINING THE 'LONELY' INTEGRASE GENETIC ELEMENT 121

in LIST OF TABLES

Table 1 - PCR primers used for PCR in this study 55

Table 2 - The distribution of intll gene, class 1 integron and 'lonely integrase' in generic

E. coli in regards to type of sampling, timing of sample and feedlot 56

Table 3 - Gene cassettes present within a class 1 integron in generic E. coli from beef cattle in feedlots collected at entry, >60 DOF and at exit 57

Table 4- Distribution of gene cassettes and variants within class 1 integrons of generic E. coli from beef cattle from four different feedlot operations in Alberta, Canada 58

Table 5 - Distribution of the number of gene cassettes present within class 1 integrons in generic E. coli from beef cattle in feedlots collected at entry, >60 DOF and at exit 59

Table 6 - Susceptibility testing results and genotypes for major gene cassette types identified in class 1 integrons 60

Table 7 - The logistic regression analysis outputs for associations between class 1 integrons, sulfonamide resistance genes, major gene cassettes and phenotypic resistance 61

Table 8 - The logistic regression analysis output for the 'lonely integrase' specifically calculated for the sulfonamide resistance genes, major gene cassettes and phenotypic resistance 63

Table 9 - The Simpson's Diversity Index calculated for all gene cassettes and their variants 65

Table 10 - Primers, PCR conditions and PCR products for the investigation of the 'lonely integrase' 77

IV Table 11 - Primers used in overlapping PCR sets for characterization of the 'lonely integrase' isolates 80

Table 12 - The eight 'lonely' integrase isolate groups based on phenotypic resistance to chloramphenicol, streptomycin, sulfizoxazole and tetracycline 81

Table 13 - The PCR results spanning the entire sequenced 'lonely' integrase region for

247 isolates 82

Table 14 - The adjusted prevalence, standard error and 95% confidence interval

calculated for intll, class 1 integrons and 'lonely integrase' Ill

Table 15 - The plasmid sequencing results for plasmid p260cD (120 kb).- 121

Table 16 - The plasmid sequencing results for plasmid p299cD (140 kb) 124

v LIST OF FIGURES

Figure 1 - (A) General structure of class 1 integrons. (B) General structure of class 2 integrons 18

Figure 2 - The theoretical model of evolution and divergence of clinical class 1 integrons 31

Figure 3 - Southern blot analysis to determine the location of the 'lonely integrase' 83

Figure 4 - The immediate genetic environment of the 'lonely integrase' genetic environment 85

Figure 5 - The Stata 11 coding and output for the logistic regression of class 1 integrons 112

Figure 6 - The Stata 11 coding and output for the logistic regression of 'lonely' integrase isolates 113

Figure 7 - The alignment of the nucleotide sequence of the three variants of the aadAl gene cassette identified in E. co/z in a class 1 integron 114

Figure 8 - The alignment of three variants of the amino acid sequence of aadAl gene products identified in E. coli in a class 1 integron 116

Figure 9 - The alignment of the nucleotide sequence of the three variants of the aadB- aadA2 gene cassette identified in E. coli in a class 1 integron 117

Figure 10 -The alignment of three variants of the amino acid sequence of the expressed aadB gene cassette identified in E. coli in a class 1 integron 119

Figure 11 - The alignment of three variants of the amino acid sequence of the expressed aadA2 gene cassette identified inE coli in a class 1 integron 120

vi LIST OF ABBREVIATIONS

AAC Aminoglycoside acetyltransferase ABC ATP-binding cassette ACAAF Advancing Canadian Agriculture and Agrifood ACID Annotation of Cassette and Integron Data AMC Amoxicillin-clavulanic acid AMP Ampicillin AMR Antimicrobial resistance ANT Aminoglycoside nucleotidyltransferase APH Aminoglycoside phosphotransferase ATP Adenosine triphosphate bp Base pair BRD Bovine respiratory disorder CAT Chloramphenicol acetyltransferase CHL Chloramphenicol CI Confidence interval CIPARS Canadian Integrated Program for Antimicrobial Resistance Surveillance CRO Ceftriaxone CS Conserved segment DI Diversity index DIG Digoxigenin DNA Deoxyribonucleic acid DOF Days on feed ESBL Extended spectrum pMactamase FHMS Feedlot Health Management Services FOX Cefoxitin GEN Gentamicin ID Identification KAN Kanamycin kbp Kilo base pair LB Luria-Bertani MIC Minimum inhibitory concentration NAL Nalidixic acid ORF Open reading frame PBP Penicillin binding protein PCR Polymerase chain reaction RFLP Restriction fragment length polymorphism RNA Ribonucleic acid SOS Save our souls vii SOX Sulfixoxazole SSC Saline sodium-citrate STR Streptomycin SXT Trimethoprim - sulphamethoxazole TCY Tetracycline

viii DECLARATION OF WORK

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

1. The collections of fecal samples and nasal swabs from cattle at feedlots was performed by Feedlot Health Management Services (FHMS) in Okotoks, Alberta and the isolation of E. coli and M. haemolytica was performed by Tim McAllister and collaborators at Agriculture and Agrifood Canada in Lethbridge, Alberta. 2. Antimicrobial susceptibility testing was performed by the Antimicrobial Resistance Surveillance Laboratories, Laboratory for Foodborne Zoonoses, Public Health Agency of Canada in Guelph, Ontario and Saint-Hyacinthe, Quebec. 3. The sequencing reactions were performed at the Guelph Molecular Supercentre, Laboratory Services Division, University of Guelph, Guelph, Ontario and by Macrogen Inc, Seoul, South Korea. 4. Screening of approximately half of the feedlot E. coli for the integrase genes, sulfonamide resistance genes and gene cassettes was performed by Elizabeth Hillyer. 5. Sulfonamide multiplex PCR conducted on approximately 900 feedlot E. coli isolates and screening of approximately 130 E. coli containing the 'lonely integrase' using overlapping PCRs was performed by Fiona Coutinho and Jennie Pouget respectively in the Boerlin Lab, University of Guelph, Guelph, Ontario. 6. Plasmid sequencing was conducted at the McGill University and Genome Quebec Innovation Centre facilities in Montreal, Quebec. 7. Replicon typing of two plasmids was conducted by Gabhan Chalmers in the Boerlin Lab, University of Guelph, Guelph, Ontario.

IX Due to an error in pagination, there is no text on this page.

1 INTRODUCTION

Antimicrobial agents are used in the prevention and treatment of disease in both human and veterinary medicine. On beef cattle feedlots, antimicrobials may be administered to sick animals individually or administered as a preventative measure to entire groups of animals (McEwen and Fedorka-Cray 2002). Over time, bacteria adapt to the selective pressures of antimicrobial agents and undergo genetic modifications to become resistant to antimicrobials. Resistance may arise from mutations in pre-existing genes or from acquiring new genes via horizontal gene transfer. The spread of genes which confer resistance is accelerated by mobile genetic elements, such as transposons, phages and plasmids (Courvalin 2005). Antimicrobial resistance reduces treatment efficiency, limits treatment options and may increase the costs of treating and preventing disease. Resistant bacteria, found in food animals, enter the human food chain via the consumption of animal products. Although these bacteria may not be animal or human pathogens and may not even be capable of colonizing a human host, their genes can still be acquired by the bacteria which make up the normal microbiota of the gastrointestinal tract. The majority of the microflora of the gastrointestinal tract is composed of commensal bacteria, which can become reservoirs of these antimicrobial resistance determinants for pathogenic bacteria (Salyers et al. 2002, Yang et al. 2010). Non pathogenic commensal bacteria, such as "generic" fecal E. coli, may be used as indicator organisms to determine the level of antimicrobial resistance present in a given bacterial population (Aarestrup and Wegener 1999).

Some bacteria are capable of acquiring multiple genes which in turn confer resistance to a wide range of antimicrobials. The integron, which provides a site-specific

2 recombination system that captures mobile gene cassettes, is an important genetic element in the accumulation of antimicrobial resistance genes and the development of multi-resistance. Class 1 integrons are often embedded within highly mobile genetic elements and are therefore distributed widely among many different species of bacteria.

The structure of a typical class 1 integron contains in the 3'conserved segment the

AqacEDl and sull genes, which confer resistance to quaternary ammonium compounds and to sulfonamides, respectively (Fluit and Schmitz 1999). Gene cassettes are incorporated into the class 1 integron structure, between the integrase gene and the 3' conserved segment. These cassettes can confer resistance to pMactams, aminoglycosides, chloramphenicol, erythromycin, fosfomycin, lincomycin, quaternary ammonium compound family antiseptics, rifampin, streptothricin and trimethoprim (Mazel 2006).

Up to seven gene cassettes have been observed within a single class 1 integron (Carattoli

2001).

Although a generally well-defined and conserved genetic structure, the class 1 integron is still subject to occasional changes. Remodelling of the class 1 integron usually occurs within the 3' conserved segment and can be due to an insertion sequence, transposon or a recombination event (Hall et al. 1994, Bischoff et al. 2005). A recent study by Hillyer and collaborators, described an intll gene which was not associated with the 3' conserved segment of integrons and possessed no detectable variable region. This

'lonely intll' gene was present in 11% of generic fecal E. coli from beef cattle in Alberta and was statistically associated with resistance to tetracyclines, sulfonamides and streptomycin (Hillyer 2009). The high frequency of the 'lonely integrase' and its association with antimicrobial resistance genes not usually part of integrons, suggests a

3 potentially important role in the genetic adaptation of E. coli required to colonize cattle or to withstand the antimicrobials used to treat cattle. This warrants further investigations to improve our knowledge of antimicrobial resistance epidemiology in bacteria from cattle, in order to better investigate the impact of resistance on animal and human health. By understanding the evolution of integrons, we may develop better surveillance practices for monitoring the dissemination of antimicrobial resistance determinants as well as predicting important emerging resistance patterns.

A variety of molecular methods were used in this thesis to determine the distribution, frequency and diversity of class 1 integrons in beef cattle in Western

Canada. The first objective was to determine the frequency and distribution of class 1 integrons in M. haemolytica and generic fecal E. coli isolated from beef cattle. Gene cassettes were compared between the two species to determine if E. coli from the gastrointestinal tract could serve as a reservoir for M. haemolytica residing in the nasopharynx of the same host. Statistical analysis was used to determine if associations exist between the presence of a specific gene cassette, the amount of time animals spent on the feedlot and the presence of multiple gene cassettes within an integron. The second objective was to characterize the surrounding genetic environment of the 'lonely intlV gene and determine whether it is an evolutionary ancestor to the class 1 integron or what genetic remodelling has occurred for the typical class 1 integron to lose its 3' conserved segment.

4 CHAPTER ONE: LITERATURE REVIEW

1. Antimicrobial Use in Food Animal Production and Antimicrobial Resistance

Antimicrobial agents are defined as 'any substance of natural, semi-synthetic or synthetic origin that kills or inhibits the growth of a microorganism but causes little or no damage to the host'. Antimicrobial agents often exploit the differences between prokaryotic and eukaryotic cells (Giguere 2006a). In order for these agents to interfere with cellular growth or survival, they must interact with a vital component of the microbial cell or block a specific metabolic pathway. The main classes of antimicrobial drugs usually target cell wall biosynthesis and integrity, protein synthesis or DNA replication and repair (Walsh 2000).

Antimicrobial agents are used in food animals to prevent and/or treat disease and sometimes for growth promotion (McEwen and Fedorka-Cray 2002). Individual animals that display clinical signs may be individually treated. However, entire groups of animals are often medicated at the same time, rather than as individuals. This practice, commonly referred to as metaphylaxis, is aimed at both treating sick individuals and preventing infection in those at risk within a group of animals difficult to handle separately. To treat and prevent commonly occurring diseases in cattle, the following families of antimicrobials are the most often used: P-lactams, aminoglycosides, macrolides, quinolones, sulfonamides and tetracycline (McEwen and Fedorka-Cray 2002).

Acquired antimicrobial resistance (AMR) can be defined as the temporary or permanent ability of a microorganism to remain viable and/or multiply in the presence of antimicrobial agents which normally inhibit growth or kill other members of the species

5 (Walsh 2000). AMR can arise from mutations in housekeeping, structural, or regulatory genes or from the acquisition of new genes through horizontal gene transfer.

Dissemination of genes conferring resistance may occur via mobile genetic elements, which are infectious and may spread exponentially (Courvalin 2005). Bacterial cells use basic strategies to overcome antimicrobials, three major examples being:

1. Antimicrobial efflux pumps, which reduce the antimicrobial concentration within the cell. 2. Enzymatic modification of antimicrobial agents, which causes them to be ineffective. 3. Reprogramming of target structures to block antimicrobial entry into the cell or block antimicrobial function (Walsh 2000). Specific examples of these mechanisms and their association with integrons will be discussed in greater detail later in this literature review.

AMR is important in both human and veterinary medicine as it may reduce treatment efficacy, increase costs of treatments and limit the therapeutic options. In a review on the use of antimicrobials in veterinary medicine, Piddock hypothesises that there are three possible ways for use of antimicrobial agents in animals to pose a risk to human health: 1. Antimicrobial resistant bacteria from animals that are pathogenic to humans contaminate food and are consumed by humans; 2. Antimicrobial resistant bacteria from animals that are non-pathogenic to humans contaminate food and are consumed by humans, then AMR genes are transferred to pathogenic bacteria within the host gut; 3. Residues of antimicrobial agents used in animals remain within food products which creates a selective environment within the human gut after consumption (Piddock

1996). In Canada a withdrawal time, between antimicrobial treatment of the animal and slaughter, is required to reduce the risk of antimicrobial residues being consumed in meat

6 (McEwen and Fedorka-Cray 2002). Therefore, the probability of antimicrobial residues from food animals causing a selective environment for resistant bacteria among the human intestinal commensal population is small. The real risk is the transfer of resistant bacteria, which may either directly cause disease in humans (zoonotic agents) or may be capable of transferring AMR determinants to members of the human intestinal microflora and to human pathogens (Salyers et al. 2002). This study focuses on two organisms from cattle, a commensal of the gastrointestinal tract (Escherichia coli) and an opportunistic pathogen of the upper respiratory tract (Mannheimia haemolytica). Escherichia coli was

chosen as it is easily isolated and identified and may be used as an indicator organism when studying the dynamics of AMR, whereas M. haemolytica was chosen as it is

implicated in a disease which generates major economic loss in the cattle industry.

2. Escherichia coli as a Commensal and Reservoir for AMR Genes

E. coli is a Gram-negative rod and member of the bacterial family

Enterobacteriaceae (Holt et al. 1994). It is the major facultative anaerobe found in the

intestinal tract of many animal species, at approximately 10-10 colony forming units

per gram of feces. E. coli is one of the first organisms to colonize the sterile intestinal

tract of newborns, and remains part of the normal microflora, usually in the capacity of a

commensal organism (Gyles et al. 2004a). As with other commensal bacteria that live

within the gastrointestinal tract of animals it is thought to provide the host with essential

nutrients, promote healthy immune responses from the gut, and prevent colonization by

pathogens (Farthing 2004). While these functions are beneficial to the host, they are

likely secondary to the function of survival by E. coli in this habitat.

7 Commensal strains of E. coli within the gastrointestinal tract rarely cause disease unless certain predisposing factors are present such as a breach in the mucosal barriers or

an impaired immune system (Russo and Johnson 2000). However, some strains of E. coli have acquired virulence factors which can inflict damage to their host causing these

strains to become pathogenic (Farthing 2004). Virulence genes are frequently located on mobile genetic elements, including plasmids, lysogenic bacteriophages, and pathogenicity islands (Gyles et al. 2004a). Some of the pathogenic E. coli remain in the

gut (Boucher et al. 2007) and are the cause of enteric disease or of systemic disease

associated with the spread of toxins to other body compartments, whereas other pathogenic strains of E. coli are able to invade the host and cause extra-intestinal

infection such as urinary tract infections, septicaemia and meningitis (Russo and Johnson

2000).

Populations of E. coli exist in the gastrointestinal tract as either resident or

transient strains. Resident strains remain within the gut for long periods of time, months

to years, whereas transient strains remain within the gut for shorter periods of time, days

to weeks (Caugant et al. 1981). As illustrated by the characterization of E. coli from fecal

samples of hospital patients (Cooke et al. 1969), the intestinal populations are capable of

dramatic changes over a short period of time. For instance, multilocus enzyme

electrophoresis identified 12 different E. coli types in a single human fecal sample and 53

E. coli types from one source over an 11 month period (Caugant et al. 1981). These

findings indicate that new strains of E. coli and other bacteria are constantly being

introduced into the gastrointestinal tract fromth e environment and food, but factors

which determine their persistence are not well described.

8 The presence of antimicrobial agents in the gastrointestinal tract creates a selective environment which may increase the frequency of AMR in commensal organisms (van den Bogaard and Stobberingh 2000). To assess the level of selective pressure and its effect on bacterial populations in general, indicator organisms are used to determine a baseline of resistance which can be extrapolated to less frequent bacteria such as pathogens which are more difficult to detect and isolate. "Generic E. coir refers to strains that may or may not act as commensal organisms within the gastrointestinal tract but are not usually pathogenic (Van Donkersgoed et al. 2003). Such strains are considered useful indicators of AMR and antimicrobial selective pressure as they can be consistently isolated from healthy animals and humans and can thus serve as a comparison basis across a variety of species and environments. In addition, generic E. coli are also hypothesised to be a reservoir of AMR genes that may be acquired by pathogenic bacteria (Aarestrup and Wegener 1999, Yang et al. 2010). The incoming and transient strains may carry new transferable genes capable of conferring AMR to pathogenic bacteria within the gastrointestinal tract (Salyers et al. 2002). Although differences in the proportional prevalence of AMR genes among indicator organisms and pathogenic strains have been observed (Travis et al. 2006, Kozak et al. 2009b), the same genes are often found in indicator organisms and pathogenic strains. A study conducted by Gow and collaborators determined the prevalence of AMR in generic fecal E. coli from western Canadian beef calves. Resistance to at least one antimicrobial was observed in 31% of the isolates collected. However, the generic fecal E. coli isolates in this study were rarely resistant to antimicrobials of very high importance to human health

(Gow et al. 2008c). Another study examined the level of AMR in E. coli isolated from a

9 commercial beef processing plant and observed resistance to tetracycline, sulfisoxazole, streptomycin and ampicillin (Aslam et al. 2009).

3. Mannheimia haemolytica: Opportunistic Pathogen of Ruminants

M. haemolytica is a Gram-negative rod, a member of the bacterial family

Pasteurellaceae, and the main aetiological agent of bovine pneumonic pasteurellosis commonly referred to as shipping fever or bovine respiratory disease (BRD) (Holt et al.

1994, Quinn et al. 2002, Klima et al. 2010a). M. haemolytica was formerly named

Pasteurella haemolytica and was subdivided into 16 serotypes (twelve A and four T serotypes). DNA-DNA hybridization and 16S RNA sequencing analysis allowed eleven of the A serotypes to be assigned into the species M. haemolytica. The four P. haemolytica T serotypes were renamed Bibersteinia trehalosi while the last A serotype was renamed Mannheimia glucosida (Rice et al. 2007). Twelve serotypes of M. haemolytica now exist; the Al and A2 serotypes are most prevalent around the world with M. haemolytica Al being the main causative agent of BRD (Zecchinon et al. 2005).

