38 dummy Obanda - PhD whole thesis draft 2 - 10.7.2018.doc correcrion 1.doc material method.doc 180. - Molecular epidemiology and methicillin-resistance

38 dummy Obanda - PhD whole thesis draft 2 - 10.7.2018.doc correcrion 1.doc material method.doc 180.

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Unformatted text preview: Molecular epidemiology and methicillin-resistance of Staphylococcus aureus isolated from hospital patients, abattoir workers and animals in Busia County Kenya POSSIBILITY OF HIV-POSITIVE ABATTOIR WORKERS BEING SOURCES OF LIVESTOCK-ASSOCIATED –MRSA FOR HUMAN INPATIENTS IN BUSIA, KENYA 1 Table of contents Contents Abbreviations...............................................................................................128 ABSTRACT..................................................................................................1410 INTRODUCTION..........................................................................................1713 1.0: Introduction.....................................................................................1713 1.1. Research questions..........................................................................2016 1.2. Hypotheses......................................................................................2016 1.2.2. Hypotheses................................................................................2016 1.2.2. Null hypotheses.........................................................................2016 1.3. Objectives........................................................................................2117 LITERATURE REVIEW..................................................................................2117 2.1 History of Staphylococcus aureus....................................................2117 2.2 Nomenclature of Staphylococcus aureus.........................................2218 2.3 Colonial Morphology and biochemical characterization of Staphylococcus aureus...........................................................................2319 2.4 Phenotypic MRSA detection..............................................................2319 2.5 Identification of Staphylococcus aureus..........................................2420 2.6: Diagnosis of Staphylococcus aureus................................................2420 2.6.1 Growth requirements and characteristics...................................2420 2.7 Specimens for diagnosis humans and livestock................................2622 2.8 Virulence factors and pathogenesis.................................................2723 2.8 .1 Background of Virulence factors and pathogenesis...................2723 2.8 .2 Cell surface factors....................................................................2824 2.8 .3 Secreted factors.........................................................................2925 2.9 Pathology caused by Staphylococcus aureus.................................3430 2.9 .1 Skin abscess..............................................................................3430 2.9 .2 Septicemia (blood poisoning).....................................................3632 2.9 .3 Staphylococcus aureus and Pneumonia....................................3733 2 2.9 .4 Osteomyelitis.............................................................................4440 2.10 . Staphylococcus aureus in veterinary medicine............................4743 2.10 .1 Introduction of Staphylococcus aureus in veterinary medicine .............................................................................................................4743 2.10 .2 Staphylococcus aureus infections in animals...........................4743 2.10 .3 Staphylococcus aureus infections in animals...........................4844 2.10 .4. Sources of Staph. aureus contamination................................5147 2.10 .6 The infective process of mastitis.............................................5248 2.10 .7 Prophylactic and control methods for mastitis.........................5349 2.10 .8 Prevention of S aureus in cattle...............................................5349 2.10 .9 Disinfecting surfaces or objects...............................................5349 2.10 .10 Hand washing........................................................................5450 2.10 .11 Staphylococcus aureus decolonization in human beings......5450 2.10 .12 Treatment in Animals.............................................................5551 2.11 Antibiotic target and mode of Resistance......................................5652 2.11 .1 Evolutional emergence of antibiotic resistance in Staph. aureus .............................................................................................................5652 2.11 .2 Sulphamethoxazole and trimethoprim (SXT)...........................5753 2.11 .3 Penicillin and Methicillin...........................................................6056 2.11 .4 Quinolones...............................................................................6359 2.11 .5 Vancomycin..............................................................................6561 2.11 .6 Daptomycin..............................................................................6965 2.11 .7 Oxazolidinones.........................................................................7066 2.11 .8 Tetracyclines............................................................................7167 2.11 .9 New generation of Tetracycline :Tigecycline............................7268 2.11 .9.1 Tigecycline Mode of action....................................................7268 2.11 .9.2 Tigecycline resistance mechanisms.....................................7268 2.11 .10 Macrolides and lincosamides.................................................7268 2.12 Molecular typing techniques used for characterisation..................7470 2.12 .1 Staphylococcal cassette chromosome mec (SCCmec) typing..7571 2.12 .2 Multilocus sequence typing......................................................7571 3 2.12 .3 Pulsed-field gel electrophoresis (PFGE)....................................7773 2.12 .4 Staphylococcal protein A (Spa) typing.....................................7975 2.12 .5 Whole genome analysis...........................................................8076 2.13 Phylogenetic tree...........................................................................8278 2.14 Evolution of MRSA clones................................................................8581 2.14 .1 Emergence of antibiotic resistant Staphylocccus aureus clones .............................................................................................................8581 2.14 .2 International spread of MRSA clones.......................................8682 2.14 .3 Hospital associated MRSA........................................................8783 