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 of The University of Guelph by MATTHEW LESLIE In partial fulfillment of requirements for the degree of Master of Science May, 2011 © Matthew Leslie, 2011 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Vote reference ISBN: 978-0-494-82791-8 Our We Notre r6f6rence ISBN: 978-0-494-82791-8 NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciaies ou autres, sur worldwide, for commercial or non support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 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