Mannheimia haemolytica is a commensal organism living on the mucosal membranes of the nasopharynx of healthy ruminants. Under normal conditions when small numbers of M. haemolytica are inhaled in aerosolized droplets, alveolar clearing mechanisms remove the bacteria fromth e lungs (Rice et al. 2007). If the host's immune system becomes compromised by "stress" or concurrent infection, and the efficiency of the alveolar clearing mechanisms decreases, the opportunistic M. haemolytica may settle within the lungs and cause pneumonia or exacerbate a more severe disease initiated by other pathogens already present within the lung (Quinn et al. 2002, Gyles et al. 2004b).

10 A study conducted in 1992 noted that BRD was responsible for approximately one billion dollars in annual economic loss to the North American beef cattle industry (Whiteley et al. 1992), and it continues to be a source of considerable economic loss today (Klima et al. 2010a). Several pathogenic species of bacteria and viruses may contribute to the development of BRD which normally affects post-weaning calves after transport over long distances, assembling of animals of different origins into feedlots, and close confinement. These conditions provide excellent opportunities for pathogens to be exchanged among the immunologically naive calves (Lillie 1974, Quinn et al. 2002,

Srikumaran et al. 2007). Along with the possibly psychological stress effects associated with the changes mentioned above, physical factors such as dust, cold, humidity, sudden and extreme changes in temperature, hypoxia as well as intercurrent infections by viruses and the host immune response to viral infection, may all reduce the clearance of inhaled bacteria and create a selective growth environment for M. haemolytica (Lillie 1974,

Whiteley et al. 1992). Calves suffering from BRD usually display a combination of depression, fever, nasal discharge, cough and weight loss (Dabo et al. 2007). The major cause of death in BRD is acute fibrinous pleuropneumonia, which is a result of the accumulation of fibrinous exudates within the bronchioles and the accumulation of lymphocytes and fibrin within the alveoli that leads to obstruction (Rice et al. 2007).

Vaccines are available which provide protection against M. haemolytica; however, the protection is not absolute (Katsuda et al. 2009). Calves displaying clinical

signs of BRD must be isolated and treated early, to avoid spread of the infection to

susceptible calves. Infected calves are moved to a low stress environment and are given antimicrobial treatments, which differ from country to country (Quinn et al. 2002). For

11 example, in North America, oxytetracycline, potentiated sulfonamides and ampicillin are

likely to be used for first line treatment of bovine respiratory diseases, whereas in Japan the most popular antimicrobial agents are tetracycline, penicillin and streptomycin

(Quinn et al. 2002, Katsuda et al. 2009).

Katsuda and collaborators assessed the susceptibility of M. haemolytica isolated

from cattle showing clinical signs of BRD, to 14 antimicrobial agents. Of the 229 isolates

sampled, approximately 50% displayed resistance to at least one antimicrobial (Katsuda

et al. 2009). A separate study looking at the prevalence of AMR of pathogens of cattle,

identified resistance to ampicillin, tetracycline, trimethoprim and sulfonamides in M.

haemolytica in European isolates (Hendriksen et al. 2008). Until recently no work had

been conducted on examining the genetic diversity of AMR in M. haemolytica from

cattle in feedlots. A study by Klima and collaborators described that there are low levels

of AMR in M. haemolytica isolated from Canadian beef cattle. Resistance was observed

to the following antimicrobials: oxytetracycline, ampicillin, and amoxicillin/clavulanic

acid. It is interesting that neither amoxicillin nor clavulanic acid were used in the

treatment of the cattle during this study, suggesting that AMR may be acquired before

entry to the feedlot (Klima et al. 2010a).

Although this organism does not pose a threat to human health, it is of great

economic importance in cattle. By understanding the molecular mechanisms

M. haemolytica uses to acquire AMR genes, treatment strategies may be developed which

may decrease the morbidity and mortality of infected calves and this may help prevent

the future development of resistance, as well as to reduce antimicrobial use in cattle.

12 4. Integrons and Their Role in AMR Integrons were first identified because of their association with AMR genes and mobile genetic elements, such as transposons and plasmids. They were subsequently observed on the chromosome of many bacterial species (Partridge et al. 2009). An integron is a genetic element which contains a site-specific recombination system capable of recognizing and capturing mobile gene cassettes (Fluit and Schmitz 1999). Integrons are composed of an intl gene which encodes an integrase enzyme, the attl recombination site and Pc, an outward-oriented promoter. These three elements are necessary for the capture and integration of exogenous genes. The integrase enzyme inserts gene cassettes into the integron downstream of the Pc promoter, via site-specific recombination, allowing expression of the genes within the cassette (Mazel 2006). Gene cassettes are inserted into the integron at the attl recombination site within a 7 base pair sequence

(GTTRRRY) referred to as a core site (Hall et al. 1999). This system of recombination and expression promotes the dissemination of AMR genes throughout bacterial populations (Ploy et al. 2000). Although integrons are not independently mobile, their association with mobile genetic elements has lead to their global distribution (Fluit and

Schmitz 1999).

Integrons can be divided into two major groups, the resistance integrons and the super integrons. Resistance integrons can be located on plasmid or chromosomal DNA, and contain gene cassettes which encode resistance to disinfectants or antimicrobials.

Super integrons are much larger than resistance integrons, located on chromosomal DNA and contain gene cassettes with a much broader variety of functions (Fluit and Schmitz

2004). Super integrons are stable in phylogenetic lineages however, over evolutionary

13 time frames they have been observed to exhibit intragenomic, intercellular and interspecies movement (Gillings et al. 2008). The intl gene is the determinant characteristic in classifying integrons. Classes 1, 2 and 3 of integrons are believed to be the most important in the dissemination of AMR genes (Ploy et al. 2000). Class 1 and class 2 integrons are often embedded within highly mobile genetic elements which account for their broad distribution among bacteria (Boucher et al. 2007). Class 1 integrons are frequently associated with transposons derivatives of Tn402 which are sometimes embedded within larger transposons such as Tn27. Class 2 integrons have only been observed to be associated with Tn7 transposons (Mazel 2006).

Typical class 1 integrons (Figure 1) contain two different conserved segments

(CS) at the 5' and 3' ends (Partridge et al. 2009). The 5'CS region contains the intll gene which encodes the integrase enzyme as well as the promoter region. This promoter region includes two potential promoters PI and P2, which allow for the expression of integrated gene cassettes, one being the aforementioned Pc promoter. Four different PI promoters and two different P2 promoters have been described, and vary in their ability to promote expression of integrated gene cassettes (Fluit and Schmitz 1999). The P2 promoter is activated by the insertion of three guanine residues, which creates an optimal spacing of 17 base pairs between the potential -35 and -10 sites (Partridge et al. 2009).

The 3'CS region of class 1 integrons normally contains the following three genes

AqacEDl, sull and or/5 (Fluit and Schmitz 1999). The AqacEDl gene is a truncated sequence, which confers resistance to quaternary ammonium compounds, the sull gene confers resistance to sulfonamides and the or/5 is an open reading frame of unknown function (Ploy et al. 2000). Gene cassettes are integrated between the 5' and 3' CS

14 regions. Class 1 integrons have been detected carrying between zero and seven gene cassettes, and in some cases multiple copies of the same cassette have been detected

(Carattoli 2001). Gene cassettes have been described from class 1 integrons, which confer resistance to P-lactams, aminoglycosides, erythromycin, fosfomycin phenicols, lincomycin, quaternary ammonium compound family antiseptics, rifampin, streptothricin and trimethoprim (Mazel 2006).

Class 1 integrons from bacteria isolated in clinical settings contain a highly conserved intll gene, the distribution and abundance of the intll gene in nonclinical environmental samples is not well understood, however, intll seems to be widespread in environmental bacteria. Real time quantitative PCR showed the presence of intll in

2.65% of all bacterial cells from 26 creek sediment samples in Australia (Hardwick et al.

2008). It is the general belief that the use of antimicrobials in human and veterinary medicine and agriculture have exerted a selective pressure for bacteria to possess a class

1 integron; however they have been observed in bacteria isolated from relatively remote areas (Yang et al. 2010). A study by Yang and collaborators explored the frequency of class 1 integrons in 'commensal' isolates from four locations; a beef ranch, a dairy farm, a city park and the Rocky Mountain National Park. The highest frequency of class 1 integrons was observed within the beef ranch and dairy farm, suggesting that while class

1 integrons can be found in natural environments, the exposure to antimicrobials does positively select for this genetic element (Yang et al. 2010).

Recently an online database was launched which contains all publicly available sequence information regarding integrons and gene cassettes. This database contains over 5622 gene cassettes and 471 intll sequences, which provides evidence that there is

15 much diversity among the intll gene present in the nonclinical environment (Joss et al.

2009). To date the most widely used tool for identification of integrons in clinical and nonclinical environments is PCR, which relies on the use of specific primers to amplify the intll gene, however this approach needs to be re-evaluated. By using the specific primers, it may not be possible to identify all intll gene variants, especially those that have base pair mutations within the regions to which the primers anneal. Using a PCR based approach, Schwarz was unsuccessful in describing class 1 integrons in a sample of

M. haemolytica isolates (personal communication, 2009). It may therefore be wiser to use a DNA-DNA hybridization approach, using labelled probes for the intll gene, rather than PCR since the stringency of the reaction can be manipulated to capture less closely related gene variants.

Class 2 integrons (Figure 1) are similar in structure to class 1 integrons in the 5'

CS region but are considerably different in the 3' CS region. The intI2 gene produces a non-functional protein due to a truncation of 12 amino acids and shares 46% homology with the intll (Recchia and Hall 1995). The 3'CS region contain tnsE and tnsD genes which are required for the transposition mechanism of Tn7 (Ramirez et al. 2005). Six gene cassettes associated with resistance to erythromycin (ereA), streptothricin

(satl/sat2), aminoglycoside {aadAl) and trimethoprim (dfrAl/dfrAlb) have been detected within class 2 integrons (Biskri and Mazel 2003, Ramirez et al. 2005). The relative lack of diversity of gene cassettes is likely due to a nonsense mutation within the class 2 integrase gene, intll (Mazel 2006).

Gene cassettes are mobile elements which can be captured by integrons (Partridge et al. 2009). Each gene cassette consists of one promoter-less open reading frame and a

16 recombination site (attC) located at its 3' end (Ploy et al. 2000, Partridge et al. 2009).

Cassette length ranges between approximately 260 to 1550 base pairs, with most of this variation caused by the differences in gene size, since there are very few non-coding

sequences flanking the genes (Recchia and Hall 1995). Each gene is associated with a

specific attC recombination site, which differs in both sequence and length (Carattoli

2001). Despite this variation, the attC element share conserved regions at their ends which are normally imperfect inverted repeats predicted to form stem-loop structures

(Partridge et al. 2009). The integration and excision of gene cassettes occur via site-

specific recombination catalyzed by integrases (Ploy et al. 2000). Gene cassettes are

always integrated in the same orientation with the 5' segment of the gene closest to the

inti gene. Although gene cassettes are most commonly found within integrons, they also

have been found as free circular molecules which are formed by excision of the cassette

from within an integron (Recchia and Hall 1995).

17 5' Conserved Segment 3' Conserved Segment

attl attC

att/ crttC

Figure 1 - (A) General structure of class 1 integrons. (B) General structure of class 2 integrons.

Figure adapted from Levesque et al. 1995, Recchia & Hall 1995, Fluit & Schmitz 1999 and Ramirez et al. 2005.

18 Approximately 130 different cassettes which confer AMR have been identified to date (Partridge et al. 2009). Of the gene cassettes which are known to confer AMR; 39 have been identified which confer resistance to P-lactams, 43 confer resistance to aminoglycosides, 11 confer resistance to chloramphenicol, 24 confer resistance to trimethoprim, 1 confers resistance to streptothricin, 3 confer resistance to rifampin, 2 confer resistance to erythromycin, 5 confer resistance to fosfomycin, 2 confer resistance to lincomycin, 5 confer resistance to quaternary ammonium compounds and 2 confer resistance to quinolones (Partridge et al. 2009). Although most of the known gene cassettes encode resistance to older antimicrobials, cassettes which confer resistance to recent antimicrobials have been described, such as the blaoxA and blaviM cassettes (Fluit and Schmitz 1999). As integrons may contain multiple cassettes at once, a variety of cassette combinations have been detected (Fluit and Schmitz 2004). A single class 1 integron is capable of capturing multiple gene cassettes (Carattoli 2001). When multiple gene cassettes are present within a single integron, an array is formed, with all cassettes in the same orientation. The position of the gene cassette within the array, in relation with its distance to the promoter may play an important role in its level of expression

(Partridge etal. 2009).

5. Predominant AMR Genes in Integrons and the Resistance Mechanisms They Confer The major antimicrobials that are of significance due to their use in cattle, their frequency of resistance and their association with integrons are: P-lactams, phenicols, aminoglycosides, quinolones, sulfonamides, trimethoprim, macrolides and lincosamides

(Partridge et al. 2009).

19 I. p-Lactams - Map, blasEL, blaoESt blavEB, blamp, blaGM> blasm, blayiM and blaoxA- The P-lactam antimicrobials consist of penicillins, cephalosporins, carbapenems, monobactams and penams (Prescott 2006). These agents represent more than 65% of the world antimicrobial market and are widely used because of their selectivity, low toxicity and versatility. The P-lactam antimicrobials interfere with the final stage of peptidoglycan synthesis by inhibiting the activity of penicillin-binding proteins (PBPs), which causes cell death, through mechanisms not yet clearly defined (Poole 2004).

Resistance to the P-lactam antimicrobials may result from P-lactamase drug inactivation via a hydrolytic cleavage of the characteristic four-membered P-lactam ring, alterations in

PBPs, or reduced permeability and increased efflux (Poole 2004). Plasmid mediated

p-lactamases are widespread among Gram-negative primary and opportunistic bacterial pathogens. Generally, these enzymes are expressed constitutively and are located within the periplasm, which allows for a high-level of resistance (Prescott 2006). Two

classification systems have been developed for the P-lactamase family of enzymes, the

Bush-Jacoby-Medeiros classification and the Ambler classification. The Bush-Jacoby-

Medeiros classification system contains four groups (Group 1-4) of enzymes (Bush et al.

1995), while the Ambler classification system contains four classes (Class A-D) of

enzymes (Ambler 1980). Three classes of P-lactamase genes have been detected within the variable region of integrons. Class A P-lactamases contain the MOBEL, MaoEs, blavEB

genes as well as three variants of the Map genes. Class B metallo-P-lactamases contain

the blciGiM and blasm genes, five blavm gene variants and 14 blaiMP gene variants. Class

D P-lactamases contain at leastl2 blaoxA gene variants (Partridge et al. 2009). The Map

genes confer resistance to extended-spectrum cephalosporins while the blaoxA genes

20 encode resistance to most penicillins. The blavm genes confer resistance to carbapenems and most (^-lactams (Riccio et al. 2001). Extended spectrum P-lactamases (ESBLs) provide resistance to a variety of P-lactams, including third generation cephalosporins, but remain susceptible to inhibitors such as clavulanic acid (Martinez-Freijo et al. 1998).

The SHV and TEM enzymes encoded by MCITEM and blasHv are the major P-lactamases found in E. coli in North American farm animals, blasHv being less frequent than MCITEM

(Li et al. 2007). The blarm and Was/^have not yet been detected within the variable regions of integrons. However, there is a significant increase in resistance to ceftazidime, aztreonam and ceftriaxone in isolates that contain integrons, which may suggest a physical link between these genes and integrons (Martinez-Freijo et al. 1998).

II. Phenicols - cat, cm1A,floR. Chloramphenicol, thiamphenicol and florfenicol are broad spectrum phenicols antimicrobials that are effective against Gram-positive or

Gram-negative bacteria which are either aerobic or anaerobic (Lai et al. 2009). This group of bacteriostatic antimicrobials binds irreversibly to the 5 OS subunit of the bacterial ribosome which prevents peptide transfer thus inhibiting protein synthesis (Schwarz et al.

2004, Dowling 2006b). The first observed resistance mechanism to chloramphenicol was via the chloramphenicol acetyltransferase (CAT) which inactivates the antimicrobial by acetylation. The CAT enzymes are encoded by the cat genes which are present on bacterial chromosomes, plasmids and within transposons (Schwarz et al. 2004). The cat genes encode resistance to chloramphenicol and thiamphenicol but not to florfenicol

(Dowling 2006b). Resistance to chloramphenicol can also be conferred by efflux pumps encoded by cml genes, first observed in Pseudomonas aeruginosa but now also found in other bacterial species. The cml genes are commonly located on plasmids and within

21 transposons and integrons and have been observed in members of the family

Enterobacteriaceae (Bissonnette et al. 1991, Schwarz et al. 2004). The cat gene is more frequently detected in Salmonella whereas the cmlA gene is more frequently detected in

E. coli (Chen et al. 2005). Among 28 chloramphenicol resistant E. coli isolates from cattle, swine and poultry, catA genes were detected in 19 isolates and cmlA genes in 10 isolates; however this study did not search for their presence within integrons (Guerra et al. 2003). Eight gene variants of the cat gene and three gene variants of the cml gene have been detected within the variable region of integrons (Partridge et al. 2009). The floR gene confers resistance to chloramphenicol and florfenicol via an efflux pump.

While it has not been detected as part of the variable region of class 1 integrons, floR has occasionally been associated with integrons in E. coli and Salmonella (Schwarz et al.

2004).

III. Aminoglycosides - aacA, aacC, aadA, aadB, aphAlS. The aminoglycosides are bactericidal antimicrobials used to treat infections caused by aerobic

Gram-negative bacteria and staphylococci. The main antimicrobial agents of the aminoglycoside/aminocyclitol family that are used in veterinary medicine are streptomycin, spectinomycin, kanamycin, neomycin, gentamicin, tobramycin and amikacin. Aminoglycosides exploit oxygen dependant transporters to enter the bacterial cell. This group of antimicrobials bind to the 30S ribosomal subunit and cause misreadings of the genetic code which leads to the interruption of normal bacterial protein synthesis (Dowling 2006a). The most common resistance mechanism to aminoglycosides is by enzymatic modification and inactivation. The three families of enzymes that confer resistance to aminoglycosides are aminoglycoside

22 phosphotransferases (APHs), aminoglycoside acetyltransferases (AACs) and aminoglycoside nucleotidyltransferases (ANTs) (Shaw et al. 1993, Vakulenko and

Mobashery 2003). The aacA and aacC genes each encode for two different AACs. The

AAC encoded by aacA confers resistance to gentamicin and tobramycin, while the AAC encoded by aacC confers resistance to gentamicin, sisomicin and fortimicin (Rouch et al.

1987, Levings et al. 2005). The aadA and aadB genes encode two different ANTs. The aadA ANT confers resistance to streptomycin and spectinomycin, while the ANT encoded by aadB confers resistance to kanamycin/neomycin, tobramycin and gentamicin

(Cameron et al. 1986, Vanhoof et al. 1992, Binh et al. 2009). The aphA 15 gene encodes an APH, which decreases sensitivity to kanamycin/neomycin, amikacin, netilmicin and

streptomycin (Riccio et al. 2001). Genes of all three families have been detected as gene cassettes within integrons. There are 21 variants of the aacA genes, 8 variants of the aacC genes and 12 variants of the aadA genes which have been detected within the variable region of integrons (Partridge et al. 2009).

