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Detection of Antibiotic Resistance in Swine Production

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Detection of Antibiotic Resistance in Swine Production Detection of Antibiotic Resistance in Swine Production By: SEPIDEH PAKPOUR Department of Food Science and Agricultural Chemistry Macdonald Campus, McGill University Montreal, Quebec A thesis submitted to the Office of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the degree of Master of Science. August, 2010 © SEPIDEH PAKPOUR This thesis is dedicated to my first and beloved teachers: My parents Mr. Mohammad Reza Pakpour and Mrs. Esmat Abolmasomi. TABLE OF CONTENT TABLE OF CONTENT I LIST OF TABLES VII LIST OF FIGURES VIII LIST OF ABBREVIATIONS IX ABSTRACT X RÉSUMÉ XI ACKNOWLEDGMENTS XIII INTRODUCTION XV Hypotheses XVI Objectives XVII Experimental Approach XVII CONTRIBUTION TO RESEARCH XIX LIST OF PUBLICATIONS AND PRESENTATIONS XX CHAPTER 1. LITERATURE REVIEW 1 1.1 General Introduction to Antibiotics 1 1.1.1 Definition 1 1.1.2 History of Antibiotics 1 1.1.3 Classifications 2 1.1.4 How do Antibiotics Work? 2 1.1.5 Applications of Antibiotics 3 1.2 General Introduction to Antibiotic Resistance 4 I 1.2.1 Definition 5 1.2.2 Classification 6 1.2.2.1 Innate (Intrinsic) Resistance 6 1.2.2.2 Acquired (Extrinsic) Resistance 6 1.2.2.3 Adaptation 7 1.3 General Introduction to Swine Rearing 9 1.4 Antibiotic Resistance in Swine Production 11 1.5 Tetracyclines 12 1.5.1 Discovery and Development of Tetracyclines 12 1.5.2 Structure of Tetracyclines 14 1.5.3 Mode of Action 14 1.5.4 Resistance to Tetracyclines 17 1.5.4.1 Protection of the Ribosome 17 1.5.4.2 Efflux Proteins 19 1.5.4.3 Enzymatic Inactivation 19 1.5.4.4 Other/Unknown Mechanisms of Resistance 21 1.6 Macrolides 21 1.6.1 Discovery and Development of Macrolides 21 1.6.2 Structure of Macrolides 23 1.6.3 Mechanisms of Action of Macrolides 23 1.6.4 Resistance to Macrolides 23 1.6.4.1 Ribosomal Methylation 23 1.6.4.2 Efflux Systems 25 II 1.6.4.3 Enzymatic Inactivation 25 1.6.4.4 Mutational Resistance to Macrolides 25 1.7 General Introduction to Tylosin 26 1.8 Food-Borne Pathogens 27 CONNECTING STATEMENT TO CHAPTER 2 29 CHAPTER 2. PRELIMINARY STUDIES 29 2.1 Abstract 29 2.2 Introduction 30 2.3 Material and Methods 31 2.3.1 Swine Rearing and Sampling 31 2.3.2 Enumeration of Total Aerobic Bacteria on Solid 32 Media 2.3.2.1 Preparation of Aerobic Dilution Water 32 2.3.2.2 Preparation of Solid Media 32 2.3.2.3 Dilution of Fecal Samples 32 2.3.2.4 Inoculation of Solid Media by Spread-Plate 33 2.3.3 Enumeration of Total Anaerobic Bacteria on Solid 33 Media 2.3.3.1 Preparation of Anaerobic Dilution Water 33 2.3.3.2 Preparation of Solid Media 35 2.3.3.3 Dilution of Fecal Samples 35 2.3.3.4 Inoculation of Solid Media by Spread-Plate 35 2.3.4 DNA Extraction 36 2.3.4.1 Extraction of Total DNA From Bacterial 36 III Strains Used as Positive Control for PCR 2.3.4.2 Extraction of Total Community DNA From 36 Swine Feces 2.3.4.3 DNA Integrity, Concentration and Purity 37 2.3.5 PCR for Bacteria 16S rDNA 37 2.3.6 Real-Time PCR for tet (O) 41 2.3.6.1 Primers and Probes 41 2.3.6.2 Preparation of DNA Dilution Series 41 2.3.6.3 Real-Time PCR for tet (O) 42 2.4 Results 44 2.4.1 Anaerobic Dilution Water 44 2.4.2 Enumeration of Total Aerobic and Anaerobic 44 Bacterial Populations 2.4.3 DNA Extraction 44 2.4.3.1 Extraction of Total DNA From Bacterial Strains 44 Used as Positive Control for PCR 2.4.3.2 Extraction of Total Community DNA From 46 Swine Feces 2.4.4 PCR for Bacteria 16S rDNA 46 2.4.5 Real-time PCR for tet (O) 48 2.5 Discussion 51 2.5.1 Anaerobic Dilution Water 51 2.5.2 Bacterial Enumerations 51 IV 2.5.3 Extraction of Total Community DNA from Swine 52 Feces 2.5.4 PCR Reaction Mixture for Bacteria 16S rDNA 53 2.5.5 PCR Conditions for Bacteria 16S rDNA 54 2.5.6 Real_Time PCR for tet (O) 55 2.6 Conclusions 57 2.7 Acknowledgments 57 CONNECTING STATEMENT TO CHAPTER 3 58 CHAPTER 3. ANTIBIOTIC RESISTANCE IN SWINE 58 PRODUCTION 2.5 YEARS AFTER DISCONTINUATION OF ANTIBIOTIC USE. 3.1 Abstract 58 3.2 Introduction 59 3.3 Material and Methods 61 3.3.1 Swine Rearing and Sampling 61 3.3.2 Bacterial Enumerations 61 3.3.3 DNA Extraction 63 3.3.4 PCR Amplification 64 3.3.5 Standard for Real-Time PCR 65 3.3.6 Optimization of Real-Time PCR 65 3.4 Results 67 3.4.1 Bacterial Enumerations. 