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Molecular Characterization of Aeromonas Hydrophila

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Molecular Characterization of Aeromonas Hydrophila MOLECULAR CHARACTERIZATION OF AEROMONAS HYDROPHILA AND ANTIMICROBIAL ACTIVITIES OF SELECTED MEDICINAL PLANTS AGAINST PATHOGENIC ISOLATES FROM WATER AND STOOL SAMPLES IN THE ERA OF HIV/AIDS IN LIMPOPO PROVINCE, SOUTH AFRICA by NALEDZANI JEOFFRY RAMALIVHANA submitted in accordance with the requirements for the degree of DOCTOR OF PHILOSOPHY in the subject ENVIRONMETAL SCIENCE at the UNIVERSITY OF SOUTH AFRICA SUPERVISOR: PROF C L OBI MAY 2010 i TITLE PAGE DECLARATION DEDICATION ACKNOWLEDGEMENTS SUMMARY TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF SYMBOLS AND ABBREVIATIONS PUBLISHED AND UNPUBLISHED ARTICLES CHAPTER 1: GENERAL INTRODUCTION AND LITERATURE REVIEW CHAPTER2:ANTIMICROBIAL SUSCEPTIBILITY TESTING OF AEROMONAS HYDROPHILA ISOLATED FROM LIMPOPO PROVINCE, SOUTH AFRICA USING VITEK 2 SYSTEM, MICROSCAN WALKAWAY, DISK DIFFUSION AND E-TEST METHODS CHAPTER 3: EXTENDED-SPECTRUM Β-LACTAMASES IN AEROMONAS HYDROPHILA ISOLATED FROM STOOL SAMPLES IN THE LIMPOPO PROVINCE, SOUTH AFRICA CHAPTER 4: ISOLATION AND CHARACTERIZATION OF CLASS 1 INTEGRONS GENES IN CLINICAL AEROMONAS HYDROPHILA ISOLATES FROM LIMPOPO PROVINCE, SOUTH AFRICA CHAPTER 5: RANDOM AMPLIFIED POLYMORPHIC DNA TYPING OF CLINICAL AND ENVIRONMENTAL OF AEROMONAS HYDROPHILA ISOLATED IN THE LIMPOPO PROVINCE, SOUTH AFRICA CHAPTER 6: ANTIBACTERIAL ACTIVITY PROFILES OF PYRENACANTHA GRANDIFLORA BAILL AND FICUS SYCOMORUS USED IN RURAL VENDA COMMUNITIES IN SOUTH AFRICA AGAINST AEROMONAS HYDROPHILIA CHAPTER 7: GENERAL CONCLUSIONS AND RECOMMENDATIONS ii APPENDICES mm Milimiter µl Microlitre µg Microgram °C Degree celcius % Percentage AIDS Acquired immune deficiency syndrome AMK Amikacin AMP Ampicillin AFLP Amplified fragment length polymorphism AST Antimicrobial susceptibility testing ATCC American type culture collection AZT Aztreonam Bp Based pair BA Blood agar BHI Brain heart infusion BPW Buffered peptone water Bfp Bundle-forming pili CF Cefazolin CFP Cefepime CTX-M Cefotaximase CFT Cefotaxime CTZ Ceftazidime CTX Ceftriaxone CPM Cefuroxime CPN Cephalothin CphA Cephalosporinase A CIP Ciprofloxacin iii CA Clavulanic Acid CFU/mL Colony forming units per millilier CMT Complex mutants of TEM CS Conserved segment CLED Cysteine Lactose Electrolytes Deficient CLSI Clinical and Laboratory Standards Institute DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic acid EDTA Ethylene diamine tetraacetic acid ERT Ertapenem ESBLs Extended-spectrum ß-lactamases GCAT Glycerophospholipid cholesterol acyltransferase GES Guiana extended spectrum GM Gentamicin GNB Gram-negative bacilli HCL Hydrochlroric acid HGs Hybridization groups HGT Horizontal gene transfer HIV Human immunodeficiency virus IMI Imipenem Kda Kilodaltons LCR Ligase chain reaction LTD Limited MBC Minimum Bactericidal Concentrations MDR Multiple drug resistance MEM Meropenem MIC Minimum inhibitory contraction MIN Minutes iv MLST Multilocus sequence typing Mm Milimolar N-CO Nitrogen-carboxyl NCCLS National Committee for Clinical Laboratory Standards N Number ND Not Done NI Nitrofurantoin O Somatic antigen Omps Outer membrane proteins OXA Oxacillinase PRL Piperacillin PIP Pipemidic acid PBP Penicillin bending protein PCR Polymerase chain reaction PER Beta -lactamse named after (P.Nordmann, E .Ronco,R.Labia) PFGE Pulsed-filed gel electrophoresis PG Prostaglandin PNA Peptide nucleic acid RFLP Restriction fragment length polymorphism RPM Revolutions per minute RAPD Random amplified polymorphic DNA RFLP PCR-Restriction fragment length polymorphism Rrna Ribosomal ribonucleic acid SAS Statistical analysis system SDS Sodium dodecyl sulfate Sesbl Stool extended spectrum beta-lactames SHV Sulphydryl variabr SSCP Single–strand conformational polymorphism v TCBS Thiosulfate citrate bile salts sucrose TE Tris-EDTA TEM Temoniera TN Tobramycin VEB Vietnamese extended spectrum betalactames WHO World health organisation XDCA Xylose deoxycholate citrate agar vi LIST OF TABLES Table 1.1 Summarizes important molecular techniques that can be used for the 51 detection of ESBLs in Gram-negative bacteria Table 1.2 Various techniques, principle, advantages and disadvantages 53 Table 2.1 Comparison of three different susceptibility methods on isolates of 104 Aeromonas hydrophila isolated from stool samples Table 2.2 Percentage susceptibility of isolates of Aeromonas hydrophila from stool 107 samples Table 2.3 MICs of isolates Aeromonas hydrophila from stool samples 110 Table 2.4 Comparison of three different methods for characterisation of 112 Aeromonas hydrophila from water samples Table 2.5 Percentage of antibiotic susceptibility of Aeromonas hydrophila from 115 water samples Table 2.6 MIC (µg/ml) of Aeromonas hydrophila from water samples 118 Table 3.1. Primers