IV. Quinolones - qnr. Quinolones are a synthetic class of antimicrobial agents that are active against a wide range of Gram-negative bacteria. The clinically most relevant antimicrobial agents that belong to the quinolone family of antimicrobials are fluoroquinolones: ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, ibafloxacin, marbofloxacin, orbifloxacin and pradofloxacin (Walker and Dowling 2006). Nalidixic

acid was the first quinolone used in clinical practice; however its moderate antibacterial

activity limited its application. The addition of a fluorine molecule to the quinolone nucleus gave rise to the fluoroquinolones, which have a much broader antibacterial

activity (Ball 2000). After gaining entry into the bacterial cell, the quinolones target

23 DNA gyrase and DNA topoisomerase IV, inhibiting their normal function and disrupting

DNA synthesis and replication (Walker and Dowling 2006). Resistance to quinolones may arise from chromosomal mutations that alter the quinolone binding site of DNA gyrase and/or DNA topoisomerase IV, production of quinolone target protection proteins, to changes in membrane permeability, or increased efflux of quinolone leading to reduced concentration within the cell (Martinez-Martinez et al. 1998, Hata et al. 2005).

DNA gyrase and topoisomerase IV are each composed of four subunits encoded by gyrA, gyrB, mdparC, parE respectively. In Enterobacteriaceae, fully resistant isolates usually contain multiple mutations within the gyrA orparC genes. The gyr and par genes are located within the bacterial chromosome and have not been associated with integrons

(Piddock 1998). The qnr genes encode a protein of the pentapeptide family which binds to the DNA gyrase which physically blocks the quinolones from binding to and thus from inhibiting the DNA gyrase (Tran et al. 2005, Wang et al. 2008). In Enterobacteriaceae the qnr genes have been located within plasmids, implying that horizontal gene transfer of resistance to quinolones and fluoroquinolones is occurring (Park et al. 2009). Two variants of the qnr gene have been associated with integrons (Partridge et al. 2009).

However, the qnr genes are not associated with the 59-bp element and are found in uniquely modified integrons (Mammeri et al. 2005).

V. Sulfonamides - sull, sul2, sul3. Sulfonamides are broad spectrum antimicrobial agents capable of inhibiting bacterial and protozoan growth. Sulfonamides interfere with the synthesis of folic acid in bacterial cells by competitively blocking para- aminobenzoic acid from entering the dihydropteroate synthase active site. This interference results in a dramatic decrease of thymine synthesis, ultimately causing an

24 inhibition of DNA synthesis and repair (Skold 2001). Sulfonamides are among the oldest chemotherapeutic agents. Widespread sulfonamide resistance is a current issue because of extensive use since their discovery (Then 1982). Three genes which confer resistance to sulfonamides, sul\, sull and su!3, often associated with integrons or located on plasmids, have been described. Each of these genes encodes a slightly different low affinity sulfonamide dihydropteroate synthase, and allows bacterial cells to produce folic acid in the presence of sulfonamides (Skold 2001, Perreten and Boerlin 2003). The sull gene is normally found within the 3'CS of class 1 integrons, while the sul2 gene is typically detected within plasmids but not associated with integrons. The suB gene has been observed to sometimes replace the sull in modified class 1 integrons (Bischoff et al.

2005).

VI. Trimethoprim -dfr. Trimethoprim is a synthetic antimicrobial, active against both Gram-positive and Gram-negative aerobic bacteria. Trimethoprim interferes with bacterial dihydrofolate reductases, indirectly inhibiting the synthesis of folic acid and purines, and thus interfering with DNA synthesis and repair in ways similar to sulfonamides (Skold 2001). The dfr genes which confer resistance to trimethoprim are frequently encoded on plasmids. Twenty-four dfr gene variants have been described to date and have been shown to be located within the variable region of integrons (Skold

2001, Partridge et al. 2009). In a study conducted on generic fecal E. coli from Canadian beef cattle, 27 out of 33 trimethoprim resistant isolates carried the dfr A gene (Gow et al.

2008a).

VII. Erythromycin - ereA. Erythromycin belongs to the macrolide family of antimicrobials, which inhibit protein synthesis by reversibly binding to the 50S subunit of

25 the ribosome. Erythromycin is effective against a range of Gram-positive and some

Gram-negative bacteria; however M. haemolytica and E. coli isolates are intrinsically resistant, because of the relative impermeability of the outer membrane caused by the hydrophobic nature of erythromycin, which limits the use of this antimicrobial in treating

M. haemolytica related respiratory disease (Pechere 2001, Giguere 2006c). The erm genes have been detected in Gram-positive bacteria. These genes encode an erythromycin resistant methylase, which methylates ribosomal RNA and blocks the binding of erythromycin to the 50S ribosomal subunit. However, erm genes have not been associated with integrons (Pechere 2001) and because of intrinsic ineffectiveness are unlikely to play a major role in AMR in Gram-negative bacteria, particularly in

Enter obacteriaceae. Resistance to erythromycin can also be achieved by the inactivation of the antimicrobial via erythromycin esterase, which is encoded by the ereA gene (Plante

et al. 2003). Despite the intrinsic resistance of Enterobacteriaceae to erythromycin, the ereA gene was originally detected within a plasmid which provided low levels of resistance to erythromycin in E. coli (Ounissi and Courvalin 1985). Two variants of the

ereA gene have been detected within the variable region of integrons (Partridge et al.

2009).

VIII. Lincomycin - tin. Lincomycin belongs to the lincosamide family of

antimicrobials which are moderate-spectrum drugs. Similarly to macrolides, lincomycin

inhibits protein synthesis by binding to the 5 OS ribosomal subunit. Lincomycin is used in

the treatment of respiratory disease in cattle, often in combination with spectinomycin

(Giguere 2006b). Lincomycin nucleotidyltransferases, which are encoded by lin genes

confer resistance to lincomycin by the inactivation of the antimicrobial (Bozdogan et al.

26 1999). Two variants of the lin gene have been detected within the variable region of integrons (Partridge et al. 2009).

IX. Annotation of Cassette and Integron Data (ACID). The ACID database makes sequence information regarding the integrase genes, attC recombination sites and gene cassettes publically available. Users have the ability to download annotated sequences of interest as well as submit and automatically annotate their own sequences.

The ACID database allows for better understanding of the frequency and diversity of integrons (Joss et al. 2009).

6. AMR in Bacteria Isolated from Canadian Beef Cattle

Although reports have been made that antimicrobial resistant bacteria are present in beef, few have attempted to link these bacteria to those entering the food supply. A study conducted by Alexander and collaborators examined the effect that the administration of antimicrobials on cattle feedlots as growth promoters had on the prevalence of AMR E. coli isolated at a slaughter. They observed that, despite the antimicrobial withdrawal period that occurs before the cattle reach the abattoir, those cattle which were treated with antimicrobial growth promoters were more likely to shed antimicrobial resistant E. coli

(Alexander et al. 2010, Alexander et al. 2011).

Antimicrobial use in animals is generally thought to be associated with selection of

AMR. However, this seems not always to be the case. An association between the amount of antimicrobials used to treat disease and the changes in AMR (Checkley et al.

2008) was attempted by studying fecal E. coli isolates from calves on arrival at the feedlot. Of the 153 cattle sampled at arrival, 36.6% had E. coli that were susceptible to

27 all antimicrobials and 5.9% had E. coli that were resistant to at least three antimicrobials.

The most frequentAMR s were for sulphamethoxazole, ampicillin and tetracycline. The

cattle received antimicrobial metaphylaxis treatment during their entire stay at the

feedlot. Although some cattle which initially tested negative for AMR gave positive

AMR results at the end of the study, no significant association between antimicrobial use

and AMR was observed (Checkley et al. 2008).

Another study conducted by Gow and collaborators examined the prevalence of AMR

in generic fecal E. coli frombee f calves and cow-calf pairs in Western Canada. Over

1600 E. coli isolates from 480 beef calves were analyzed for AMR. This study found that

48.8% of the isolates were resistant to at least one antimicrobial; the most common being tetracycline, sulphamethoxazole and streptomycin (Gow et al. 2008c). A second study

by the same authors found that 12.7% of 2185 E. coli isolates from calves and cows were

resistant to two or more antimicrobials (Gow et al. 2008b). A third study analyzed 207

generic fecal E. coli isolates from 77 cow-calf herds in Western Canada for AMR genes.

Sixteen different patterns of multi-resistance were observed and statistical analysis was

done to determine the associations between genes. The strongest associations were

observed between: cati and dfrl, sull and cati, aadA and dfrl, sull and aadA and tetB and

sul2 (Gow et al. 2008a). Although this study did not search for their presence, integrons

may be responsible for capturing some of these genes resulting in the strong associations

observed between some of the genes investigated. These studies provide evidence that

AMR is widely present in E. coli in Canadian beef cattle. Although these studies did not

determine the presence of integrons or the location of the AMR genes, many of the genes

reported have been observed in association with integrons. All of the above genes,

28 except catl, sul2 and tetB, have been detected as gene cassettes or as part of integron structures (Antunes et al. 2005, Partridge et al. 2009). Further studies need to be conducted to determine where the AMR genes are located, how these genes are being dispersed, whether new AMR genes are emerging and how genetic elements which confer resistance to antimicrobials have emerged in the past.

7. Ancestral Integrons, Modified Integrons and the 'Lonely Integrase'

Class 1 integrons have been isolated from bacteria occurring in soil and sediment, in the absence of antimicrobial selection pressures. However, in contrast to integrons from clinical isolates, these class 1 integron lack the typical Tn402-\ike features and are located within the bacterial chromosome, suggesting that the common ancestors of class

1 integrons were chromosomally located (Stokes et al. 2006). The attC sites within super integrons display a high level of sequence similarity to the attC sites within resistance integrons. The cassettes within the V. cholerae super integron are also substrates for the class 1 integrase of resistance integrons (Mazel et al. 1998). Consequently, it has been hypothesized that resistance integrons evolved from super integrons through the entrapment of intll and the attl site onto mobile genetic structures. Experimental evidence has indicated that the integrase of super integrons is active, that some of the gene cassettes present in super integrons provide adaptive functions and that super integrons are widespread among the proteobacteria (Rowe-Magnus et al. 2001). A super integron has been detected in an isolate of Vibrio metschonikovii from1888 . This isolate predates antimicrobial usage and supports the hypothesis that integrons are ancient structures and did not only arise to provide an adaptive advantage for AMR (Mazel et al.

1998). The boundaries of the majority of class 1 integron isolates are defined by the

29 presence of inverted repeats. These inverted repeats are the recognition sites needed for

Tn¥02-mediated transposition. It has therefore been suggested that chromosomal class 1 integrons became plasmid-borne during a mobilization event involving the Tn402 transposon (Gillings et al. 2008). Figure 2 displays the theoretical evolution of the clinical class 1 integron.

Modifications in the 3' end of integrons were first reported in 1994 (Hall et al.

1994), when comparing the structure of six independently located integrons using restriction enzyme and nucleotide sequence analysis. It was observed that the 5' region was generally constant, whereas the 3' regions of these six integrons were occasionally variable. Modifications to the 3' region included insertion sequences and inverted repeats (Hall et al. 1994). Three unique integrons were identified from Pseudomonas aeruginosa isolates from Russia, India and the United States. These integrons all lacked the 3' CS found in class 1 integrons and instead contained the tniC gene, which encodes the resolvase of Tn5090 (Toleman et al. 2007). Bischoff and collaborators observed 20

integrons isolated from E. coli in swine in which the sull gene present in class 1

integrons was replaced with sul3. A representative isolate was chosen for sequencing and

a unique integron structure was observed. Although the 5' region of this integron was

similar to other isolates, the 3' region possessed a uniquely modified structure. Rather than possessing the sull and qacEAl sequences, this isolate contained qacH and sul3

(Bischoff ef a/. 2005).

30 intlt cassette array chromosome A Moblzation of intH B ^^Zfl, and cassette arrays Insertion into tni module C Tn4G2 backbone s#« i qacE? D Insertion of sul and arte cassettes i 3 qacE sull oris

qacE deletion event 1 a p qacEA orfS a mmmm 2. Acquisition/excision of cassettes; deletions of tni trM G Insertion into transposons & plasmids; acquisition & excision of cassettes H

Figure 2 - The theoretical model of evolution and divergence of clinical class 1 integrons.

(A and B) The common ancestor of the clinical class 1 integron was a member of an

integron pool which was acquired by Betaproteobacteria but not other lineages. (C and

D) One chromosomal integron recombined into a Tn402-like element. The capture of the qacE gene likely occurred at a similar time. (E) The sull gene was captured. (F) The

3'-conserved segment was formed due to insertions, deletions and rearrangements of the

qacE, sull genes and adjacent sequences. (G) Deletions and insertions involving tni

generated Tn402-transposition incompetent integrons, which possessed diverse cassette

arrays conferring various AMR phenotypes. (H) A Jrara-mediated Tn¥02-like transposition event moved integrons into diverse plasmids and other transposons. Figure

and legend adapted from Gillings et al. 2008.

31 Another example of modified class 1 integrons exists in the complex In4 family.

In¥ integrons are also known as complex-sw/i integrons and possess duplicated sull and qacEAl which surround or/513. This or/513 may act as a recombinase system for AMR genes that are located nearby (Mammeri et al. 2005). Class 1 integrons which lack the characteristic 3'CS have been detected inE. coli isolated from food, animals and healthy humans. One study investigated 13 E. coli isolates from the previously mentioned sources and observed no clonal relationships between the isolates but found that while the isolates lacked the typical sull gene, all carried a sul3 (Saenz et al. 2010). Other studies present examples of modifications occurring in the 3'CS, describing the sull being replaced with a sul3 (Sunde et al. 2008), the entire 3'CS being replaced with a qacH-

IS440-sul3 (Soufi et al. 2009) and intll genes which was associated with gene cassettes but completely lack a 3'CS (Vinue et al. 2008). These studies provide reminders that the genetic environment within bacteria is capable of being remodelled. However, changes to the genetic code which cause unfavourable outcomes normally are not selected for.

This may be why modifications to the 5' region of integrons are observed less frequently, than modifications to the 3' region. If the 5' region was to be dramatically changed, there is risk of losing the intlgene, the attl site and/or the PI and P2 promoters, which are all necessary structures for gene capture and expression.

Hillyer and collaborators recently conducted a study to assess the prevalence of cassettes in the variable region of class 1 integrons. This group conducted PCR on 1258 generic fecal E. coli samples fromabattoir s and feedlots of beef cattle in Western

Canada. The isolates were screened for the presence of intll, sull and gene cassettes in the variable region of class 1 integrons. One hundred and seventy-three isolates tested

32 positive for the intll gene, however only 34 of these isolates were typical class 1 integrons. The remaining 139 isolates had no sull gene and no detectable gene cassettes.

The intll in these isolates was termed 'lonely integrase'. Although the 'lonely integrase' lacked the link with the sull gene, the majority of these isolates carried sull or sul3. The isolates containing the 'lonely integrase' were more frequently resistant to chloramphenicol and tetracycline than expected. No integron associated tetracycline resistance cassette has been reported to date (Hillyer 2009). Further studies are needed to determine where the lonely integrase gene is located and if it is a modified integron or an ancestor to the modern class 1 integron.

8. The Dissemination of Class 1 Integrons

Class 1 integrons have many mechanisms by which they spread throughout bacterial populations. They have been frequently observed within plasmids and transposons, which often have broad host ranges and allow for interspecies spread (Liebert et al.

1999). Plasmids are extra-chromosomal DNA elements that range in size from 300 bp-

2400 kbp and are found in both Gram-positive and Gram-negative bacteria (Kado 1998).

Transposons are mobile genetic elements which are composed of two sets of inverted repeats, genes required for transposition and other accessory genes of variable function.

These genetic elements have the capacity to move from one location to another within and between chromosomal and extrachromosomal DNA and can often mobilize adjacent pieces of DNA thus aiding in horizontal gene transfer (Harbottle et al. 2006). Integrons have been observed within transposons, making transposition of transposons an important mechanism in the transfer of AMR and gene cassettes between bacteria as well as in the accumulation of multi-resistance within a bacterial population (Fluit and Schmitz 1999).

33 9. Proposal

This project consists of three parts; 1) a study looking for the presence of class 1 and/or class 2 integrons in M. haemolytica from beef cattle at feedlots in Western

Canada, 2) a study which characterizes the variable regions of class 1 integrons in generic fecal E. coli from beef cattle at feedlots in Western Canada and analyzes the frequency and distribution of the gene cassettes, and 3) a study to characterize the genetic environment surrounding the 'lonely integrase' in generic fecal E. coli from beef cattle at feedlots in Western Canada.

Objectives. The first objective is to determine if class 1 and/or class 2 integrons are present in M. haemolytica isolated from beef cattle. If class 1 or class 2 integrons are found to be present within M. haemolytica they will be characterized to determine which gene cassettes are present and what gene variants may exist. The gene cassettes and gene variants will be compared to those that are found in generic fecal E. coli to assess what similarities exist. The second objective is to characterize the variable regions of class 1 integrons found in generic fecal E. coli isolated from beef cattle, to determine which gene cassettes and what gene variants may exist and what their distribution is. The third objective is to characterize the surrounding gene environment of the 'lonely integrase', assessing its location and linkage to antimicrobial resistance genes. This information will be used to help determine if the 'lonely integrase' is ancestral to the class 1 integrons or if the gene is part of a modified integron which derives from class 1 integrons and lacks the typical 3' conserved region.

34 Rationale. Integrons are natural genetic cloning and expression structures, capable of harbouring and transferring genes which confer AMR to bacteria. Integrons play a very important role in the dissemination of AMR gene cassettes and multiresistance (Fluit and Schmitz 1999), however currently little is known about the role of class 1 and class 2 integrons in the spread of AMR in beef cattle. By determining the presence of integrons in M. haemolytica, an opportunistic pathogen of cattle, new insights may be gained on the spread of AMR in M. haemolytica and on the transfer or lack of transfer of resistance determinants between unrelated bacterial genera and families may be gained. It may also provide some insight on the relationships and exchange of genetic material between bacteria from the respiratory and gastro-intestinal tract of cattle. The characterization of gene cassettes can be used to study trends in the dissemination of

AMR genes within generic fecal E. coli and pathogenic bacteria. The gene cassettes can also be compared at the sequence level to assess the frequency of recombinations within integrons and the spread of integrons across broad bacterial populations as well as assess if the sequence diversity is high enough to be used as a tool to trace integron distribution and dispersal. Understanding the genetic environment of the 'lonely integrase' may give insight into integron evolution and/or integron modifications to the 3' end. Since the

'lonely integrase' appears to be much more frequent in beef cattle than the integrase of standard class 1 integrons, this research may provide an explanation for this widespread distribution.

Approach. For the first part of this study, the collection of M. haemolytica from nasal contents of beef cattle in feedlots of Western Canada, collected between 2007 and

2009, was sampled. The sample (n=200) consisted of 45 M. haemolytica isolates selected

35 using a formal randomisation process and 155 M. haemolytica isolates that had an MIC to sulfonamides of > 128 ng/uL using broth microdilution or an inhibition zone diameter of

6 mm on a sulfonamide disk diffusion plate. By selecting isolates that display resistance to sulfonamides it is more likely to select for the presence of integrons, due to the association between sul genes and integrons. This sample will be used to determine the presence of class 1 and class 2 integrons by DNA-DNA hybridization. PCR was not chosen for this approach, as this method relies on the use of specific DNA primers for the amplification of the gene of interest. By using the specific primers, it may not be possible to identify all intll gene variants, especially those that have base pair mutations within the regions to which the primers anneal. By using the DNA-DNA hybridization techniques, with DIG-labelled probes for the intll and int!2 genes, the stringency of the reaction can be manipulated to capture less closely related gene variants. For the second and third parts of this study, generic E. coli recovered from feces of beef cattle in feedlots in Western Canada, collected between 2008 and 2009, was sampled (n=1000). This sample will be used to determine the presence of the class 1 integrase gene, intll.