2.14 .4 Community-associated MRSA..................................................9490 2.15 Antibiotics resistance.................................................................105101 2.16 Human Immunodeficiency Virus/ Acquired Immune Deficiency Syndrome and MRSA..........................................................................108104 2.17 Risk factors associated with Staphylococcus aureus infection in Human Immunodeficiency Virus/ Acquired Immune Deficiency Syndrome individuals...........................................................................................109105 MATERIALS AND METHODS....................................................................110106 3.0 Sampling design..........................................................................110106 3.1 Sample collection..........................................................................110106 3.2 Processing of collected samples...................................................111107 3.2.1 Identification of Staphylococcus aureus.................................111107 3.2.2 MRSA antibiotic susceptibility testing....................................111107 3.2.3. Molecular detection of Methicillin resistant (MecA) gene.......112108 3.3. PVL gene detection......................................................................118114 3.4. Multi Locus Sequence Typing.......................................................118114 3.5 Whole Genome sequencing..........................................................121117 3.5.1. Wet Lab..................................................................................121117 3.5.2 Dry-lab....................................................................................121117 3.6 Data Management........................................................................122118 3.7 Statistical analysis........................................................................122118 3.8 Ethical consideration....................................................................123119 RESULTS.................................................................................................125121 4 4.0 BCH monthly total admissions during study.................................125121 4.1 Descriptions of the Study Population Stratified by antibiotic empirical therapy...............................................................................................127123 4.2 Inpatients rearing various livestock in their household................129125 4.3 Staphylococcus aureus antibiotic resistance among inpatients admitted at BCH.................................................................................................132128 4.4 Multi drug resistance patterns of Staphylococcus aureus isolates obtained from nares of inpatients admitted at BCH............................135131 4.7 Antibiotic categories tested at BCH.............................................138134 4.8 Resistance of Staphylococcus aureus and Staphylococcus sciuiri nasopharyngeal isolates to Linezolid. Teicoplanin, Vancomycin in Busia County................................................................................................140136 4.9 MLST STs,PVL and TST genes identified amongst inpatients admitted at BCH.....................................................................................................142138 4.10 Difference of alleles found in ST 508 and a new ~ST 508 sequence type of single allele............................................................................143139 4.11 Comparison of MRSA new sequence type isolates from inpatients admitted at BCH with other known sequence types...........................144140 4.12 The prevalence of MRSA and MSSA amongst Abattoir workers in Busia County................................................................................................146142 4.13 Prevalence of antibiotic resistant MSSA and MRSA strains among HIVpositive and HIV-negative abattoir workers in Busia County.............149145 4.14 Multidrug resistance patterns of MSSA and MRSA isolates recovered from HIV - positive and HIV – negative abattoir workers in Busia County ............................................................................................................151147 4.15 Sequence types, PVL and TST genes identified amongst Abattoir workers in Busia County.....................................................................155151 4.16 Difference in association of MSSA, SXT resistance and associated STs among HIV - positive and HIV – negative abattoir workers..............159155 4.17 Novel clonal complex and new sequence type MSSA strains from Abattoir workers working in Busia County..........................................160156 4.18 Staphylococcus aureus isolated from animal nasopharyngeal samples in Busia County...................................................................................162158 4.19 Genetic diversity of MSSA STs in Busia Kenya in relation to other sequence types found in other African countries................................164160 5 4.20 Genetic diversity of MRSA STs in Busia Kenya in relation to other sequence type found in other African countries.................................166162 4.21 Phylogenetic analysis and transmission amongst livestock, inpatient and abattoir workers...........................................................................170166 4.22 Ancestral phylogenetic analysis of PVL gene..............................172168 4.23 Phylogenetic analysis of ST8 more possible transmission in BCH ............................................................................................................174170 4.24 Ancestral phylogenetic analysis of dfrG – trimethoprim resistance gene in Busia County..........................................................................176172 4.25 Phylogenetic analysis of ST CC1 more possible transmission in Busia County................................................................................................181177 DISCUSSION...........................................................................................183179 References.............................................................................................233229 Contents Abbreviations...................................................................................................3 ABSTRACT........................................................................................................3 INTRODUCTION................................................................................................3 1.0: Introduction...........................................................................................3 1.1. Research questions................................................................................3 