used in the study 130 Table 3.2 PCR products after amplification with the TEM primers 135 Table 3.3 PCR products after amplification with the SHV primers 137 Table 3.4 PCR products after amplification with the CMY-2 primers 139 Table 3.5 PCR products after amplification with the CTX-M primers 141 Table 5.1 Minimum inhibitory concentration ranges of the isolates to Antibiotics 195 Table 5.2 Random Amplified Polymorphic DNA profiles 196 Table 6.1 Ethnobotanical information of selected Venda medicinal plants used in 210 the study Table 6.2 Antimicrobial Activity of Medicinal Plant Extracts against isolates of 212 Aeromonas hydrophila Table 6.3 Percentage susceptible of all tested species of aeromonas hydrophila 212 against medicinal plants vii List of Figures Figure 1.1 Illustration of the mechanisms involved in genetic exchange among bacteria showing transformation, conJugation and transduction 18 Figure 1.2 Illustration of bacterial mechanisms of antibiotic resistance in Staphylococcus aureus , Escheriachia coli , Pseudomonas aeroginosa and 20 lactobacillus latis Figure 1.3 Illustration of mechanisms of antibiotic resistance. 22 Figure 1.4 Illustration different aspects of beta-lactamases in Gram-positive and Gram-negative cell wall structures 29 Figure 1.5 Illustration the Structure of Peptidoglycan in beta-lactam antibiotic 29 Figure 1.6 Illustration of Antibiotic Targets Sites 30 Figure 1.7 The β-Lactam Ring 30 Figure 1.8 The Structure of Penicillin 31 Figure 1.9 Structure of Penicillin-binding Proteins 31 Figure 1.10 The Structure of Clavulanic Acid 32 Figure 1.11 NCCLS-style disc diffusion confirmatory test for ESBL production using cefpodoxime 45 Figure 1.12 Double-disc diffusion test for the detection of ESBL production in Klebsiella pneumoniae ATCC 700603 (ESBL-positive control strain). 46 Figure 1.13 Clear cut ESBL positive: MIC ratio of ceftazidime/ceftazidime + clavulanate 46 viii Figure 1.14 A rounded ‘phantom’ inhibition zone below cefepime end indicative of ESBL 47 Figure 1.15 MIC values and interpretation parameters 47 Figure 1.16 Deformation of the cefepime inhibition ellipse indicative of ESBL 48 Figure 3.1 Detection and identification of A. hydrophila Tem 63 genes by amplification of fragments 134 Figure 3.2 Detection and identification of A. hydrophila SHV genes by amplification of fragments 136 Figure 3.3 Detection and identification of A. hydrophila CMY-2 genes by amplification of fragments 138 Figure 3.4 Detection and identification of A. hydrophila CTX-M genes by amplification of fragments 140 Figure 4.1 consensus sequence 169 Figure 4.2 Class 1 integron sequence data maps 170 Figure 4.5.1 The integron deposited in the genbank under accession number 171 Figure 5.1 Gel electrophoresis depicting the RAPD banding pattern obtained with 197 primer H1 Figure 5.2 Antimicrobial activtites of all tested isolates from water and stool 198 samples Figure 5.3 Dendogram obtained through RAPD typing with primers H1, 272 and 275 depicting the relationship between A.hydrophila stool isolates and water 199 isolates obtained from Limpopo Province, South Africa Figure 5.4 Dendogram obtained through RAPD typing with primers 277, H1 and 275 200 depicting the relationship between A.hydrophila stool isolates and water isolates obtained from Limpopo Province, South Africa Figure 6.1 Activity of varying concentrations of P.grandiflora extracts against selected isolates of A. hydrophila using methanol extract 213 ix Figure 6.2 Activity of varying concentrations of P.grandiflora extracts against selected isolates of A. hydrophila using hexane extract 213 Figure 6.3 Activity of varying concentrations of P.grandiflora extracts against selected isolates of A. hydrophila using acetone extract 213 Figure 6.4 Activity of varying concentrations of F.sycomorus extracts against selected isolates of A. hydrophila using acetone extract 214 Figure 6.5 Activity of varying concentrations of F.sycomorus extracts against selected isolates of A. hydrophila using hexane extract 214 Figure 6.6 Activity of varying concentrations of F.sycomorus extracts against selected isolates of A. hydrophila using methanol extract 215 x Chapter one: General Introduction and Literature review 1.1 Introduction and Literature Review 1 1.2 Description of the Genus 1 1.3 Occurrence 4 1.4 Virulence associated and health effects in humans 4 1.5 Health Effects in Animals 7 1.6 Risk Assessment 7 1.7 History of Aeromonas species 8 1.8 Treatment of Gastrointestinal Aeromonas infections 13 1.8.1 Use of Antibiotics 16 1.8.2 Antibiotic resistance 17 1.8.2.1 Development and acquisition of resistance 17 1.8.2.2 Mechanisms of antibiotic resistance 19 1.9 Reduced permeability 20 1.10 Efflux 21 1.11 The extended –spectrum β-Lactamases and β-Lactam antibiotics in bacteria 22 1.11.1 Types of ESBLs in bacteria 27 1.12 Role of plasmids in resistance 32 1.13
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