Isolates which contain the intll gene will be further studied to determine if they carry either typical class 1 integrons or the 'lonely integrases' element. For the former ones, the gene cassettes present in the variable region of the integrons will be characterized by

DNA sequencing of PCR products. The genetic environment of lonely integrase genes will be analyzed and comparisons between isolates will be done to assess its diversity.

Materials and Methods. Dot-blot DNA hybridization will be used to detect the presence of class 1 and/or class 2 integrons within the M. haemolytica isolates with DIG- labelled intll and intll probes using standard protocols. The stringency of the

36 hybridization reactions will be lowered in order to detect intll/intU genes which are not closely related to those of E. coli. Isolates which are positive for intll and/or intI2 will undergo further genetic characterization, leading to the sequencing of the variable regions to determine which AMR genes are present. Polymerase chain reaction (PCR) will be used to detect the presence of classical intll within generic fecal E. coli isolates, using primers and protocols from literature (Sandvang et al. 2002). The variable region of isolates testing positive for intll will be determined as above. DNA sequencing of the amplified variable regions will be performed to identify the gene cassettes and their variants inserted in these integrons. From the isolates which are positive for intll but have no amplifiable variable region, a sample will be further analyzed to determine the location of the intll gene and the genetic environment surrounding the 'lonely integrase'.

Southern blots will provide information on the presence of the 'lonely integrase', whether it is chromosomal or plasmid-borne. If the gene is found to be plasmid-borne, then primer walking of the plasmids it is located on will be conducted. If the gene is found to be within the chromosome, then various cloning techniques will be employed to determine the surrounding genetic environment.

Expected Outcome. This study will provide insight into the dissemination of integrons and the gene cassettes which confer AMR in generic fecal E. coli frombee f cattle at feedlots in Western Canada. It will provide more information on the potential for the spread of integrons between the enteric bacterial reservoir and bacteria from the respiratory tract such as M. haemolytica. The AMR gene cassettes which are found in this study will be compared to AMR genes found in E. coli in other food animals and in the environment to attempt to track and monitor their spread and distribution at a national

37 and global level. Understanding the genetic environment of the lonely integrase gene, will further improve our overall understanding of integron evolution and/or integron modification. Understanding the dissemination of integrons among different bacterial species, the epidemiology of AMR gene cassettes and the evolution of integrons may eventually aid in the development of possible AMR prevention and intervention strategies.

38 CHAPTER TWO: AN INVESTIGATION AND

CHARACTERIZATION OF CLASS ONE INTEGRONS IN

ESCHERICHIA COLI AND MANNHEIMIA HAEMOLYTICA

ISOLATED FROM FEEDLOT CATTLE IN WESTERN CANADA

ABSTRACT Objectives: To determine the frequencyan d characterize the diversity of gene cassettes of class 1 integrons in generic fecal E. coli and in M. haemolytica isolated from beef cattle in four feedlots in Western Canada.

Methods: DNA-DNA hybridizations were used to detect the intll and intll genes in

M. haemolytica. PCR was used to determine the frequency of the integrase (intll) gene and of class 1 integron in E. coli. The variable regions of the class 1 integrons were characterized by DNA sequence analysis.

Results: Whereas the intll gene was detected at a high frequency in E. coli, a low prevalence of complete typical class 1 integrons was observed. Neither intll nor intI2 were detected in M. haemolytica. A diversity of gene cassettes and gene arrays were identified inE. coli: aadAl, aadA2, aadA7, aadA12, aadB-aadA2, blaoxA-2-orfD, dfrAl- aadAl, dfrAl-aadA5, and dfrA12-orfF-aadA2. The intll and class 1 integrons were found in 11.8% and 3.1%, respectively, of E. coli from beef cattle.

Conclusions: This study provides baseline information on the prevalence of class 1 integrons and the diversity of gene cassettes in Canadian beef cattle as well as insight to class 1 integron dynamics within beef cattle feedlots. No correlation was observed between M. haemolytica and E. coli. The frequentdetectio n of the intll gene in the absence of the typical 3' conserved segment warrants further examination. 39 INTRODUCTION

Integrons are dynamic genetic elements capable of capturing and expressing a variety of mobile gene cassettes (Fluit and Schmitz 1999, Partridge et al. 2009). The major antimicrobial resistance (AMR) integrons (i.e. class 1 and 2 integrons) are frequently associated with highly mobile genetic elements, which accounts for their

global distribution within a broad range of bacterial hosts (Partridge et al. 2009). A large variety of gene cassettes have been described from class 1 integrons, that confer resistance to P-lactams, aminoglycosides, chloramphenicol, erythromycin, fosfomycin,

lincomycin, quaternary ammonium compound family antiseptics, rifampin,streptothrici n

and trimethoprim (Mazel 2006, Partridge et al. 2009). Gene cassettes associated with

class 2 integrons confer resistance to erythromycin, streptothricin, aminoglycosides and trimethoprim (Mazel et al. 1998, Ramirez et al. 2005). Integrons are major players in the

development and spread of multi-drug resistance among bacteria and up to seven gene

cassettes have been observed simultaneously in a single class 1 integron (Carattoli 2001).

E. coli is a useful indicator of AMR since it can be isolated consistently from the

feces or intestine of healthy as well as diseased animals. It has been shown that E. coli

can also act as a reservoir of AMR genes, and that these genes may be transferred to

pathogenic bacteria within the gastrointestinal microflora (Aarestrup and Wegener 1999).

Mannheimia haemolytica is a commensal organism which colonizes the mucosal

membranes of the nasopharynx of healthy ruminants (Rice et al. 2007). As key a

component of the BRD complex, it may also cause pneumonia or exacerbate a current

infection (Quinn et al. 2002).

40 Resistance to a variety of antimicrobials that are associated with integrons such as ampicillin, sulfonamides and trimethoprim, has been observed in M. haemolytica isolates

(Hendriksen et al. 2008). Although integrons are widespread in E. coli, they have never been reported in M. haemolytica. Both M, haemolytica and E. coli can be isolated from beef cattle and although they colonize separate niches there is some opportunity for contact and genetic transfer in the upper respiratory and gastrointestinal tracts.

The Canadian Integrated Program for Antimicrobial Resistance Surveillance

(CIPARS) monitors the frequencyo f resistance in enteric bacteria of Canadian farm animals. However, there is little information available about the frequenciesan d

distributions of resistance genes in bacteria from cattle in general and, to date, only one

study has provided information on these genes in Canadian cattle (Gow et al. 2008a).

This study focused on calf-cow operations and no data are available at all on beef cattle.

To define baseline frequenciesan d to better understand AMR and its evolution in bacteria fromCanadia n beef and to complement the calf-cow operations data, genotypic

data, regarding AMR determinants, on beef cattle at feedlot operations are needed.

The objectives of this study were to first investigate the presence of class 1 and class 2 integrons in M. haemolytica and to then assess the frequency and diversity of gene

cassettes and of their variants within class 1 integron in generic fecal E. coli isolated from

feedlot beef cattle in Western Canada. Defining the diversity and frequencyo f gene

cassettes and combinations thereof in bacterial populations fromdivers e origins is a prerequisite to understanding their movements and distributions.

41 MATERIALS AND METHODS

Isolate selection. The isolates used in this study were systematically recovered from nasal swabs (M. haemolytica), and from composite fecal samples within feedlot pens

(975 E. coli) and from rectal samples of individual animals (1010 E. coli) collected by

Feedlot Health Management Services (Okotoks, Alberta, Canada) at four feedlots in

Alberta between 2007 and 2009. In some instances, the vast majority being from composite samples, several isolates were analyzed per sample. The isolation of M. haemolytica and E. coli from these samples has been described previously (Klima et al.

2010a, Klima et al. 2010b). A total of 200 M. haemolytica and 1991 E. coli isolates were used for the present study. Of these isolates 155 M. haemolytica and 725 E. coli were selected on the basis of reduced susceptibility to sulfisoxazole, as detected by either disk diffusion or minimum inhibitory concentration analysis (Klima et al. 2010a). All remaining isolates were selected using a formal randomization procedure from a culture collection of 1058 M. haemolytica and 3890 E. coli isolates after excluding the sulfisoxazole resistant bacteria from the list. Sulfonamide resistant isolates were purposely overrepresented in order to increase the likelihood of detecting class 1 integrons.

Preparation of DNA. The M. haemolytica isolates were grown overnight aerobically without shaking at 37°C in 1 mL of Brain heart infusion broth (Difco, Mississauga,

Ontario, Canada). Cultures were centrifuged and resuspended in the remaining 200 uL of broth before DNA extraction and purification. The M. haemolytica DNA for blotting and

PCR was prepared using the Agencourt® Genfind™ v2 Genomic DNA Isolation Kit, following manufacturer's instructions. The final concentration of DNA was standardized 42 to 100 ng/uL. The E. coli template DNA for PCR was prepared using a previously described method (Miserez et al. 1998), with 25 uL of an overnight culture grown at

37°C in Luria-Bertani broth (Becton Dickinson, Sparks, MD) mixed with 200 uL of lysis buffer. Both purified DNA and lysates were stored at -20°C until use.

Screening for integrase genes and integrons. Dot-blot hybridizations (Russell and

Sambrook 2001) using 100 ng total genomic DNA per dot were used to detect the intll

and intI2 genes in the M. haemolytica isolates. E. coli isolates were used as controls for the hybridization reactions, using strains which were known to possess or lack the intll

or intI2 genes. DIG-labelled probes were prepared using primers outlined in Table 1 and the Roche PCR-DIG probe synthesis kit. The stringency of the dot-blot hybridization

was lowered by increasing the salt concentrations of the washing solutions (final

concentration of 5x SSC), to allow probes to anneal to DNA sequences with a sequence

similarity as low as 73%. The intll gene of class 1 integrons was detected in E. coli

using a previously described PCR protocol [(Sandvang et al. 2002); Table 1], and lysates

prepared as mentioned above as template. All isolates which contained the intll gene

were further screened using previously described PCRs for the sulfonamide resistance

genes sull, sul2, and sul3 [(Kozak et al. 2009a); Table 1], and gene cassettes in the

variable regions of class 1 integrons [(Levesque et al. 1995); Table 1]. For negative

controls, isolates known to lack the intll, in all reactions E. coli DH10B and E. coli

ATCC25922 strains were used. Four previously characterized strains of E. coli were

used as positive controls; strains RL079 and 1329A possessing the intll and strains

EC01AB011052 and EC01AB040373 possessing the intI2.

43 Sequencing of class 1 integron variable regions. The PCR products of the integron variable regions were purified using the QIAquick PCR purification kit (Qiagen). Both strands were sequenced by a commercial service provider (Macrogen, Seoul, South

Korea), using the 5'-CS and 3'-CS primers (Table 1) followed by primer walking where needed. Sequence data was assembled using Sequencher Software version 4.5 (Gene

Codes Corporation, Ann Arbor, MI). Individual genes within gene cassettes were identified by searching the GenBank database of the National Centre for Biotechnology

Information using the blastn network service (Altschul et al. 1990). Gene variants were identified by aligning the nucleotide sequences using clustalW

(http://www.ebi.ac.uk/Tools/clustal2w/index.html).

Statistics. A Simpson's Diversity Index (Hunter and Gaston 1988) was calculated for the gene cassettes and their variants across all four feedlots. For statistical analysis, isolates from individual and composite samples were analyzed separately. The statistics software

Stata 11 (StataCorp LP, College Station, TX) was used to perform logistic regression analysis. The following independent variables were used in these models as fixed effects: feedlot, sulfonamide resistance selection (i.e. isolates selected on the basis of sulfonamide resistance vs. isolates selected using a formal randomization procedure), and sampling time, while pen ID was used as a random variable. Occasionally pens were mixed and/or combined after the >60 days on feed (DOF) time point, which created complications when analyzing composite fecal sample. To correct for this the >60 DOF samples were pooled together with exit samples so that the 'sampling time' variable consisted of two time points: entry, >60 DOF.

44 RESULTS

Distribution of the intll and intI2 genes. Neither the intll nor the intI2 were detected among the 200 M. haemolytica tested despite the low stringency conditions used for hybridization, which would detect genes sharing at least 73% sequence identity. Thus, class 1 or 2 integrons are absent or at best rare in M. haemolytica from cattle in the feedlots under investigation (95% confidence interval is 0-1.8%). The intll gene was detected in 17.9% of the E. coli isolates tested. Isolates which contained both the intll gene and sull gene (n=l 10) were classified as containing a typical class 1 integron and represented 5.5% of all the isolates tested. After correction for over-representation of sulfonamide-resistant isolates (Appendix 1) 3.1% of all E. coli isolates from cattle in the four feedlots were carrying a class 1 integron (95% confidence interval is 3.103%-

3.105% (Dohoo et al. 2003) (Table 14)). No variable region could be amplified from eight of these 110 isolates possessing a typical class 1 integron (7.3%), while 247 intll - positive isolates (69.5%) did not contain a 5' conserved segment and lacked detectable gene cassettes, and is estimated to be found in 8.7% of E. coli isolated from these beef cattle. Further details outlining the frequencyo f these elements among the feedlots are shown in Table 2.

Cassette types and subtypes. Ten types of class 1 integrons with ten different resistance gene cassettes were identified in E. coli. These cassettes included the dihydrofolate reductases genes dfrA 1, dfrAll and dfrAl 7 conferring resistance to trimethoprim; the aminoglycoside adenyltransferase genes aadAl, aadA2, aadA5, aadAl and aadA12 for resistance to streptomycin and spectinomycin; the aadB gene for resistance to kanamycin,

45 neomycin, gentamicin, and tobramycin, and the pMactamase gene blaoxA-2 conferring resistance to aminopenicillins, isoxazolyl penicillins, and to penicillinase inhibitors. Two gene cassettes of unknown function, orfD and orfF, were also identified and matched

100% with gene sequences catalogued in GenBank (AJ295229.1 and FJ855131.1 respectively). The frequency of class 1 integrons and the gene cassettes detected in this study appeared not uniformly distributed among the four feedlots (Table 4). Isolates which possessed either the blaoxA-2 or dfrA cassettes were more frequently detected in samples taken at entry, 80% and 61.5% respectively, whereas isolates which possessed the aadA or aadB gene cassettes were more frequently detected in samples taken at exit,

55.7% and 79.6% respectively (Table 3). Gene cassettes can be subdivided into three levels. The most general is the generic cassette category derived from the function of its product (e.g. aadA, aadB, dfrA, etc.), then the type of cassette associated with variants of this gene product (e.g. aadAl, aadA2, dfrAl, dfrA2, etc.) and finally the most specific subtypes of each gene variant, based on its nucleotide sequence (e.g. variants 1, 2, 3 of aadAl). This study detected five types of aadA genes and three types of dfrA genes.

Three variants were identified within the aadAl type, two variants within aadAl and two within aadB.

Cassette Combinations. Four different cassettes were seen alone within a single integron (4 generic cassette types, 35 isolates). Five combinations of two cassettes (8 generic cassette types, 64 isolates) and 1 combination of three cassettes (3 isolates) were observed. More information regarding frequencies and number of gene cassettes in relation to the sampling time point can be seen in Table 4 and 5. The most frequent

46 cassette combinations were in decreasing order: aadB-aadA2; aadAl alone; aadA12 alone; dfrAl-aadAl; and blaoxA2-orjD.

Gene Associations. The presence of class 1 integron in E. coli was positively associated with the presence of sull (p<0.0001), both in composite and in individual samples and was negatively associated with sul2 (p=0.035) in isolates from individual samples.

However, as expected because of the association between sull and integrons, class 1 integrons weremore likely to be detected in the isolates that were selected based on resistance to sulfonamides (p<0.0001) (Table 7, Appendix 2 Figure 5). An association was also observed between isolates selected based on resistance to sulfonamides

(p<0.0001) and the presence of the intll in the absence of a sull and 3' conserved

segment. The intll found in the absence of sull and the 3' conserved segment was positively associated with the sul2 gene (p<0.0001) (Table 8, Appendix 2 Figure 6).

Diversity of Cassette Types. The overall Simpson's Diversity Index for gene cassette

combination diversity for all four feedlots was 76.6%, with the lowest diversity found on

feedlot 42 (DI=44.2%) and the highest diversity on feedlot 21 (DI=87.3%; Table 9).

Three variants of the aadAl gene were identified. Variant one was the most frequent, followed by variant two, and variant three (Table 4). Variants one and two differed by only one nucleotide and their amino acid sequences were identical. These two variants

are identical to the sequences with accession number GU731079.1 and EF539213.1 on

GenBank, respectively. Variant three, identical to the GenBank sequence with accession number HM989924.1, differed from variants one and two at 14 and 13 nucleotide positions, respectively. The amino acid sequence of variant three was truncated by two amino acids at the C terminus and had five substitutions within its first ten amino acids

47 (Appendix 3 Figures 7 and 8). Differences in amino acid sequence, particularly of such

extent, warrant a specific denomination other than aadAl and a new aadA type should probably be attributed to this variant. Three variants of the aadB-aadA2 gene array were

identified. Variant one (accession no. FJ855133.1) was most frequent while both variant two (accession no. HQ148722.1) and three (accession no. FJ855134.1) were found in

only one isolate each (Table 4). The nucleotide sequences of the three variants were

almost identical, each with one single unique nucleotide substitution. Variant one's

substitution was within the aadB gene, variant two's substitution was not within a coding

region and occurred before the aadB start codon and variant three's substitution was within the aadA2 gene (Appendix 3 Figure 9). The AadB amino acid sequences were

identical for all three variants. The AadA2 amino acid sequences of variants 1 and 2 were

identical, but variant three contained a threonine residue at position 88, which both

variant one and two lacked (Appendix 3 Figures 10 and 11).

Correlation between genotype and phenotype. The vast majority (109/110) of the

intll positive isolates with a sull gene were resistant to sulfisoxazole (SOX). In a few

instances, resistance cassettes were identified in isolates that were susceptible to the

corresponding antimicrobial. The main discrepancy was between the presence of an

aadA gene and resistance to streptomycin, where 27 of 97 isolates were classified as

susceptible. The presence of the aadB gene conferred resistance to kanamycin in all but

three isolates (3/49) and resistance to gentamicin in all but four isolates (4/49). All five

isolates carrying the blaoxA-2 gene were susceptible to amoxicillin-clavulanic acid, yet

resistant to ampicillin. One out of thirteen isolates carrying the dfrA gene was susceptible

to trimethoprim/sulphamethoxazole (Table 6).