67 3.4.2 Antibiotic Resistance Genes 68 3.4.3 Optimization and Standard Curve for Real-Time 72 PCR V 3.4.4 Real-Time PCR for tet (O) 74 3.5 Discussion 77 3.5.1 Bacterial Enumerations 77 3.5.2 Gender Effect 78 3.5.3 Antibiotic resistance Genes 80 3.5.4 Abundance of tet (O) 82 3.6 Conclusions 83 3.7 Acknowledgments 83 CHAPTER 4. GENERAL CONCLUSIONS 85 4.1 Antibioic Resistance in Swine Production 86 4.2 Experimental Approach 86 4.3 Methodology 87 4.3.1 Culture-Dependent Methods 87 4.3.2 Molecular Biology Techniques 88 4.3.2.1 Polymerase Chain Reaction 88 4.3.2.2. Quantitative Real-Time PCR 88 4.4 Conclusions 89 REFERENCES 90 APPENDIX. ANIMAL USE PROTOCOLS & BIOHAZARDS 102 CERTIFICATES VI LIST OF TABLES Table 1.1 Principal members of the tetracycline class. 13 Table 1.2 Structures of the principal members of the tetracycline class 15 Table 1.3 Mechanisms of resistance for characterized tet and otr genes. 22 Table 2.1 Oligonucleotide primers, amplicon size, annealing temperatures 39 and positive used for PCR amplification of bacterial genes. Table 2.2 Optimization of the PCR reaction mixture and conditions. 40 Table 2.3 Annealing temperatures and primer and probe concentration 43 used for real-time PCR amplification of tet (O). Table 2.4 Abundance of total aerobic and anaerobic bacterial populations 45 in swine feces. Brain Heart Infusion Agar (BHIA) and Tryptic Soy agar (TSA) were compared. Table 2.5 Yield and quality of total DNA extracted from bacterial strains 45 used as positive controls for PCR. Strains were preserved at -20 oC in 15% glycerol. Table 2.6 Yield and quality of total community DNA extracted from swine 47 feces. Table 2.7 Optimization of the PCR reaction mixture and conditions. 47 Table 2.8 Precision of standard curves during optimization of real-time 49 PCR reaction mixture and conditions for tet (O). Table 2.9 Linearity and efficiency of standard curves during optimization 50 of real-time PCR reaction mixture and conditions for tet (O). Table 3.1 Swine rearing and sampling. 62 Table 3.2 PCR detection of selected tetracycline [ tet (ABCDEKLMOSY)] 71 and macrolide [ erm (ABC)] resistance genes among bacterial populations in individual pigs during suckling, weanling, growing and finishing. Only genes detected are indicated. Table 3.3 Accuracy of standard curves for the real-time PCR assay for 73 tet (O) quantification. VII LIST OF FIGURES Figure 1.1 Transformation 8 Figure 1.2 Transduction 8 Figure 1.3 Conjugation 8 Figure 1.4 Pig inventories in Canada in quarterly year-to-year change 10 Figure 1.5 Uptake of tetracycline by Escherichia coli 16 Figure 1.6 Ribosome protection 18 Figure 1.7 Efflux proteins 20 Figure 1.8 Enzymatic inactivation 20 Figure 1.9 Chemical structures of some macrolide antibiotics 24 Figure 1.10 Food-borne illness 28 Figure 3.1 Enumeration of (A) Tot, (B) Tet R and (C) Tyl R anaerobic bacterial 69 populations in individual pigs during suckling, weanling, growing and finishing Figure 3.2 Abundance (A and B), evolution (C and D) and percentage (E 70 and F) of Tot, Tet R and Tyl R anaerobic bacterial populations in males (average for 6 males in panels A, C, E) and females (average for 4 females in panels B, D, F) during suckling (S), weanling (W), growing (G) and finishing (F) Figure 3.3 Abundance of tet (O) among bacterial populations in individual 75 pigs during suckling, weanling, growing and finishing. Figure 3.4 Average abundance of tet (O) among bacterial populations in 6 76 males and 4 females during suckling (S), weanling (W), growing (G) and finishing (F). VIII LIST OF ABBREVIATIONS 16S rRNA 16S subunit of ribosomal ribonucleic acid BHIA Brian-Heart Infusion Agar BSA Bovine Serum Albumin CFU Colony Forming Unit CO 2 Carbon Dioxide DNA Deoxyribonucleic Acid dNTP Deoxyribonucleotide Triphosphate EU European Union FQRNT Fonds Quebecois de Recherche sur la Nature et les Technologies H2 Hydrogen KCl Potassium Chloride LB Luria-Bertani MgCl 2 Magnesium Chloride MIC Minimal Inhibitory Concentration mRNA Messenger Ribonucleic Acid N2 Nitrogen Na 2S.9H 2O Sodium Sulfide Nonahydrate NADPH Nicotinamide Adenine Dinucleotide Phosphate NSERC Natural Sciences and Engineering Research Council PCR Polymerase Chain Reaction rRNA Ribosomal Ribonucleic Acid Tet R Tetracycline-Resistant Tris-HCl Tris (hydroxymethyl) Aminomethane tRNA Transfer Ribonucleic Acid TSA Tryptic Soy Agar Tyl R Tylosin-Resistant USDA United States Department of Agriculture IX ABSTRACT Since antibiotics have been added to animal feed for decades, food animals and their wastes constitute a reservoir of antibiotic-resistant bacteria.
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