Molecular detection and study of Campylobacter and related microorganisms Nisreen Hoosain A thesis submitted in partial fulfillment of the requirements for the degree of Doctor Philosophiae in the Department of Biotechnology, University of the Western Cape. Supervisor: Professor Pieter Andries Gouws Co-supervisor: Professor Albert Joseph Lastovica November 2010 i DECLARATION I declare that “Molecular detection and study of Campylobacter and related microorganisms” is my own work, that it has not been submitted before for any degree or examination in any other university, and that all the sources I have used or quoted have been indicated and acknowledged as complete references. Nisreen Hoosain November 2010 Signed:………………………. ii ACKNOWLEDGEMENTS First and foremost, I wish to thank Allah for giving me the strength, wisdom and guidance during my PhD and allowing me to obtain my PhD degree. I would like to thank my supervisor, Prof. P.A. Gouws, and my co-supervisor, Prof. A.J. Lastovica for their support, guidance, encouragement and advice. A special thanks to my colleagues in the laboratory as well as all staff in the Biotechnology Department at the University of the Western Cape. Thank you for your friendship, advice and making my stay an enjoyable one. To my beloved parents, brother, sister in-law, niece, nephew and other family members: thank you for your prayers, love, understanding, encouragement and support through the years. It is greatly appreciated, I am truly blessed! In addition, I would like to extend my sincere gratitude to my financial assistants: Department of Biotechnology at the University of the Western Cape, the National Research Foundation (NRF) of South Africa, Muslim Hands of South Africa and the South African National Zakah Fund (SANZAF). iii CONTENTS Title page i Declaration ii Acknowledgements iii Contents iv-xiv List of figures xv-xvi List of tables xvii-xviii List of abbreviations xix-xxi List of units xxii-xxiii List of publications/conference contributions emanating from the work reported in this thesis xxiv-xxvii Abstract xxviii-xxix Chapter 1: INTRODUCTION INTRODUCTION 1 OUTLINE OF THESIS 1 IMPORTANCE OF STUDIES INCLUDED 1-2 REASON FOR CHOSEN STUDIES 2-4 AIMS OF CHAPTERS 4-5 FRAMEWORK OF CHAPTERS 5 iv RESEARCH DESIGN OF STUDIES 5-7 CONCLUSIONS 7-8 REFERENCES 8-11 Chapter 2 (Literature Review) Identification of Campylobacter and related microorganisms in chicken and in vitro testing of strains SUMMARY 12 INTRODUCTION 12-13 HISTORY 13-16 BRIEF DISEASE SPECTRUM 17-18 ISOLATION 18-22 DETECTION 22-38 Genus-specific and species-specific PCR 22-24 Multiplex PCR 24-26 Real-time PCR 26-28 Polymerase Chain Reaction Enzyme Linked Immunosorbent Assay (PCR-ELISA) 28-29 DNA oligonucleotide array and Loop mediated isothermal Amplification (LAMP) assays 29-30 Commercial Assays 30-32 PCR inhibitors 32-34 v Pre-PCR processing strategies 34-37 Internal Amplification Control 37-38 TYPING 38-43 Multilocus Sequence Typing 39 Restriction Fragment Length Polymorphism 39-40 Enterobacterial Repetitive Intergenic Consensus and Randomly Amplified Polymorphic DNA PCR 40 Pulsed Field Gel Electrophoresis 41 Amplified Fragment Length Polymorphism 41-42 Multiple typing techniques 42-43 SURVIVAL CAPACITY OF CAMPYLOBACTER, ARCOBACTER AND HELICOBACTER 44-50 Survival in liquids and on surfaces 44-47 Survival in poultry produce, diary and water 47-50 TREATMENT STRATEGIES 50-69 Antibiotics 50-52 Preservatives 53 Bacteriophage therapy 53-55 Bacteriocin treatment 55-56 Competitive exclusion 56-58 In vitro and in vivo inhibitory effects of various culinary products 58-69 Garlic and onions 58-60 vi Ginger 60-62 Parsley 62 Thyme 62 Cardamom 62 Lemon 63 Honey 64-65 Black cumin seeds 65 Chilies 65-66 Black pepper 66 Cinnamon 66-68 Allspice 68 Clove 68 Turmeric 68-69 CONCLUSION 69 REFERENCES 70-113 Chapter 3 Evaluation of a new method to efficiently remove PCR inhibitors from poultry DNA samples ABSTRACT 114 INTRODUCTION 114-117 MATERIALS AND METHODS 117-123 vii Preparation of genomic DNA 117-118 Hexadecyltrimethyl ammonium bromide (CTAB) method 118-119 Sample DNA extractions 119-120 Construction of a competitive Internal Amplification Control (IAC) 120 Screening of competitive IAC clones 120-121 Removal of PCR inhibitors 121-122 Multiplex PCR 122-123 Sequence analysis 123 RESULTS 124-128 Ability to detect possible multiple infections of Arcobacter, Campylobacter and Helicobacter in poultry DNA samples 124 Minimum Inhibitory Concentration (MIC) for the competitive IAC 124-125 Amplification of gDNA samples 125-126 Removal of PCR inhibitors 126-128 DISCUSSION 128-131 Conclusion 131 ACKNOWLEDGEMENTS 131 REFERENCES 131-136 viii Chapter 4 Detection and isolation of Campylobacteraceae in retail and “Free- range” organic chicken ABSTRACT 