48 Logistic regression analysis. Class 1 integrons seemed to be in the highest frequency in samples taken after the >60 DOF time point. These genetic elements were observed on all four feedlots, however it appeared that their distribution was somewhat biased towards feedlot 42. When taking other cofactors and variables into account, no significant association was observed between class 1 integron frequency and feedlot (p>0.098) or the amount of time spent on a feedlot prior to sampling (p>0.92) in either composite or individual samples (Table 2, Appendix 2 Figure 5). The intll gene which was not associated with a 3'CS was significantly more frequent in samples taken at more than 60

DOF (p<0.004) in both composite and fecal samples and was negatively associated with feedlot 31 (p=0.001) in composite samples. No significant statistical association was observed between isolates possessing an intll gene in the absence of the 3'CS and resistance to chloramphenicol, streptomycin, sulfizoxazole or tetracycline frombot h individual and composite samples. (Table 2, Appendix 2 Figure 6).

DISCUSSION

Class 1 integrons are important in the study of the epidemiology of resistance because of their ability to harbour multiple AMR genes and the ability to disseminate

AMR. Despite an estimated overall intll prevalence of 11.8% in E. coli from animals of the four feedlots investigated, neither intll nor the intI2 genes were detected in M. haemolytica. We originally hypothesized that there might be genetic exchange, which would include integrons, between these two organisms since they colonize the same host.

However this study provides no supporting evidence and is in agreement with similar negative results from other researchers (S. Schwarz, personal communication). The reason for the lack of integrons in M. haemolytica is unknown but may be a result of 49 plasmid host range and incompatibility. Another possibility is that the selective pressures which drive the spread of AMR and/or integrons in E. coli are not common in M. haemolytica and therefore integrons and associated AMR cassettes are not important for the survival of M. haemolytica in its host. This later explanation is supported by the overall low prevalence of AMR observed in the M. haemolytica isolated fromth e feedlots investigated here (T. McAllister, personal communication). In vitro conjugation experiments between E. coli and M. haemolytica isolates could be conducted to elucidate parts of these questions. This would help assess for the potential of integrons to spread into this organism and to assess the need to devise guidelines to maintain the present situation in M. haemolytica with regards to integrons and multiresistance.

The overall prevalence in E. coli of the intll gene which lacked detectable gene cassettes and was not associated with a sull gene was unexpectedly high, in this study.

To the best of our knowledge, this phenomenon has not been described at such a high frequency but has been observed in a few isolates (Guerra et al. 2006, Saenz et al. 2010).

Modified class one integrons lacking only the sull gene (Carattoli 2001) or completely lacking the 3' CS have been observed in both primitive integrons (Gillings et al. 2008) and integrons from porcine isolates (Bischoff et al. 2005). While the 'lonely integrase', an intll gene lacking a 3'CS has been reported in E. coli isolates (Vinue et al. 2008,

Hillyer 2009), it has not been observed in such high frequencies.

As depicted in Table 2, the distribution of both class 1 integrons and gene cassettes varied upon each feedlot. Only one of the gene cassettes (aadAl) was detected in all four feedlots. It is unclear why these gene cassettes follow the observed pattern of

distribution. It was also interesting that the number of empty integrons was very

50 unevenly spread, with the vast majority (seven of eight) being found on a single feedlot.

The difference in gene cassette distribution may be present at entry, however larger numbers may be needed to test this hypothesis. Further analysis needs to be conducted using antimicrobial use data, to determine whether certain antimicrobial agents are selecting and/or co-selecting for these gene cassettes.

All gene cassettes identified in the scope of this study have been previously identified in class 1 integrons from E. coli in cattle (Hillyer 2009). Overall, three variants were observed for both the aadAl and aadB-aadA2 gene cassettes. In both cases, the majority of the variants fell within one group and were detected on three of the four feedlots. The diversity indices calculated for each feedlot separately ranged from 0.44 to

0.87. These indices indicate that the diversity of the gene cassette combinations within each feedlot is variable. Rare variants can be used to understand integron dynamics within a feedlot. Once detected, a rare variant may be used as a target, to look for the transmission and spread of resistance in E. coli fromanima l to animal within a pen but also across pens and throughout an entire feedlot. The Simpson's diversity index calculated for all gene cassette variants from all four feedlots was 0.766 which denotes a relatively high level of diversity.

Although the-majority of the isolates displayed a phenotype which matched the genotype, a number of isolates were observed to carry a resistance gene but were classified as susceptible. The main discrepancy occurred with the aadA and resistance to streptomycin, where approximately a third of the isolates possessing the aadA were observed to be susceptible. It does not appear that the position of the aadA gene was related to this discrepancy as the majority (19/27) of the susceptible isolates had class 1

51 integrons with this gene cassette in the firstposition . The majority of the aadA genes which did not confer resistance belonged to the aadAl variant subtype 1. This issue has already been identified and discussed on several occasions (Morabito et al. 2002), and is related to the short range of streptomycin dilutions for MIC determination and to a misplaced streptomycin 'breakpoint" cut off for epidemiological rather than clinical purposes. Since these isolates were not screened for additional AMR genes, which are not part of class 1 integrons; it is difficult to know whether or not multiple streptomycin- resistance determinants are needed to reach such high MICs as those used here as breakpoints for streptomycin-resistance.

Logistic regression analysis showed for both composite and individual samples a positive association between the presence of class 1 integrons and sull, as well as with resistance to chloramphenicol and to streptomycin. The latter is likely related to the frequent presence of the aadA cassettes in integrons from beef cattle. However, the former cannot be related to the presence of a cmlA cassette since none of the 110 integrons investigated carried such a gene. Further investigations are needed to explain this association, which could possibly be due to a catA or floR gene (Schwarz et al. 2004) in the vicinity of integrons. The catA gene has been identified within the variable region of a class 1 integron, but to date no reports of floR within a class 1 integron have been made (Partridge et al. 2009). A negative association with sul2 was observed, suggesting at least a partial incompatibility between class 1 integrons and this other sulfonamide resistance gene. Although feedlot and sampling time did not have statistically significant associations to the presence or absence of class 1 integrons, they appeared to have some influence on other class 1 integron related features; however observations were different

52 among isolates from composite and individual samples and such observations may be the result of spurious associations only. As expected because of the presence of sull in the conserved region of class 1 integrons, the strongest association to detecting class 1 integrons was observed with the isolates that were purposely chosen for analysis on the basis of resistance to sulfonamides. A wider and more varied sample of feedlots should be examined when monitoring class 1 integrons to better understand their distribution.

Clustering at the feedlot level should be considered in frequencyanalyse s of integron distribution in beef cattle, but it may be less important to consider the effect of pens.

Although class 1 integron frequency was not shown to be significantly associated with the duration of time spent on a feedlot, this was not the case for the 'lonely integrase'. The 'lonely integrase' was found to significantly increase over time spent on a feedlot, in both individual and composite samples. This increase in frequency over time provides evidence to the hypothesis that antimicrobial use on feedlots positively selects for genetic elements which confer AMR. While these genetic elements lack gene cassettes, they have been associated with AMR to tetracycline, streptomycin,

sulfonamides and phenicols. By comparing this observed trend with the antimicrobial usage data from each feedlot, we may be able to determine which antimicrobials are contributing to the spread of the 'lonely integrase'.

In conclusion, this documents the distribution and diversity of major gene

cassettes in class one integrons recovered from E. coli in Canadian beef cattle. Class 1 integrons have a prevalence of 3.1% among E. coli isolated from western Canadian beef

cattle and contain four major types of gene cassettes (aadA, aadB, dfrA and blaoxA-i)-

Surprisingly, a 'lonely integrase' element is present in approximately 9% of E. coli

53 isolated from Canadian beef cattle. This unexpected finding warrants further investigation to determine why this genetic element is occurring so frequently. It is likely that this 'lonely integrase' is located within a mobile genetic element carrying AMR determinants other than integron cassettes such as sul2 and spreads both vertically and horizontally.

ACKNOWLEDGEMENTS

We would like to thank the Feedlot Health Management Services for performing fecal sample collection, Tim McAllister and collaborators for providing E. coli and M. haemolytica isolates, the Public Health Agency of Canada for antimicrobial susceptibility testing and Fiona Coutinho for technical support. This research project was funded by the Advancing Canadian Agriculture and Agri-Food (ACAAF) Program

54 Table 1 - PCR primers used for PCR in this study.

Gene Primer Sequence (Forward/Reverse) Annealing Product Reference

temperature size (bp) CO intll 5 '-CGGAATGGCCGAGCAGATC 68 850 (Sandvang et

al. 2002) 5 '-AAGGTTCTGGACCAGTTGCG intI2 5'-TTAGGCGCGTGGGCAGTAG 68 400 (Sandvang et

al. 2002) 5'- GTCATCCTCAGACCATGGGC

5'-3' 5'-GCATCCAAGCAGCAAG 59 900- (Levesque et

Variable 1500 al. 1995) 5'-AAGCAGACTTGACCTGA Region sull 5'- CGGCGTGGGCTACCTGAACG 66 433 (Kozak et al.

2009a) 5'- GCCGATCGCGTGAAGTTCCG sul2 5'- CGGCATCGTCAACATAACCT 66 721 (Kozak et al.

2009a) 5'- TGTGCGGATGAAGTCAGCTC sul3 5'-CAACGGAAGTGGGCGTTGTGGA 66 244 (Kozak et al.

2009a) 5'- GCTGCACCAATTCGCTGAACG

55 Table 2-The distribution of intll gene, class 1 integron and 'lonely integrase' in generic E. coli in regards to type of sampling, timing of sample and feedlot.

Total Sampled Integrase Integron 'Lonely' Integrase

Individual 1010 162 (16%) 65 (6.4%) 97 (9.6%)

Composite 975 195 (20%) 45 (4.6%) 150(15.4%)

Entry 842 107 (12.7%) 29(3.4%) 78 (9.3%)

>60 DOF 268 64(23.9%) 21 (7.8%) 43 (16%)

Exit 874 186(21.3%) 60 (6.9%) 126 (14.4%)

Feedlot 5 679 107 (15.8%) 26 (3.8%) 81(11.9%)

Feedlot 21 683 132 (19.3%) 23 (3.4%) 109(15.9%)

Feedlot 31 279 15 (5.4%) 6 (2.2%) 9(3.2%)

Feedlot 42 344 103 (29.9%) 55 (15.9%) 48 (13.9%)

Overall 1985 357 (17.9%) 110(5.5%) 247 (12.4%)

Adjusted

Prevalence** 4890 357(11.8%) 110(3.1%) 247(8.7%)

All E. coli were isolated from beef cattle in one of four feedlots. Fecal samples were collected from individual animals or composite samples at entry, greater than 60 days on feed (DOF) and at exit.

**Prevalence was adjusted to take isolate selection based on sulfonamide resistance into consideration.

56 Table 3 - Gene cassettes present within a class 1 integron in generic E. coli from beef cattle in feedlots collected at entry, >60 DOF and at exit.

aadA aadB bldoXA-2 dfrA

Entry 24 1 4 8

>60 DOF 19 9 0 1

Exit 54 39 1 4

Feedlot 5 24 6 2 1

Feedlot 21 20 1 2 10

Feedlot 31 5 0 1 2

Feedlot 42 48 42 0 0

Overall 97 49 5 13 Numbers represent the actual numbers of integrons carrying these gene cassettes

57 Table 4- Distribution of gene cassettes and variants within class 1 integrons of

generic E. coli from beef cattle from four different feedlot operations in Alberta,

Canada.

Gene Cassette Total (n=l 10) Feedlot 5 Feedlot 21 Feedlot 31 Feedlot 42 aadAl 26 IT 8 I (5 variant 1 20 11 3 - 6 variant 2 5 -41- variant 3 1 - 1 - - aadA2 1 1 - - - aadA7 1 1 aadA12 7 5 - 2 - aadB-aadA2 49 6 1 - 42 variant 1 47 6 1 - 40 variant 2 1 - - 1 variant 3 1 - - 1 blaoxA-2-orfD 5 2 2 1 - dfrAl-aadAl 6 5 1 - dfrAl-aadA5 2 2 - - dfrAl7-aadA5 2 1 1 - - dfrAl 2-orfF-aadA2 3 2 1 - no detectable cassette 8 1 _ 7

Total: 110 26(3.8%) 23(3.4%) 6(2.2%) 55(15.8%)

Total Sampled: 1985 681 683 279 348

58 Table 5 - Distribution of the number of gene cassettes present within class 1 integrons in generic E. coli from beef cattle in feedlots collected at entry, >60 DOF and at exit.

Overall Entry >60 DOF Exit

0 cassettes 8 1 (12.5%) 2 (25%) 5 (62.5%)

1 cassette 35 15(42.9%) 9(25.7%) 11(31.4%)

2 cassettes 64 11(17.2%) 10(15.6%) 43(67.2%)

3 cassettes 3 2(66.7%) - 1(33.3%)

59 Table 6 - Susceptibility testing results and genotypes for major gene cassette types identified in class 1 integrons

Gene Cassettes AMC AMP FOX CRO CHL GEN KAN NAL STR SOX TCY SXT

aadB:aadA2 46/49 45/49* 46/49* 43/49* 49/49 45/49

aadAl 9/26 2/26 1/26 10/26* 26/26 24/26

aadA2 1/1 1/1 .* 1/1 1/1 1/1

aadAl2 5/7 6/7* 7/7 7/7 -

dfrAl: aadAl 5/6 5/6* 6/6 6/6 5/6*

blaoxA-2 5/5* - 5/5 4/5 -

dfrAl :aadA5 1/2 2/2* 2/2 2/2 2/2* dfrA12:orf:aadA2 1/3 1/3 1/3 1/3 3/3 3/3* 3/3 3/3 3/3*

dfrAl7:aadA5 1/2 2/2 1/2 1/2* 2/2 2/2 2/2*

aadAl .* 1/1 1/1 .

Numbers in each cell of the table represent isolates resistant to antimicrobial / isolates with gene cassette. Resistance

phenotypes were determined for all isolates using broth microdilutions and clinical MIC breakpoints (CLSI 2006).

* denotes an expected resistance to the antimicrobial conferred by the gene cassette.

60 Table 7 - The logistic regression analysis outputs for associations between class 1 integrons, sulfonamide resistance

genes, major gene cassettes and phenotypic resistance.

Outcome Class 1 Integron Feedlot 21 Feedlot 31 Feedlot 42 Exit Selection based on Sul

Composite

Samples

1230.1 1.4 2.9 0.7 0.8 1.8

mil (281.2-5381.0) (0.5-3.9) (1.1-7.8) (0.2-2.2) (0.4-1.5) (0.9-3.3)

0.7 1.5 2.6 1.7 2.1 152.1

sul2 (0.3-1.4) (0.6-3.85) (1.1-6.5) (0.7-4.2) (1.2-3.4) (76.6-302.0)

N/A 0.4 0.4 0.2 5.9 0.4

aadA (0-3.3) (0-7.6) (0-2.6) (0.6 -62.6) (0-4.4)

13.6 1.2 0.9 1.2 0.9 8.6

CHLR (7.8-23.8) (0.6-2.1) (0.5-2.1) (0.6-2.6) (0.6-1.3) (5.8-12.8)

3.1 1.6 1.1 1.9 0.9 8.2

STRR (1.9-5.1) (1.1-2.4) (0.7-1.8) (1.2-2.9) (0.7-1.1) (6.2-10.8)

0.7 2.4 1.8 1.5 12.8 37.7

TCYR (0.3-1.8) (1.6-3.6) (1.0-2.9) (0.8-2. 6) (9.26-17.9) (20.5-69.1)

Individual

Samples

4248.4 4.4 7.8 0.5 0.3 4.1

sull (418.8-43101.9) (0.7-26.5) (1.3-45.2) (0.1-3.9) (0.1-0.9) (1.5-11.2)

sul2 0.3 0.6 2.2 0.9 2.3 411.8

61 (0.1-0.9) (0.1-2.6) (0.5-8.9) (0.3-3.8) (0.9-6.1) (112.2-1511.2)

16.9 0.9 0.9 1.9 1.6 7.4

CHLR (7.9-36.4) (0.4-2.1) (0.3-2.9) (0.8-5.0) (0.8-3.1) (3.89-14.5)

3.9 1.6 2.1 2.9 1.8 7.4

STRR (1.9-7.6) (0.9-2.8) (1.14.1) (1.6-5.1) (1.1-2.8) (4.8-11.3)

0.3 1.1 2.0 0.9 38.6 170.6

TCYR (0-1.2) (0.6-2.2) (0.8-5.1) (0.3-2.2) (20.1-74.0) (48.2-603.2) Table displays the odds ratio and confidence interval in brackets. Confidence intervals which do not include 1.00 denote statistically significant results.

Logistic regression analysis failed for the following outcomes due to colinearity or an insufficient number: sul3, aadA, aadB, dfrA, bla0xA-2 and SULR.

62 Table 8 - The logistic regression analysis output for the 'lonely integrase' specifically calculated for the sulfonamide

resistance genes, major gene cassettes and phenotypic resistance.

Outcome Lonely' Integrase Feedlot21 Feedlot31 Feedlot 42 Exit

Composite

Samples

0.3 0.8 0.7 0.6 0.9 4.4

null (0.2-0.7) (0.36-1.87) (0.3-1.6) (0.3-1.5) (0.6-1.4) (2.7-7.0)

3.8 1.5 3.1 1.8 1.9 143.7

sul2 (1.6-8.7) (0.59-3.82) (1.2-7.9) (0.7-4.6) (1.2-3.3) (71.2-290.3)

1.8 1.0 0.9 1.5 0.9 8.5

CHLR (1.2-2.7) (0.59-1.74) (0.5-1.9) (0.8-2.9) (0.7-1.3) (5.8-12.5)

0.8 1.6 1.0 1.9 0.9 9.2

STRR (0.6-1.2) (1.08-2.37) (0.6-1.7) (1.2-3.2) (0.7-1.1) (6.9-12.1)

5.9 2.3 1.8 1.5 12.3 30.4

TCYR (2.2-15.6) (1.56-3.49) (1.1-3.1) (0.9-2.7) (8.8-17.2) (16.8-55.1)

Individual

Samples

0.8 0.6 0.7 0.3 0.6 7.8

sull (0.3-1.9) (0.2-2.3) (0.2-2.6) (0.1-1.1) (0.3-1.4) (3.5-17.2)

9.3 0.9 3.4 1.3 2.0 484.9

sul2 (2.3-37.9) (0.2-4.2) (0.7-15.9) (0.3-5.8) (0.7-5.5) (113.9-2063.7)

SULR 1.1 0.5 2.2 1.2 2.1 26126.5

63 (0.7-18.4) (0.1-4.6) (0.2-21.3) (0.1-12.6) (0.4-11.8) (3854.8-177078.(

1.2 0.7 0.8 2.1 1.5 9.2

CHLR (0.6-2.2) (0.3-1.6) (0.3-2.2) (0.9-4.9) (0.8-2.8) (4.9-17.3)

0.6 1.5 1.8 2.9 1.8 8.6

STRR (0.4-1.1) (0.9-2.5) (0.9-3.5) (1.6-5.2) (1.2-2.9) (5.6-13.1)

5.1 1.1 2.0 0.9 34.0 119.6

TCYR (0.9-29.4) (0.6-2.2) (0.8-5.0) (0.4-2.3) (17.8-64.9) (38.9-367.8)

Table displays the odds ratio and confidence interval in brackets. Confidence intervals which do not include 1.00 denote statistically significant results. Logistic regression analysis failed for the following outcomes due to colinearity or an insufficient number: sul3, aadA, aadB, dfrA and

bla0xA-2-

64 Table 9 - The Simpson's Diversity Index calculated for all gene cassettes and their variants.

Feedlots Diversity Index Lower 95% CI Upper 95% CI

All Feedlots 0.766 0.764 0.768

Feedlot 5 0.722 0.716 0.727

Feedlot21 0.873 0.872 0.875

Feedlot 31 0.778 0.769 0.786

Feedlot 42 0.442 0.431 0.453

65 CHAPTER 3: CHARACTERIZATION OF A CLASS 1 INTEGRASE

GENE ASSOCIATED WITH TETRACYLINE RESISTANCE IN

ESCHERICHIA COLI ISOLATED FROM CANADIAN BEEF CATTLE

ABSTRACT

Objectives: The objective of this study was to determine the genetic environment flanking a 'lonely integrase' (intll) gene not associated with the 3' conserved segment of typical class 1 integrons in E. coli.

