THE ISOLATION AND GENOTYPIC CHARACTERISATION OF CAMPYLOBACTER JEJUNI FROM ENVIRONMENTAL MATRICES __________________ A thesis submitted in partial fulfilment of the requirements for the Degree of Master of Science in Microbiology in the University of Canterbury By P.M.L. Devane ____________________ University of Canterbury 2006 Campylobacter in environmental matrices i Table of Contents Table of Contents i List of Figures iv List of Tables v List of Abbreviations vi Abstract ix 1 Introduction 1 1.1 The Genus Campylobacter .......................................................................................1 1.1.1 Basic morphology and physiology of Campylobacter .........................................2 1.1.2 Characteristics of the Campylobacter genome .....................................................2 1.1.3 Incidence of species of Campylobacter implicated in campylobacteriosis ..........3 1.1.4 Survival of Campylobacter...................................................................................5 1.1.5 Viable but non-culturable bacteria........................................................................7 1.1.6 Antibiotic resistance of Campylobacter strains ..................................................10 1.2 Epidemiological aspects of Campylobacter infection ............................................12 1.2.1 Incidence of campylobacteriosis.........................................................................12 1.2.2 Symptoms of campylobacteriosis .......................................................................14 1.2.3 Complications associated with campylobacteriosis............................................14 1.2.4 Seasonality of Campylobacter............................................................................17 1.2.5 Age and gender distribution................................................................................18 1.2.6 Ethnicity..............................................................................................................18 1.2.7 Estimating the economic burden of campylobacteriosis ....................................19 1.3 The pathogenesis of Campylobacter.......................................................................20 1.4 Infective dose of Campylobacter............................................................................22 1.5 Risk factors associated with campylobacteriosis....................................................25 1.5.1 Cases of human campylobacteriosis associated with chicken consumption ......25 1.5.2 Outbreaks of campylobacteriosis........................................................................27 1.6 Research Model ......................................................................................................28 1.6.1 Objectives ...........................................................................................................28 1.6.2 Hypotheses..........................................................................................................30 1.6.3 Possible outcomes...............................................................................................31 1.7 Format of the thesis.................................................................................................32 2 Method development of enrichment PCR protocol 33 2.1 Introduction.............................................................................................................33 2.1.1 Campylobacter isolation from environmental matrices......................................33 2.1.2 Campylobacter prevalence in environmental matrices.......................................36 2.1.3 Objectives ...........................................................................................................44 2.2 Materials and methods ............................................................................................45 2.2.1 Media and reagents .............................................................................................45 2.2.2 Bacterial strains and culture conditions..............................................................45 2.2.3 Development of the multiplex PCR assay ..........................................................47 2.2.4 PCR template preparation...................................................................................48 2.2.5 Determination of the sensitivity of the PCR.......................................................49 2.2.6 Determination of the optimal enrichment broth for growth of C. coli and C. jejuni in a range of food, faecal and water matrices.......................................50 2.3 Results.....................................................................................................................56 2.3.1 Characteristics of the PCR developed ................................................................56 ii Campylobacter in environmental matrices 2.3.2 The optimal enrichment broth.............................................................................57 2.3.3 Determination of the detection limit of Campylobacter in the enriched matrices .............................................................................................................................60 2.4 Discussion ...............................................................................................................62 2.4.1 Development of the enrichment-PCR method....................................................62 2.4.2 Conclusions.........................................................................................................69 3 Application of the enrichment-PCR method to field studies 71 3.1 Introduction.............................................................................................................71 3.1.1 Methodology for the detection of Campylobacter..............................................71 3.1.2 Campylobacter prevalence in live chickens and chicken meat products............72 3.1.3 Counts per chicken carcass .................................................................................73 3.1.4 Water as a transmission route for Campylobacter..............................................74 3.1.5 Subtyping methodologies....................................................................................75 3.1.6 Objectives............................................................................................................80 3.2 Materials and methods ............................................................................................81 3.2.1 Media and reagents .............................................................................................81 3.2.2 Isolation and identification of campylobacters from whole chicken carcasses ..81 3.2.3 Collection and processing of river water samples ..............................................81 3.2.4 Subtyping of Campylobacter isolates from water by pulsed-field gel electrophoresis (PFGE).......................................................................................83 3.3 Results.....................................................................................................................86 3.3.1 Identification of target campylobacters in chicken carcass samples...................86 3.3.2 Prevalence of Campylobacter in river water.......................................................87 3.3.3 Seasonality of Campylobacter prevalence in water............................................87 3.3.4 Detection of multiple Campylobacter subtypes in individual samples...............89 3.4 Discussion ...............................................................................................................91 3.4.1 The prevalence of Campylobacter in chicken produce.......................................91 3.4.2 The detection of Campylobacter in river water samples ....................................92 3.4.3 Conclusions.........................................................................................................95 4 Multiple subtypes of C. jejuni in chicken carcasses 97 4.1 Introduction.............................................................................................................97 4.1.1 The relevance of multiple subtypes of bacterial species in individual samples..97 4.1.2 Statistical significance of multiple subtypes of bacterial species present in individual samples...............................................................................................99 4.1.3 Campylobacter subtype prevalence in chicken farms.......................................100 4.1.4 PFGE subtyping techniques..............................................................................100 4.1.5 Standardisation of PFGE between laboratories ................................................101 4.1.6 The stability of the Campylobacter genome in relation to genotypic subtyping methods .............................................................................................................102 4.1.7 Objectives..........................................................................................................104 4.2 Materials and methods ..........................................................................................105 4.2.1 Media and reagents ...........................................................................................105 4.2.2 Characterisation of C. jejuni from chicken carcasses .......................................105 4.2.3 Pulsed-field gel electrophoresis (PFGE)...........................................................107