molecular characterization of aeromonas hydrophila and antimicrobial activities of selected ...

248 Pages·2011·3.94 MB·English

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

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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PUBLISHED AND UNPUBLISHED ARTICLES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

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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
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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
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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
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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

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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

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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
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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
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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

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