Methods: Long range and inverse PCRs were used to elucidate the immediate genetic environment of the 'lonely integrase'. A series of overlapping PCRs were used to screen the collection of isolates which possess a 'lonely integrase' to assess how conserved and widespread this element is.

Results: The 'lonely integrase' is a new unique genetic element containing two identical intll gene copies separated by an intact tet(M) gene. This element is flanked by a

Tni 721 transposon, which carries a tet(A) gene. Both tet(M) and tet(A) confer resistance to tetracyclines, and have not previously been reported together in E. coli.

Conclusions: This study describes a unique genetic element common in generic fecal E. coli of Canadian beef cattle that is likely a modified integron. Further investigations should assess the functionality of both tet(M) and tet(A) genes to determine whether the bacterium gains a selective resistance advantage by harbouring both genes.

66 INTRODUCTION

Integrons are genetic elements which capture and express mobile gene cassettes conferring resistance to a wide array of antimicrobial agents (Fluit and Schmitz 1999,

Partridge et al. 2009). Class 1 integrons are major players in antimicrobial (multi-) resistance and are frequently associated with mobile genetic elements, which leads to their wide distribution among a broad range of bacterial hosts (Partridge et al. 2009). A typical class 1 integron possess a 5' conserved segment, composed of the integrase gene intll and a 3' conserved segment which contains the AqacEDl and sull genes (Fluit and

Schmitz 1999).

The boundaries of many class 1 integrons are defined by the presence of inverted repeats for Tn¥02-mediated transposition, implying that the immediate common ancestor of class 1 integrons was similar to the Tn402 transposon (Gillings et al. 2008). However, some bacteria possess class 1 integrons which lack the Tn402 inverted repeats and are located within the bacterial chromosome. This suggests that the common ancestor of class 1 integrons could be chromosomal (Stokes et al. 2006) and became plasmid-borne during a mobilization event involving the Tn402 transposon (Gillings et al. 2008). A class 1 integron, designated In2, occurs within the Tn2i transposon and is flanked by imperfect inverted repeats. A 5-bp duplication of the target sequence for transposition suggests that the In2 transposed into an ancestral transposon to create the Tn2i (Liebert etal. 1999).

Modifications have been observed within the 3' conserved segment of class 1 integrons, usually, but not only, involving the replacement of the sull gene with sul3

67 and/or the replacement of the qacEAl gene with the qacH (Bischoff et al. 2005, Sunde et al. 2008, Vinue et al. 2008, Soufi et al. 2009). Thus integrase genes from class 1 integrons can occasionally be found in the absence of the typical sull, qacEAl and

ORF5.

A preliminary study on class 1 integrons in Western Canada found the intll gene in approximately 14% of fecal E. coli frombee f cattle, however close to 80% of these isolates did not possess a typical class 1 integron. The intll gene of these isolates was neither associated with the 3' conserved segment nor with common resistance cassettes of class 1 integrons and was dubbed the 'lonely integrase' gene. Although no integron associated tetracycline resistance cassettes have been reported, the isolates possessing this 'lonely integrase' were more frequently resistant to tetracycline and chloramphenicol than other isolates (Hillyer 2009).

The objective of this study was to characterize the genetic environment of the

'lonely' integrase in generic fecal E. coli from feedlot beef cattle in Western Canada. By assessing the location of the 'lonely integrase' and its linkage to AMR genes, we may gain insight into modifications and the evolution of integrons. We may also understand the reasons for the unexpected high frequencyo f the 'lonely integrase' in fecal E. coli from beef cattle.

MATERIALS AND METHODS

Bacterial isolates. All isolates used in this study were recovered frombot h composite fecal samples within feedlot pens (n=150) and fromrecta l samples of individual animals

(n=97) collected by Feedlot Health Management Services (Okotoks, Alberta, Canada) at

68 4 feedlots in Alberta during 2007-2009. For more information on bacterial isolates refer to the Materials and Methods section of Chapter 2.

Preparation of DNA. Template DNA for PCR was prepared using a previously described alkaline lysis method (Miserez et al. 1998), with 25 uL of an overnight culture grown at 37°C in Luria-Bertani broth (LB, Becton Dickinson, Sparks, MD) mixed with

200 uL of lysis buffer. The lysates were stored at -20°C. Total DNA and plasmid DNA, for Southern blot analysis, were prepared following manufacturer's instructions using the

Agencourt® Genfind™ v2 Genomic DNA Isolation Kit and the QIAGEN Plasmid Midi

Kit, respectively.

Identification of Lonely Integrase Isolates. To ensure that isolates lacked the 3' conserved segment, three overlapping PCRs were done covering the AqacEl, sull and or/5, using primers and conditions outlined in Table 10. Isolates which possessed the intll gene and lacked both an amplifiable variable region and 3' conserved segment

(n=247) were selected for further analysis. Negative controls included E. coli

ATCC25922 and DH10B and positive controls included E. coli RL079 and 1329A.

Southern blot analysis. To determine the location of the 'lonely integrase' gene,

Southern blot analysis (Sambrook and Russell 2001c) was conducted using plasmid

(QIAGEN Plasmid Midi Kit) and total DNA (Agencourt® Genfind™ v2 Genomic DNA

Isolation Kit [(Sambrook and Russell 2001c)) preparations from 10 isolates chosen to represent the different phenotypic groups of 'lonely integrase' containing isolates. DIG- labelled probes were synthesized using the PCR Probe DIG Synthesis Kit (Roche,

69 Mississauga, ON) using the intll primers (Sandvang et al. 2002) and conditions listed in

Table 10.

Preparation of transformants. Two plasmids, 120 kb (p260cD) and 140 kb (p299cD), containing the 'lonely' intll were transformed into ELECTROMAX® DH10B

Electrocompetent cells (Invitrogen) using a previously described protocol (Sambrook and

Russell 2001a). Transformants were grown overnight at 37°C on Mueller-Hinton agar

(Difco, Mississauga, Ontario Canada) containing 32 ug/mL of tetracycline.

Primer design, PCR and sequencing. All primers (Table 10 and 11) used in this study were designed using the Primer3 v.0.4.0, and sequencing reactions were conducted at the

University of Guelph Laboratory Services. Sequence data was assembled using

Sequencher Software version 4.5 (Gene Codes Corporation, Ann Arbor, MI). Genes were identified using the GenBank database of the National Centre for Biotechnology

Information using the blastn network service (Altschul et al. 1990). The 3' region of the

Tn27 transposon, common to class 1 integrons, was used as a starting point in determining the local genetic environment of the 'lonely integrase' (Table 10).

Cloning and screening clone library. The p299cD was digested with Kpnl (New

England Biolabs, Pickering, ON) following the manufacturer's instructions. The resulting plasmid fragments were inserted in the pJAZZ-OK cloning vector using the

BigEasy v2.0 Linear Cloning kit (Lucigen, Middleton, WI). Two hundred clones were selected and screened via PCR for the tet(A), floR, merA, orfAB, tniA and intll genes

(Table 10).

70 Inverse PCR. A 100ng/u.L aliquot of p299cD plasmid DNA was digested using

£co01091 (New England Biolabs) following the manufacturer's instructions and the resulting fragments circularized by overnight ligation at 4°C using T4 DNA Ligase

(Roche). An inverse PCR (Sambrook and Russell 2001b) was performed with the circularized fragments using primers in Table 10. The 6.5 kb PCR product was purified using the QIAQuick Gel Extraction Kit (Qiagen).

Screening 'lonely integrase' isolates with overlapping PCRs. Four overlapping PCRS were developed to screen the collection of 247 isolates which possessed the 'lonely' integrase, to determine how many of the isolates possessed a similar genetic element.

The conditions for each PCR reaction are listed in Table 11. Negative controls included

E. coli ATCC25922 and DH10B which lacked a class 1 integron and 'lonely integrase' and positive controls included the 260cD and 299cD isolates which were used to elucidate the structure of the 'lonely integrase'

Plasmid sequencing and Replicon Typing. Both plasmids, p260cD and p299cD, were sent for sequencing at the McGill University and Genome Quebec Innovation Centre facility in Montreal, Quebec. Replicon typing was conducted using a previously described methodology (Carattoli, 2005).

RESULTS

Correlation between phenotype and genotype. The 247 'lonely integrase' isolates were organized into eight groups, based on resistance profiles to chloramphenicol, streptomycin, sulfizoxazole and tetracycline. Resistance to tetracycline was the most frequent among these isolates (98%) whereas the least frequent resistance was to

71 chloramphenicol (29%). Twenty-one percent of the isolates were resistant to all four antimicrobials (Table 12).

Genetic environment of the 'lonely' intll. Southern blot analysis performed on plasmid and total DNA fromte n isolates showed that, depending on the strain, the 'lonely integrase' could be located on chromosomal (n=6) or plasmid DNA (n=4) (Figure 3).

The 'lonely integrase' was found on plasmids of two different sizes, (approximately

120 kb and 140 kb). Sequence data of the regions around intll (Figure 4), originating from primer walking, cloning, long range and inverse PCRs, showed that on both plasmids (p260cD and p299cD) there were two copies of the intll gene, both in the same orientation, separated by a tet(M) gene. Upstream of the integrase genes was a Tnl721 transposon which carries the tet(A) and tetR genes, while downstream of the integrase genes was a qacG gene (Figure 4).

Genetic similarity among 'lonely' intll isolates. The four overlapping PCRs covering the region shown in Figure 4 revealed that genetic differences exist around the 'lonely integrase' and that the structure elucidated in Figure 4 may not represent every intll gene that exists in the absence of the 3' conserved segment. Based on presence and absence of overlaps, the 247 isolates possessing a 'lonely integrase' formed 14 groups (Table 13). In total, 17 of the isolates did not share any similarities in the surrounding genetic environment of the 'lonely integrase', whereas 128 isolates had identical genetic environments. A total of 17 isolates shared at least one of the four regions, 23 isolates possessed at least two and 62 had at least three (Table 13). Further testing using long range PCR will further confirm the contiguity of fragments in these 14 groups.

72 Similarity between two plasmids carrying 'lonely integrase'. A list of identifiable genes found on the two partial sequences of plasmids p260cD and p299cD can been seen in Appendix 4 in Tables 15 and 16. Replicon typing revealed that both plasmids containing the 'lonely integrase' belong to different replicon types; p260cD belonging to the A/C type and p299cD belongs to the M type.

DISCUSSION

This study describes a unique genetic element and provides insight into the distribution and diversity of this genetic element in generic fecal E. coli in Canadian beef cattle. The 'lonely integrase' which has a prevalence of 8.7% among E. coli isolated from Canadian beef cattle, is associated with resistance to phenicols, streptomycin, sulfonamides and tetracyclines, and harbours two genes which confer resistance to tetracyclines, tet(A) and tet(M). It will be interesting to determine if there is a selective advantage given to a bacterium that harbours this combination of genes and if there is an interaction that occurs between these two gene products that would have a synergistic effect on tetracycline resistance.

The 'lonely integrase' is frequent in E. coli from Canadian beef cattle (Table 11;

Chapter 2). The mechanism of its dispersal is unknown, but although we did not conduct conjugation experiments to test their mobility, plasmids such as those examined here seem likely vehicles. The tnpA from the Tni 721 transposon flanking the 'lonely integrase' may also be another mobility determinant for its spread. The sequencing around the 'lonely integrase' did not reveal any inverted repeats, however these elements may be present further outside of the sequenced region. The presence of identical

73 sequences on two plasmids from different replicon types and on the chromosome of some strains strongly supports this hypothesis.

Since the tet(M) and tet(A) genes both confer resistance to tetracycline (Levy et al. 1999), it was surprising to see these two genes together on one genetic element.

Whether or not the presence of both tet genes provides a selective advantage for the host organism and therefore for the spread of the 'lonely integrase' at the same time warrants further investigation. The tet(A) gene found was linked to a Tni72i-like transposon, which is commonly observed in E. coli (Wiebauer et al. 1981, Allmeier et al. 1992).

However, the tet(M) gene is usually found within a TnPitf-like conjugative transposon, which is primarily found in Firmicutes and anaerobes (Roberts and Mullany 2009), but not frequentlyi f at all in Enterobacteriaceae. There was no trace of remnants of a Tn916 in the direct vicinity of tet(M) and of the 'lonely integrase'. It remains unclear how this gene became part of the plasmids carrying the 'lonely integrase' and what factors keep both tet genes together. However, tet(A) confers resistance to tetracyclines via an efflux pump whereas tet(M) encodes a ribosomal protection protein (Levy et al. 1999). In addition, the regulation mechanisms of these two genes are quite different (Su et al. 1992,

Saenger et al. 2000). Therefore, it is possible that if tet(M) is expressed in E. coli, some synergism may exist between these two genes by providing resistance to tetracycline in different ways and under different circumstances. The functionality and expression of both of these genes should be investigated further, and may provide clues as to why the

'lonely integrase' genetic element is so prevalent among Canadian beef cattle.

Understanding how and when these two genes are expressed and if there is a synergistic

74 interaction between the two gene products, may help better antimicrobial uses in feedlot practices.

The four overlapping PCRs and the plasmid sequencing results showed that while the 'lonely integrase' exists as a complete genetic element, as shown in Figure 4, there exists some diversity within the structure. The overlapping PCRs identified 14 groups containing different combinations of the 'lonely integrase' structure with the most frequent group being the complete 'lonely integrase' genetic element described earlier.

These variants may have been generated by recombinations, deletions or insertions which disrupted the primer's annealing site or changed the length of the PCR product. It is also possible that gene cassettes may have been inserted downstream of either of the intll genes within the attl site. This hypothesis may be tested by using isolates which possessed a 'lonely integrase' but lacked the intll-tet(M) and/or tet(M)-qacG region, and developing PCRs which would amplify the downstream region of the intll genes looking for inserted gene cassettes.

The most frequent resistances observed among the E. coli isolates possessing a

'lonely integrase' were for chloramphenicol, streptomycin, sulfonamides and tetracycline. The partial sequences of two large plasmids carrying the 'lonely integrase' both contained the floR, strA/B, sul2, in addition to the tet(A) and tet(M) genes which confer resistance exactly to the antimicrobials mentioned above. While not all of these genes are found in the sequence presented here, it is possible that these genes are flanking it and are part of the mobile element which includes the 'lonely integrase'.

75 It is likely that only a part of the mobile element associated with the 'lonely integrase' has been sequenced here, and further sequencing would be needed to assess its boundaries. Despite its apparent frequency, the 'lonely integrase' has not been described before, and it would be interesting to know how widespread this genetic element is in other sources than beef cattle in Canada. Future studies should be aimed to detect the

'lonely integrase' in other major food animals, such as poultry and swine and in beef cattle from other countries. Future studies aimed at determining where the 'lonely integrase' originates from are also important, as well as knowing if it exists in other environments or if it is a product of the specific selection pressure present within a cattle feedlot. The functionality of the intll gene products should be assessed to determine if the 'lonely integrase' is capable of integrating mobile gene cassettes commonly identified in class 1 integrons.

ACKNOWLEDGEMENTS

We would like to thank the Feedlot Health Management Services for performing fecal collections, Tim McAllister and collaborators for providing E. coli isolates, the Public

Health Agency of Canada for antimicrobial susceptibility data, Jennie Pouget and Gabhan

Chalmers for technical support. This research project was funded by the Advancing

Canadian Agriculture and Agri-Food (ACAAF) Program.

76 Table 10 - Primers, PCR conditions and PCR products for the investigation of the

'lonely integrase'.

PCR Primer Sequence (Forward/Reverse) Annealing Product product temperature size (bp)

(°Q

Class 1 Integron components of Tn2? (accession no AF071413)

AqacEl- 5'-TTCATGGGCAAAAGCTTGAT 55 847 sull 5'-AAGAAGGATTTCCGCACAC

AqacEl- 5'-CATGGTGAGTGGGGCTTT 55 989 sull-orf5

5'- GTCGATATCACCCGAGCAG sull-orfi 5'-CGTGGGCTACCTGAACGATA 55 998

5 '-AAGTGTCGACGTGGGTGAAT intll 5'-CGGAATGGCCGAGCAGATC 68 850

5'-AAGGTTCTGGACCAGTTGCG

3' region of Tn21 (accession no AF071413) istB 5'-TCCGAGAAGCTCAAGTTGGT 55 200

5'-GTCAATGCACTGGAGCAAGA istA 5'-AGTGACTCGCTTGGTTTGCT 55 366

77 5' - ATGAAGACCGCTGTGGATTC orfAB 5'-CAATACCATGCTGGATCACG 55 250

5' -CATGAGGTCTTTC AGCGTC A tniBAl 5'-GCGATTTTCCAACTGGTCAT 55 218

5'-AAATGGAGCAACTGGCTCTG tniA 5'-ATGCGACAAGGTACGGTAGG 55 172

5'-TGTCATCGACCACATCCACT urf2 5'-CGACAAAGGATTGGTCGATT 55 236

5'-GTTCGACGACGAGGTAAAGC merE 5'-GTTACGGCCAGAACGAACAA 55 165

5'-CGTTTCCGGCTACCTGTG merD 5'-CACGAAGCACAGCCGTTG 55 159

5'-ATGAGCGCCTACACGGTATC merC 5'-ACGAAGTCCCAGATCGACAC 55 239

5'-TTGAGCCAGTACGAGGGACT merP 5'-GCCTTGGTGTCGTCAAAAGT 55 287

5'-GAAACTGTTTGCCTCCCTTG

78 merT 5'-ATGAACAACGGTCGATAGGG 55 174

5'-GGGCCAACGTATGTCTGAAC merA 5'-CAGCGAGACGATTCCTAAGC 60 830

5'-TCGTCAGGTAGGGGAACAAC

PCRs used to characterize the 'lonely integrase' isolates tet(A) 5'-GGCGGTCTTCTTCATCATGC 63 502

5'-CGGCAGGCAGAGCAAGTAGA floR 5'-CGCCGTCATTCCTCACCTTC 55 215

5'-GATCACGGGCCACGCTGTGTC

Inverse 5'-CTTGCTCACCTGGGTTTGTT 60.2 6500

PCR* 5'-CGAAGGCGATACAGTGGTG

*Inverse PCR used to characterize the surrounding genetic environment of the intll gene of the 'lonely integrase'.