137-138 INTRODUCTION 138-140 MATERIALS AND METHODS 140-153 Revitalization of reference strains 140-141 Samples tested 141 Isolation of Campylobacteraceae 141-144 Phenotypic and Biochemical characterization of isolates 145-146 Preparation of genomic DNA (gDNA) 146 CTAB method 146-147 Sample gDNA extractions 147-148 Construction of a competitive Internal Amplification Control (IAC) 148-149 Screening of competitive Internal Amplification Control (IAC) clones 149 Removal of PCR inhibitors 149-150 Multiplex PCR 150-151 Sequence analysis 151-152 ix Amplified Fragment Length Polymorphism analysis of samples 152-153 RESULTS 154-159 Phenotypic and Biochemical characterization of isolates 154-155 Multiplex PCR of samples with competitive IAC 155-156 Phylogenetic tree based on the 16S rRNA gene of Arcobacter 157 Phylogenetic tree based on the 16S rRNA gene of Campylobacter 158 AFLP analysis of positive samples and isolates from chicken products and blood 158-159 DISCUSSION 160-167 Conclusion 166-167 ACKNOWLEDGEMENTS 167 REFERENCES 167-179 Chapter 5 In vitro Survival test of Campylobacteraceae and Helicobacter species in “free-range”/organic chicken blood at various temperatures ABSTRACT 180-181 INTRODUCTION 181-182 x MATERIALS AND METHODS 183-186 Revitalization of strains 183 Maintenance of pure cultures 183-184 Inoculation and incubation of strains in chicken blood 184-185 Determining the survival of strains at tested temperatures 185-186 RESULTS 186-189 DISCUSSION 190-194 Conclusion 193-194 ACKNOWLEDGEMENTS 194 REFERENCES 194-201 Chapter 6 Inhibitory effect of selected culinary products versus antibiotics against the growth of Campylobacteraceae and Helicobacter species ABSTRACT 202-203 INTRODUCTION 203-205 MATERIALS AND METHODS 205-210 Strains tested 205 Optimal method to analyze the effect of a few culinary products on the growth of Campylobacteraceae and Helicobacter strains 206-207 Preparation and testing of various culinary products 207-209 xi Antibiotic Testing 210 RESULTS 211-216 Optimal method to analyze the effect of a few culinary products on the growth of Campylobacteraceae and Helicobacter strains 211 Effects of culinary products versus antibiotics on the growth of Campylobacteraceae and Helicobacter strains 211-216 DISCUSSION 217-233 Absolute Ethanol (positive control) 217 Garlic 217-219 Ginger 219-221 Dried Parsley 221 Dried Thyme 221-222 Cardamom 222 Fresh Lemon juice 222-223 Honey 223-224 Crushed onion 224-225 Black cumin seeds 225-226 Crushed chilies 226 Ground Black Pepper 226-227 Cinnamon 227-228 Allspice and cloves 228-229 Turmeric 229-230 xii Inhibitory effects of antibiotics 230-233 Conclusion 232-233 ACKNOWLEDGEMENTS 233 REFERENCES 233-247 Chapter 7: CONCLUSION AND FUTURE PROSPECTS BACKGROUND 248-250 Isolation and detection of the target microorganisms using the IAC 248-249 Survival of Campylobacteraceae and Helicobacter strains in blood 249 Treatment of Campylobacteraceae and Helicobacter strains 249-250 ACHIEVEMENT OF AIMS 250-253 Evaluation of a new method to efficiently remove PCR inhibitors from poultry DNA samples 250-251 Detection and isolation of Campylobacteraceae in retail and “Free-range”/organic chicken 251-252 In vitro Survival test of Campylobacteraceae and Helicobacter species in “free-range” organic/chicken blood at various temperatures 252 Inhibitory effect of selected culinary products versus antibiotics against the growth of Campylobacteraceae and Helicobacter species 253 xiii FUTURE PROSPECTS 253-255 Evaluation of a new method to efficiently remove PCR inhibitors from poultry DNA samples 253-254 Detection and isolation of Campylobacteraceae in retail and “Free-range” organic chicken 254 In vitro Survival test of Campylobacteraceae and Helicobacter species in “free-range”/organic chicken blood at various temperatures 254 Inhibitory effect of selected culinary products versus antibiotics against the growth of Campylobacteraceae and Helicobacter species 255 REFERENCES 255-259 APPENDIX A: Gels and Buffers 260-262 APPENDIX B: Solutions, Media and Media Additives 263-268 APPENDIX C: Molecular weight marker and Cloning vector 269-271 APPENDIX D: The “Cape Town Protocol” 272-282 APPENDIX E: Detailed information on isolates used in this thesis 283 xiv LIST OF FIGURES CHAPTER 3: Evaluation of a new method to efficiently remove PCR inhibitors from poultry DNA samples Figure 1: A 2% agarose gel depicting the amplification of the reference fragments of Arcobacter (822-bp), Campylobacter (946-bp) and Helicobacter (1107-bp) 124 Figure 2: A 2% agarose gel depicting the amplification of the 580-bp fragment of the competitive IAC 125 Figure 3: Representative 2% agarose gel illustrating the effect of PCR inhibitors on the amplification of DNA extracted with a Qiagen DNeasy Blood and Tissue kit (Qiagen,