1.2. Hypotheses............................................................................................3 1.2.2. Hypotheses......................................................................................3 1.2.2. Null hypotheses...............................................................................3 LITERATURE REVIEW........................................................................................3 2.0 History....................................................................................................3 2.1 Nomenclature of Staphylococcus aureus................................................3 2.2 Specimens for diagnosis humans and livestock......................................3 2.3 Colonial Morphology and biochemical characterization of Staphylococcus aureus.................................................................................3 2.4 Phenotypic MRSA detection....................................................................3 6 2.5 Identification of Staphylococcus aureus.................................................3 2.6 Virulence factors and pathogenesis........................................................3 2.6.1 Background of Virulence factors and pathogenesis..........................3 2.6.2 Cell surface factors...........................................................................3 2.6.3 Secreted factors................................................................................3 2.7 Clinical microbiology of MRSA.................................................................3 2.7.1 Skin abscess........................................................................................3 2.7.2 Septicemia (blood poisoning)............................................................3 2.7.3 Methicillin-resistant Staphylococcus aureus MRSA and Lungs..........3 2.7.4 Osteomyelitis....................................................................................3 2.7. Staphylococcus aureus in veterinary medicine.....................................3 2.7.1 Introduction of Staphylococcus aureus in veterinary medicine.......3 2.7.2 Staphylococcus aureus infections in animals....................................3 2.7.3 Staphylococcus aureus infections in animals....................................3 2.7.4 Staphylococcus aureus infections in poultry.....................................3 2.7.5. Sources of contamination................................................................3 2.7.6 The infective process........................................................................3 2.7.7 Prophylactic and control methods.....................................................3 2.7.8 Prevention of S aureus in cattle........................................................3 2.7.9 Disinfecting surfaces or objects........................................................3 2.7.10 Hand washing.................................................................................3 2.7.11 Decolonization................................................................................3 2.7.12 Treatment in Animals......................................................................3 2.8 Antibiotic target and mode of Resistance...............................................3 2.8.1 The evolutional emergence of antibiotic resistance in Staph . aureus ...................................................................................................................3 2.8.2 Sulphamethoxazole and trimethoprim (SXT)....................................3 2.8.3 Penicillin and Methicillin....................................................................3 2.8.4 Quinolones........................................................................................3 2.8.5 Vancomycin.......................................................................................3 2.8.6 Daptomycin.......................................................................................3 7 2.8.7 Oxazolidinones..................................................................................3 2.8.8 Tetracycline.......................................................................................3 2.8.9 Tigecycline........................................................................................3 2.8.10 Macrolides and lincosamides..........................................................3 1.9 Molecular typing techniques...................................................................3 2.9.1 Staphylococcal cassette chromosome mec (SCCmec) typing...........3 2.9.2 Multilocus sequence typing...............................................................3 2.9.3 Pulsed-field gel electrophoresis (PFGE).............................................3 2.9.4 Staphylococcal protein A (Spa) typing..............................................3 2.9.5 Whole genome analysis....................................................................3 2.10 Phylogenetic tree thesis........................................................................3 2.11 Evolution of MRSA clones......................................................................3 2.10.1 Emergence of antibiotic resistant Staph . aureus clones................3 2.10.2 International spread of MRSA clones..............................................3 2.10.3 Hospital associated MRSA...............................................................3 2.10.4 Community-associated MRSA.........................................................3 2.11 Antibiotic, Agriculture, Hospital, Environment, Community..................3 2.12 Human Immunodeficiency Virus/ Acquired Immune Deficiency Syndrome and MRSA....................................................................................3 2.13 Risk factors associated with Staphylococcus aureus infection in Human Immunodeficiency Virus/ Acquired Immune Deficiency Syndrome individuals.....................................................................................................3 MATERIAL AND METHOD..................................................................................3 3.1 Sample collection....................................................................................3 3.2 Processing of collected samples.............................................................3 3.2.1 Identification of Staph . aureus.........................................................3 3.2.2 Antibiotic susceptibility testing for identification of MRSA................3 3.2.3. Molecular detection of Methicillin resistant (MecA) gene.................3 3.3. PVL gene detection................................................................................3 3.4. Multi Locus Sequence Typing.....
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  • Fall '18
  • dr opanga
  • Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, VE Abattoir

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