79 Table 11 - Primers used in overlapping PCR sets for characterization of the 'lonely integrase' isolates.

Overlap Region Primer Sequence (Forward/Reverse) Annealing Product size

temperature (bp) CO tet(A)-tnpA 5'-CGATATCACTGATGGCGATG 63 2119

5'-ACCTTTGATGGTGGCGTAAG tnpA-intll 5'-AGGTCGGATTCGTGGTACAG 57 4803

5 '-GCTGTTCTTCTACGGCAAGG intIl-tet{M) 5'-AGCACCTTGCCGTAGAAGAA 58.2 2800

5'-CCACATACAGGACACAATATCCA tet(U)-qacG 5'-CTACCGGTGAACCTGTTTGC 57 2500

5'-ACACCAACAAATCCCCACAT

80 Table 12 - The eight 'lonely' integrase isolate groups based on phenotypic resistance

to chloramphenicol, streptomycin, sulfizoxazole and tetracycline.

Number of

Group Number Isolates Chloramphenicol Streptomycin Sulfizoxazole Tetracycline

I 52 R R R R

II 18 R S R R

III 3 R S S R

IV 35 S R R R

V 3 S R S R

VI 80 s S R R

VII 55 s S S R

VIII 5 s s S S

"R" denotes a resistant phenotype while "S" denotes a susceptible phenotype for the

listed antimicrobial. Resistance phenotypes were determined for all isolates using broth

microdilutions and clinical MIC breakpoints (CLSI2006).

81 Table 13 - The PCR results spanning the entire sequenced 'lonely' integrase region for 247 isolates.

Group tetA-tnpA tnpA-intll intll-tetM tetM-qacG Total

I - - - - 17

II + - - - 12

III - + - - 2

IV + + - - 1

V - - + - 3

VI + - + - 4

VII - + + - 10

VIII + + + - 22

IX + - - + 1

X - + - + 1

XI - - + + 6

XII + - + + 14

XIII - + + + 26

XIV + + + + 128

+ and - denote the presence or absence of the listed overlapping PCR product, respectively.

82 Figure 3 - Southern blot analysis to determine the location of the 'lonely integrase'.

A. Total DNA preparation of E. coli carrying the 'lonely integrase'

123456789 123456789

23 kb 9.4kb 6.5 kb 4.3 kb

2.3 kb 2.0 kb

These pictures illustrate the total DNA preparations of E. coli digested with the

restriction endonuclease EcoRI and separated on an ethidium bromide stained

agarose gel (left) and the Southern blot of the total DNA preparations using the

intll probe. Lanes 1-8 contain total DNA preparations of eight E. coli isolates

characterized as containing a 'lonely integrase'. The E. coli isolate identification

numbers are as follows (from lanes 1-8) 260cD, 267cC, 299cD, 407cB, 3538C,

4575A, 5370A and 5734B. Lane 9 contains DNA Molecular Weight Marker II

(DIG-labelled) (Roche) which has DNA fragments ranging in size from 0.12-23.1

kbp. A DIG-labelled intll was used as the probe.

83 B. Plasmid preparation of E. coli carrying the 'lonely integrase'. 12345678 12345678

140kb 120kb

28 kb

8kb

These pictures illustrate undigested plasmid preparations of E. coli on an ethidium bromide stained agaraose gel (left) and the Southern blot of the same preparations using a

DIG-labelled intll probe. Lanes 1-8 contain plasmid preparations of eight E. coli isolates

(same as in panel A) containing a 'lonely integrase'.

84 Figure 4 - The immediate genetic environment of the 'lonely integrase' genetic element. 4,4+-*+**+*P2 P4 P6 P8

PI P3 P5 P7

1200 bp 651 bp 2971 bp 567 bd 1014 bp 1920 bp 1014 bp 333 bp

Tnl721

Primer (P) sets indicate the position of overlapping PCRs used to characterize the 'lonely integrase'. Primer sequences, conditions and products are further outlined in Table 11. PI and P2 represent the tet(A)-tnpA, P3 and P4 represent the tnpA- intll, P5 and P6 represent the int!l-tet(M) and P7 and P8 represent the tet(M)-qacG.

85 DISCUSSION AND CONCLUSIONS

The work presented in this thesis contributes to the understanding integron diversity and dynamics within the beef cattle feedlot environment.

The intll gene was found in approximately 12% of the E. coli isolates and was not observed in any of the 200 M. haemolytica isolates. While these two organisms prefer different niches, they both are capable of colonizing the same host and the possibility of genetic exchange seems likely. The lack of detection of both class 1 and 2 integrons in M. haemolytica is somewhat surprising. However, the low prevalence of

AMR in this organism (Hendriksen et al. 2008, Katsuda et al. 2009, Klima et al. 2010a) may suggest that the selection pressure in the respiratory tract is different than in the intestinal tract and that AMR determinants are less likely to get established in respiratory pathogens. In addition, the throat is a body compartment distinct from the intestine, and

M. haemolytica is a fastidious bacterium that is unlikely to enter the intestine where

E. coli thrives and disseminates its resistance genes. The Mannheimia isolates were made from healthy cattle and may be less resistant than clinical isolates. Plasmid incompatibilities and plasmid host spectra may also be likely explanations. Further investigation should be conducted to determine if genetic exchange and plasmid transfer in particular can occur between E. coli and M. haemolytica. If genetic exchange is possible between M. haemolytica and E. coli, it may be worth investigating more

M. haemolytica isolates collected from the same animal at the same time as an E. coli isolate which carries a class 1 or class 2 integron. It may also be worth investigating the fate of integrons transferred in vitro into M. haemolytica. The DNA-DNA hybridization

86 approach for the detection of integrons in M. haemolytica could have been flawed, as no

Mannheimia specific genes were used as probes to confirm the quality of the total genomic DNA preps used for the dot blot hybridizations. By using a gene known to be within the Mannheimia genome, we could have shown that the quality of the DNA is sufficient for hybridization reactions and be more confident in our conclusions that the class 1 and class 2 integrons are absent in M. haemolytica.

Based on our results, class 1 integrons are present in 3% of E. coli isolated from cattle in western Canadian feedlots. Although sample time was not statistically associated with the frequency of class 1 integrons, it did increase the frequency of the

'lonely integrase'. While both genetic elements confer resistance to antimicrobials, it appears as though the 'lonely integrase' is far more frequently selected for and/or transmitted among feedlot E. coli. The proliferation of the 'lonely integrase' during the time spent in the feedlot may be important in terms of multiresistance and suggests the effect of antimicrobial use in feedlots on the spread of antimicrobial resistance. This should be taken into consideration for sampling design when monitoring beef cattle for the presence of AMR. Although each feedlot follows similar health management principles, differences in geographic location, staff and cattle stock may significantly alter the distribution of class 1 integrons. To increase the number of integrons in a sample isolates should first be screened for the presence of sulfonamide resistance and that subgroup be used to detect class 1 integrons.

Although the intll gene was detected in approximately 12% of the E. coli isolates, it was a part of typical class 1 integron structures in only about one third of the samples.

The majority of the intll -positive isolates lacked sull, a detectable gene cassette, and the

87 3'CS. The existence of this 'lonely integrase' had been reported before (Guerra et al.

2006, Sunde et al. 2008, Vinue et al. 2008, Hillyer 2009, Soufi et al. 2009) but not at such great frequencies. The genetic analysis of the 'lonely integrase' shows that it is part of a genetic element formed through repeated recombination events. This genetic element was observed on plasmids, which may account for its broad distribution.

However further investigation should be conducted to determine whether such plasmids are transferable and to confirm that this new genetic element is part of a larger transposon. Despite the absence of integron cassettes, the presence of the tet(A) and tet(M) suggest that tetracycline usage may be a positive selection factor driving the spread of this genetic element. Further work needs to be done to determine the functionality of both tet(A) and tet(M) genes to understand why both of these seemingly redundant genes are present on the same genetic element. It would also be interesting to investigate the presence of this 'lonely integrase' in other bacterial species and sources of

E. coli to determine if this is an emerging antimicrobial resistance conferring genetic element or if it is confined to beef cattle. The use of sulfonamides, phenicols and tetracycline in isolation media for such investigations may help increase the sensitivity of the 'lonely integrase' detection, as these factors were strongly associated with this genetic element.

Although much can be interpreted from the results presented in this thesis, the methodologies used present a few limitations. The number of feedlots used in this study was low (n=4), they were all in a similar geographic area (Alberta, Canada) and all feedlots were operated under the same health management policies, which makes it difficult to make general statements regarding the dynamics of genetic elements which

88 confer resistance to antimicrobials. For future studies it would be useful to collect isolates from a greater number of feedlots in a wider geographic range with different management styles and policies. This would allow for more broadly relevant conclusions for the beef cattle industry. Throughout the course of the study, on all four feedlots, some pens were mixed and reorganized after the >60 DOF time point. This created a significant challenge in the analysis of composite samples, as some pens no longer consisted of the same cohort of animals at exit as they did at previous time points. To correct for this, samples taken at >60 DOF and at exit were pooled and analyzed as one time point and that may have negatively affected our analysis of time effects on resistance prevalence.

The detection and characterization for the class 1 integrons and 'lonely integrase' was largely PCR-based. While this is a sensitive tool, minor changes in the nucleotide sequence may prevent the primers from annealing to the proper target. This may explain at least in part some of the variation observed in the 'lonely integrase' structure of some isolates. Finally, antimicrobial use data was unavailable for the analysis of this work.

This data was collected throughout the sampling process and would have been interesting to analyze to determine the effect antimicrobial usage had on the prevalence and diversity of class 1 integrons, the gene cassettes and the 'lonely integrase'. The results generated with the antimicrobial use data may help in the development of evidence-based policies to ensure a more effective use of antimicrobials in veterinary medicine. Unfortunately, they will only be available after conclusion of the present thesis.

In conclusion, this study provides insight into the distribution of the intll gene, the presence and distribution of major gene cassettes and class 1 integrons in E. coli and

89 M. haemolytica isolated from beef cattle feedlots and insight into the unique and novel

'lonely integrase' genetic element. The results generated by the analysis of the data presented in this thesis provide several new opportunities for investigations to improve the knowledge of the epidemiology of AMR in E. coli and M. haemolytica in Canadian beef cattle. Class 1 integrons in E. coli with a variety of combinations of gene cassettes are present in beef cattle and contribute to the spread of resistance. The 'lonely integrase' is a newly described genetic element, which is associated with resistances to streptomycin, phenicols, sulfonamides and tetracycline, and should be investigated further to determine its role in the epidemiology of AMR and of multiresistance.

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109 APPENDIX 1: FORMULAE USED TO DETERMINE THE

ADJUSTED PREVALENCE FOR STRATIFIED RANDOM

SAMPLES

To correct the prevalence of the intll gene, class 1 integrons and the 'lonely' integrase gene to account for the stratified random sampling of isolates, the following formulae were used:

(PRXNR) + (PSXNS) N

To determine the adjusted prevalence (P), the above formula was used, where N represents the total number of isolates in the collection, NR and Ns represent the total number of isolates in each strata and PR and Ps represent the prevalence of the intll in each strata. „ M x (*^) >< fi^)> + (* - (V*) >< P&P S E - - TP

To determine the standard error (S.E.), the above formula was used, where N represents the total number of isolates in the collection, NR and Ns represent the total number of isolates in each strata, nR and ns represent the number of isolates from each strata that were tested and PR and Ps represent the estimated prevalence of the gene within the strata.

95% C./.= P ± 1.96(5. E.)

To determine the 95% Confidence Interval (C.I.), the above equation was used (Dohoo et al. 2003).

110 APPENDIX 2: STATISTICAL ANALYSES OF INTEGRASE GENE. CLASS 1 INTEGRONS AND 'LONELY INTEGRASE'

Table 14 - The adjusted prevalence, standard error and 95% confidence interval calculated for intll, class 1 integrons and 'lonely integrase'.

Standard Lower Upper

Adjusted Prevalence Error 95% CI 95% CI

Integrase 0.119 0.000030 0.118 0.119

Class lintegron 0.031 0.000005 0.031 0.031

'Lonely integrase' 0.087 0.000022 0.087 0.087

95% Confidence Intervals calculated using formula found in Dohoo et al. 2003.

Ill Figure 5 - The Stata 11 coding and output for the logistic regression of class 1 integrons.

Logistic Regression: Class 1 Integron (Composite Samples)

Code: xi:xtmelogit class lintegron i.feedlot i.entryexit i.chosenbasedonsulr ||pen:,or

Independent Odds P U 95%

Output Variable ratio value L95% CI CI

Class 1 Integron Feedlot 21 0.7599014 0.564 0.2990402 1.931012

Class 1 Integron Feedlot31 0.3609131 0.121 0.0994507 1.309777

Class 1 Integron Feedlot 42 2.377945 0.098 0.8525162 6.6.32862

Class 1 Integron >60 DOF/Exit 1.014568 0.960 0.5804273 1.773431

Class 1 Integron Chosen based on sulR 8.449175 <0.001 4.436557 16.09098

Logistic Regression: Class 1 Integron (Individual Samples)

Code: xi:xtmelogit class lintegron i.feedlot i.entryexit i.chosenbasedonsulr ||pen:,or

Independent Odds P U 95%

Output Variable ratio value L95% CI CI

Class 1 Integron Feedlot 21 0.330514 0.093 0.0908711 1.202137

Class 1 Integron Feedlot 31 0.2244526 0.105 0.0368591 1.366797

Class 1 Integron Feedlot 42 1.679037 0.363 0.5502023 5.123871

Class 1 Integron >60 DOF/Exit 0.9526049 0.920 0.3692159 2.457793

Class 1 Integron Chosen based on sulR 13.34008 O.001 4.668976 38.11491

112 Figure 6 - The Stata 11 coding and output for the logistic regression of 'lonely'

integrase isolates.

Logistic Regression: Lonely Integrase (Composite Samples)

Code: xi:xtmelogit lonelyintegrase i.feedlot i.entryexit i.chosenbasedonsulr ||pen: ||fhmscase:,or

Odds

Output Independent Variable ratio P value L95% CI U 95% CI

Lonely Integrase Feedlot21 1.914514 0.072 0.9431244 3.886407

Lonely Integrase Feedlot31 0.1467216 0.001 0.0468254 0.459734

Lonely Integrase Feedlot 42 0.7560468 0.533 0.3003155 1.903354

Lonely Integrase >60 DOF/Exit 2.199398 0.004 1.284467 3.76604

Lonely Integrase Chosen based on sulR 7.625893 O.001 4.740782 12.2668

Logistic Regression: Lonely Integrase (Individual Samples)

Code: xi:xtmelogit lonely integrase i.feedlot i.entryexit i.chosenbasedonsulr ||pen: ||fhmscase:,or

Odds

Output Independent Variable ratio P value L95% CI U 95% CI

Lonely Integrase Feedlot 21 0.8631494 0.787 0.296301 2.514429

Lonely Integrase Feedlot 31 0.3370194 0.165 0.072686 1.562638

Lonely Integrase Feedlot 42 0.3553779 0.117 0.097585 1.29419

Lonely Integrase >60 DOF/Exit 58.0449 O.001 9.761758 345.1439

Lonely Integrase Chosen based on sulR 5.469823 0.001 1.999994 14.95953

113 APPENDIX 3: NUCLEOTIDE AND AMINO ACID ALIGNMENTS

FOR VARIANTS OF THE AADAl AND AADB-AADA2 GENE

CASSETTES

Figure 7 - The alignment of the nucleotide sequence of the three variants of the aadAl gene cassette identified in E. coli in a class 1 integron.

Variant1 GTGAiCHCGAAiTpCGAHCAACTATCAGAGGlWlMGCGTCATiGAGCGCCATCTC 6 0 Variant2 GTGAicBcGAAJJTfc 6 0 Variant3 GTGA|cBcGAA§rfTCGA8c^UiCTATCAGAGGTBl»;CGTCAT|GAGCGCCATCTC 6 0 **** * **** * **** ************** * ******* ************

Variant1 GAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCA 120 Variant2 GAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCA 120 Variant3 GAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCA 120 ************************************************************

Variant1 CACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGA 180 Variant2 CACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGA 180 Variant3 CACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGA 180 ************************************************************

Variant1 GCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGC 240 Variant2 GCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGC 240 Variant3 GCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGC 24 0 ************************************************************

Variant1 GCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAG 300 Variant2 GCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAG 300 Variant3 GCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAG 300 ************************************************************

Variant1 CGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCA 360 Variant2 CGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCA 360 Variant3 CGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCA 360 ************************************************************

Variantl GCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCC 420 Variant2 GCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCC 420 Variant3 GCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCC 420 ************************************************************

Variantl TTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAG 480 Variant2 TTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAG 480 Variant3 TTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAG 480 ************************************************************

Variantl GCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGA 540 Variant2 GCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGA 540 Variant3 GCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGA 540 ************************************************************

Variantl AATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCG 600 Variant2 AATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCG 600 Variant3 AATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCG 600 ************************************************************

Variantl AAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATA 660

114 Variant2 AAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATA 660 Variant3 AAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATA 660 ************************************************************

Variant1 CTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCiCGCGCAGAT 720 Variant2 CTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTcicGCGCAGAT 720 Variant3 CTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTcJcGCGCAGAT 720 ************************************************** *********

Variant1 CAGTTGGAAGAATTTGTjCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAA 780 Variant2 CAGTTGGAAGAATTTG11CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAA 780 Variant3 CAGTTGGAAGAATTTG'IICACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAA 780 ***************** ****************************************** Highlights indicate changes in nucleotide sequence between variants

115 Figure 8 - The alignment of three variants of the amino acid sequence of aadAl gene products identified in E. coli in a class 1 integron.

Variantl VAVSTSVVGVRHTAVHYGSAVDGGKHSDDVTVTVRDTTRRANDTSASGSRAWTWHDDW Variant2 VAVSTSVVGVRHTAVHYGSAVDGGKHSDDVTVTVRDTTRRANDTSASGSRAWTWHDDW Varaant3 —VTSNSVSVRHTAVHYGSAVDGGKHSDDVTVTVRDTTRRANDTSASGSRAVVTVVHDDW

Variantl RYAi^GWRNDAGATDDATKARHSVAVGAADVDANT'TWNSDWAGDRNVVTSRWYSAVTGKA Variant2 RYAKRGWRNDAGATDDATKARHSVAVGAADVDANTTWNSDWAGDRNWTSRWYSAVTGKA Variant3 RYAi^GWRNDAGATDDATKARHSVAVGAADVDANTTWNSDWAGDRNVVTSRWYSAVTGKA

Variantl KDVAADWAMTRAYVARAYGDRASRADVHYVKGTKWGKST Variant2 IU}VAADWAMTRAYVARAYGDRASRADVHYVKGTKVVGKST Variant3 KDVAADWAMTRAYVARAYGDRASRADVHYVKGTKVVGKST

116 Figure 9 - The alignment of the nucleotide sequence of the three variants of the aadB-aadA2 gene cassette identified in E. coli in a class 1 integron.

Variantl GCGCGTTACGCCGTGGGTCJATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAG 6 0 Variant2 GCGCGTTACGCCGTGGGTC1ATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAG 6 0 Variant3 GCGCGTTACGCCGTGGGTC|ATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAG 6 0 ******************* ****************************************

Variantl CAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGCCGCATGGACACAAC 120 Variant2 CAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGCCGCATGGACACAAC 120 CAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGCCGCATGGACACAAC 120 Variant3 ************************************************************

Variantl GCAGGTCACATTGATACACAAAATTCTAGCTGCGGCAGATGAGCGAAATCTGCCGCTCTG 180 Variant2 GCAGGTCACATTGATACACAAAATTCTAGCTGCGGCAGATGAGCGAAATCTGCCGCTCTG 180 GCAGGTCACATTGATACACAAAATTCTAGCTGCGGCAGATGAGCGAAATCTGCCGCTCTG 180 Variant3 ************************************************************

Variantl GATCGGTGGGGGCTGGGCGATCGATGCACGGCTAGGGCGTGTAACACGCAAGCACGATGA 240 Variant2 GATCGGTGGGGGCTGGGCGATCGATGCACGGCTAGGGCGTGTAACACGCAAGCACGATGA 240 Variant3 GATCGGTGGGGGCTGGGCGATCGATGCACGGCTAGGGCGTGTAACACGCAAGCACGATGA 240 ************************************************************

Variantl TATTGATCTGACGTTTCCCGGCGAGAGGCGCGGCGAGCTCGAGGCAATAGTTGAAATGCT 300 Variant2 TATTGATCTGACGTTTCCCGGCGAGAGGCGCGGCGAGCTCGAGGCAATAGTTGAAATGCT 300 Variant3 TATTGATCTGACGTTTCCCGGCGAGAGGCGCGGCGAGCTCGAGGCAATAGTTGAAATGCT 300 ************************************************************

Variantl CGGCGGGCGCGTCATGGAGGAGTTGGACTATGGATTCTTAGCGGAGATCGGGGATGAGTT 360 Variant2 CGGCGGGCGCGTCATGGAGGAGTTGGACTATGGATTCTTAGCGGAGATCGGGGATGAGTT 360 Variant3 CGGCGGGCGCGTCATGGAGGAGTTGGACTATGGATTCTTAGCGGAGATCGGGGATGAGTT 360 ************************************************************

Variantl ACTTGACTGCGAACCTGCTTGGTGGGCAGACGAAGCGTATGAAATCGCGGAGGCTCCGCA 420 Variant2 ACTTGACTGCGAACCTGCTTGGTGGGCAGACGAAGCGTATGAAATCGCGGAGGCTCCGCA 420 Variant3 ACTTGACTGCGAACCTGCTTGGTGGGCAGACGAAGCGTATGAAATCGCGGAGGCTCCGCA 420 ************************************************************

Variantl GGGCTCGTGCCCAGAGGCGGCTGAGGGCGTCATCGCCGGGCGGCCAGTCCGTTGTAACAG 480 Variant2 GGGCTCGTGCCCAGAGGCGGCTGAGGGCGTCATCGCCGGGCGGCCAGTCCGTTGTAACAG 480 Variant3 GGGCTCGTGCCCAGAGGCGGCTGAGGGCGTCATCGCCGGGCGGCCAGTCCGTTGTAACAG 480 ************************************************************ Variantl CTGGGAGGCGATCATCTGGGATTACTTTTACTATGCCGATGAAGTACCACCAGTGGACTG 540 Variant2 CTGGGAGGCGATCATCTGGGATTACTTTTACTATGCCGATGAAGTACCACCAGTGGACTG 540 Variant3 CTGGGAGGCGATCATCTGGGATTACTTTTACTATGCCGATGAAGTACCACCAGTGGACTG 540 ************************************************************ Variantl GCCTACAAAGCACATAGAGTCCTACAGGCTCGCATGCAClCACTCGGGGCGGAAAAGGTT 600 Variant2 GCCTACAAAGCACATAGAGTCCTACAGGCTCGCATGCAclcACTCGGGGCGGAAAAGGTT 600 GCCTACAAAGCACATAGAGTCCTACAGGCTCGCATGCAC|CACTCGGGGCGGAAAAGGTT 600 Variant3 *************************************** ********************

Variantl GAGGTCTTGCGTGCCGCTTTCAGGTCGCGATATGCGGCCTAACAATTCGTCCAAGCCGAC 660 Variant2 GAGGTCTTGCGTGCCGCTTTCAGGTCGCGATATGCGGCCTAACAATTCGTCCAAGCCGAC 660 Variant3 GAGGTCTTGCGTGCCGCTTTCAGGTCGCGATATGCGGCCTAACAATTCGTCCAAGCCGAC 660 ************************************************************ Variantl GCCGCTTCGCGGCGCGGCTTAACTCAGGTGTTAGACATCATGAGGGAAGCGGTGACCATC 720 Variant2 GCCGCTTCGCGGCGCGGCTTAACTCAGGTGTTAGACATCATGAGGGAAGCGGTGACCATC 720 Variant3 GCCGCTTCGCGGCGCGGCTTAACTCAGGTGTTAGACATCATGAGGGAAGCGGTGACCATC 720 ************************************************************

Variantl GAAATTTCGAACCAACTATCAGAGGTGCTAAGCGTCATTGAGCGCCATCTGGAATCAACG 780 Variant2 GAAATTTCGAACCAACTATCAGAGGTGCTAAGCGTCATTGAGCGCCATCTGGAATCAACG 780 Variant3 GAAATTTCGAACCAACTATCAGAGGTGCTAAGCGTCATTGAGCGCCATCTGGAATCAACG 780 ************************************************************

Variantl TTGCTGGCCGTGCATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCATACAGCGAT 840 Variant2 TTGCTGGCCGTGCATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCATACAGCGAT 840 TTGCTGGCCGTGCATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCATACAGCGAT 840 Variant3 ************************************************************

117 Variant 1 ATTGATTTGTTGGTTACTGTGGCCGTAAAGCTTGATGAAACGACGCGGCGAGCATTGCTC 900 Variant2 ATTGATTTGTTGGTTACTGTGGCCGTAAAGCTTGATGAAACGACGCGGCGAGCATTGCTC 900 Variant3 ATTGATTTGTTGGTTACTGTGGCCGTAAAGCTTGATGAAACGACGCGGCGAGCATTGCTC 900 ************************************************************ Variant1 AATGATCTTATGGAGGCTTCGGCTTTCCCTGGCGAGAGCGAGACGCTCCGCGCTATAGAA 960 Variant2 AATGATCTTATGGAGGCTTCGGCTTTCCCTGGCGAGAGCGAGACGCTCCGCGCTATAGAA 960 Variant3 AATGATCTTATGGAGGCTTCGGCTTTCCCTGGCGAGAGCGAGACGCTCCGCGCTATAGAA 960 ************************************************************

Variantl GTCACCCTTGTCGTGCATGACGACATCATCCCGTGGCGTTATCCGGCTAAGCGCGAGCTG 1020 Variant2 GTCACCCTTGTCGTGCATGACGACATCATCCCGTGGCGTTATCCGGCTAAGCGCGAGCTG 1020 GTCACCCTTGTCGTGCATGACGACATCATCCCGTGGCGTTATCCGGCTAAGCGCGAGCTG 1020 Variant3 ************************************************************

Variantl CAATTTGGAGAATGGCAGCGCAATGACATTCTTGCGGGTATCTTCGAG|CAGCCATGATC 1080 Variant2 CAATTTGGAGAATGGCAGCGCAATGACATTCTTGCGGGTATCTTCGAG|CAGCCATGATC 1080 Variant3 CAATTTGGAGAATGGCAGCGCAATGACATTCTTGCGGGTATCTTCGAG|CAGCCATGATC 1080 ************************************************ t ***********

Variantl GACATTGATCTAGCTATCCTGCTTACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGT 1140 Variant2 GACATTGATCTAGCTATCCTGCTTACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGT 1140 Variant3 GACATTGATCTAGCTATCCTGCTTACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGT 1140 ************************************************************

Variantl CCGGCAGCGGAGGAATTCTTTGACCCGGTTCCTGAACAGGATCTATTCGAGGCGCTGAGG 1200 Variant2 CCGGCAGCGGAGGAATTCTTTGACCCGGTTCCTGAACAGGATCTATTCGAGGCGCTGAGG 1200 Variant3 CCGGCAGCGGAGGAATTCTTTGACCCGGTTCCTGAACAGGATCTATTCGAGGCGCTGAGG 1200 ************************************************************

Variantl GAAACCTTGAAGCTATGGAACTCGCAGCCCGACTGGGCCGGCGATGAGCGAAATGTAGTG 1260 Variant2 GAAACCTTGAAGCTATGGAACTCGCAGCCCGACTGGGCCGGCGATGAGCGAAATGTAGTG 1260 GAAACCTTGAAGCTATGGAACTCGCAGCCCGACTGGGCCGGCGATGAGCGAAATGTAGTG 1260 Variant3 ************************************************************

Variantl CTTACGTTGTCCCGCATTTGGTACAGCGCAATAACCGGCAAAATCGCGCCGAAGGATGTC 1320 Variant2 CTTACGTTGTCCCGCATTTGGTACAGCGCAATAACCGGCAAAATCGCGCCGAAGGATGTC 1320 Variant3 CTTACGTTGTCCCGCATTTGGTACAGCGCAATAACCGGCAAAATCGCGCCGAAGGATGTC 1320 ************************************************************ Variantl GCTGCCGACTGGGCAATAAAACGCCTACCTGCCCAGTATCAGCCCGTCTTACTTGAAGCT 1380 Variant2 GCTGCCGACTGGGCAATAAAACGCCTACCTGCCCAGTATCAGCCCGTCTTACTTGAAGCT 1380 GCTGCCGACTGGGCAATAAAACGCCTACCTGCCCAGTATCAGCCCGTCTTACTTGAAGCT 1380 Variant3 ************************************************************

Variantl AAGCAAGCTTATCTGGGACAAAAAGAAGATCACTTGGCCTCACGCGCAGATCACTTGGAA 1440 Variant2 AAGCAAGCTTATCTGGGACAAAAAGAAGATCACTTGGCCTCACGCGCAGATCACTTGGAA 1440 Variant3 AAGCAAGCTTATCTGGGACAAAAAGAAGATCACTTGGCCTCACGCGCAGATCACTTGGAA 1440 ************************************************************

Variantl GAATTTATTCGCTTTGTGAAAGGCGAGATCATCAAGTCAGTTGGTAAATGATGTCTAACA 1500 Variant2 GAATTTATTCGCTTTGTGAAAGGCGAGATCATCAAGTCAGTTGGTAAATGATGTCTAACA 1500 Variant3 GAATTTATTCGCTTTGTGAAAGGCGAGATCATCAAGTCAGTTGGTAAATGATGTCTAACA 1500 ************************************************************

Variantl ATTCGTTCAAGCCGACCGCGCTACGCGCGGCGGCTTAACTCCGGCGTTAGATGCACTAAG 1560 Variant2 ATTCGTTCAAGCCGACCGCGCTACGCGCGGCGGCTTAACTCCGGCGTTAGATGCACTAAG 1560 Variant3 ATTCGTTCAAGCCGACCGCGCTACGCGCGGCGGCTTAACTCCGGCGTTAGATGCACTAAG 1560 ************************************************************

Variantl CACATAATTGCTCACAGCCAAACTATCA 1588 Variant2 CACATAATTGCTCACAGCCAAACTATCA 1588 Variant3 CACATAATTGCTCACAGCCAAACTATCA 1588 ****************************

Highlights indicate changes in nucleotide sequence between variants. The aadB gene

sequence begins at nucleotide 109 and ends at 643. The aadA2 gene sequence begins at nucleotide 699 and ends at 1497.

118 Figure 10 -The alignment of three variants of the amino acid sequence of the expressed aadB gene cassette identified in E. coli in a class 1 integron

Variantl CYGAATHTRSSNDVTGSRKTKGRHTDTTVTHKAAADRNWGGGUADARGRVTRKHDDDTGR Variant2 CYGAATHTRSSNDVTGSRKTKGRHTDTTVTHKAAADRNHGGGtfADARGRVTRKHDDDTGR Variant3 CYGAATHTRSSNDVTGSRKTKGRHTDTTVTHKAAADRMTGGGUADARGRVTRKHDDDTGR ************************************************************

Variantl RGAVHTGGRVHTDYGAGDDCAWliJADAYAAGSCAAGVAGRVRCNSWAWDYYYADVVDWTKH Variant2 RG AVHTGGRVHTD YG AGD D C ATO AD A YAAGS C AAGV AGRVRCNS WAWDYYYADWDWTKH Variant3 RGAVHTGGRVHTDYGAGDDCATOADAYAAGSCAAGVAGRVRCNSWAWDYYYADVVDWTKH ************************************************************

Variantl SYRACTHSGRKRRSCVSGRDHTRMNSSKTRGAA Variant2 SYRACTHSGRKRRSCVSGRDHTRNMSSKTRGAA Variant3 SYRACTHSGRKRRSCVSGRDHTRNNSSKTRGAA *********************************

119 Figure 11 - The alignment of three variants of the amino acid sequence of the expressed aadA2 gene cassette identified in E. coli in a class 1 integron.

Variantl VAD AAS RRGTVD MTRAVTSNSVSVRHS TAVHYGS AVD GGKYS D DVTVAVKD TTRRAND HT Variant2 VAD AAS RRGTVD HTRAVTSNSVSVRHS TAVHYGSAVD GGKYS D DVTVAVKDTTRRAND HT Var iant3 VADAASRRGTVDHTRAVTSNSVSVRHSTAVHYGSAVDGGKYSDDVTVAVKDTTRRANDHT ************************************************************

Variantl ASAGSTRAVTWHDDURYAKRGWRNDAG-AHTDDATKARHSVAVGAADVDARTKliJNSDliirA Variant2 ASAGSTRAVTWHDDHRYAKRGWRNDAG-AHTDDATKARHSVAVGAADVDARTKTilNSDTirA Var iant3 ASAGSTRAVTVVHDDURYAKRGURNDAGTAHTDDATKARHSVAVGAADVDARTKUNSDHA **************************** *******************************

Variantl GDRNWTSRUYSATGKAKDVAADWAKRAYVAKAYGKDHASRADHRVKGKSVGK Variant2 GDRNWTSRWYSATGKAKDVAADUAKRAYVAKAYGKDHASRADHRVKGKSVGK Variant3 GDRNWTSRUYSATGKAKDVAADUAKRAYVAKAYGKDHASRADHRVKGKSVGK *****************************************************

120 APPENDIX 4 PLASMID SEQUENCING RESULTS OF TWO LARGE

PLASMIDS CONTAINING THE 'LONELY' INTEGRASE GENETIC

ELEMENT

Table 15 - The plasmid sequencing results for plasmid p260cD (120 kb).-

Size Accession

Contig (bp) Genes Number Function

00002 3698 MR CP000971.1 Tetracycline resistance regulator

tet(A) Tetracycline efflux pump

00461 47125 yubP EU935738.1 Hypothetical protein

yeiA Dihydropyrimidine dehyrdogenase, NADH-dependent subunit B

yehA Putative Yeh fimbrial adhesin YehA precursor

traM CP001065.1 Type 4 secretion-like conjugation transfer system protein

flnP Putative transglycosylase

traA Type 4 conjugative transfer system pilin

traL Type 4 secretion-like conjugation transfer system protein

traE Type 4 secretion-like conjugation transfer system protein

traK CP00971.1 Type 4 secretion-like conjugation transfer system protein

traB CP001162.1 Type 4 secretion-like conjugation transfer system protein

traP Conjugal transfer protein

traR CP000971.1 Type 4 secretion-like conjugation transfer system protein

traV Type 4 secretion-like conjugation transfer system protein

traC AB255435.1 Conjugal transfer protein

121 trbl Conjugal transfer pilus assembly protein

traW Conjugal transfer pilus assembly protein

traU EU330199.1 Conjugal transfer peptidase

traF Conjugal transfer protein

trbE Conjugal transfer mating pair stabilization protein

traN AY214164.3 Putative conjugal transfer protein

trbC Conjugal transfer mating pair stabilization protein

traG Conjugal transfer pilus assempbly protein

traH Conjugal transfer transcriptional regulator

trhJ Conjugal transfer pilin chaperone

traO Conjugal transfer protein

trbA Conjugal transfer protein

traD Conjugal transfer protein

traT AM886293.1 Conjugal transfer surface exclusion protein

traG Conjugal transfer mating pair stabilization protein

tral Conjugal transfer nickase/helicase

traX Conjugal transfer pilus acetylation protein

flnO Conjugal transfer fertility inhibition protein

yigA Hypothetical protein

repA2 Replication protein

repA6 Replication protein

repAl Replication protein

repA4 Replication protein

00467 27351 stbA HQ 114282.1 Plasmid stability and partitioning protein

stbB Plasmid stability and partitioning protein

122 umuC UV protection protein

ssb Single stranded DNA-binding protein

00469 1333 intll EU935739.1 Class 1 integrase

00470 1796 tnpA X61367.1 Tn7 721 transposase

tnpR Tn/ 721 transposase repressor

00472 5249 psiA GU371926.1 Putative SOS inhibition protein

psiB Putative SOS inhibition protein

parB Chromosome partitioning protein

ssb Single stranded DNA-binding protein

00473 2421 tetM U09422.1 Ribosomal binding protein (TET resistance)

123 Table 16 - The plasmid sequencing results for plasmid p299cD (140 kb).

Plasmid Contig Size (bp) Genes Accession Number Function

00001 3698 DQ464880.1 Tetracycline resistance regulator

Tetracycline efflux pump

Hypothetical protein

Tnl721 transposase •Mi l

00003 7404 ychA AP001918.1 Putative transcriptional regulator

ompP Outer membrane protease

yddA Inner membrane ABC transporter ATP-binding pr

int Phage integrase

02000 9646 ccdA AP009379.1 Plasmid maintenance protein

ccdB Plasmid maintenance protein

02001 9417 ydiA AP001918.1 Hypothetical protein

ydiB Hypothetical protein

repB Replication protein

ydhA Outer membrane lipoprotein

ydiA Putative phosphotransferase

Florfenicol/chloramphenicol resistance

02008 floR DQ206638.1 protein

02024 tet(M) U09422.1 Ribosomal binding protein (TET resistance)

02009 traG AP001918.1 F pilus assembly and aggregate stability

traS Conjugal transfer entry exclusion protein

traT Conjugal transfer surface exclusion protein

traD Conjugal transfer protein

124 trbH Conjugal transfer protein

tral Conjugal transfer nickase/helicase

traX F pilin acetylation

finO Conjugal transfer fertility inhibition protein

yacA Regulator of plasmid replication

Modulator of post-segregation killing

srnB protein

srnC Post-segregational killing toxin

repA2 Replication protein

repL Replication protein

repAl Replication protein

yfhB Hypothetical protein

ssb Single stranded DNA-binding protein

flmC Flagellin modification protein

flmA Flagellin modification protein

ygdA Ribosome associated factor

ygeA Racemase

ygeB Hypothetical protein

02538 11937 ybhB AP001918.1 Kinase inhibitor homolog

ycbA Hypothetical protein

ybaA Hypothetical protein

ybbA ABC transporter ATP-binding protein

ybcA Putative DNA-binding transcriptional regulator

ybdA Enterobactin exporter

125 ybdB Esterase

ybgA Hypothetical protein

ybhA Phosphatase

02543 14027 tniC GQ857074.1 Tn402 transposition protein

tniQ Tn402 transposition protein

MB Tn402 transposition protein

tniA Tn402 transposition protein

02547 6158 sul2 CP000971.1 Dihydropteroate synthase protein (SUL resistance

strA Aminoglycoside phosphotransferase (STR resistai

strB Aminoglycoside resistance protein (STR resistanc

126