Chau Sze-lok. / Thesis submitted in: December 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 221-245). / Abstracts in English and Chinese. / ABSTRACT (English) --- p.i / ABSTRACT (Chinese) --- p.iii / ACKNOWLEDGMENT --- p.v / LIST OF CONTENTS --- p.vii / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xvii / ABBREVIATIONS --- p.xx / TERMS --- p.xxi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / (PART A) / Chapter 1.1 --- Acinetobacter spp --- p.1 / Chapter 1.2 --- Clinical importance of Acinetobacter --- p.4 / Chapter 1.3 --- Resistance mechanisms / Chapter 1.3.1 --- Intrinsic resistance --- p.7 / Chapter 1.3.2 --- Acquired resistance --- p.15 / Chapter 1.4 --- Resistance in Acinetobacter --- p.21 / Chapter 1.4.1 --- The efflux system in Acinetobacter --- p.22 / Chapter 1.4.2 --- Other antibiotic resistance mechanisms in Acinetobacter --- p.23 / (PART B) / Chapter 1.5 --- Methods used in this study --- p.29 / Chapter 1.6 --- Rationale of this study --- p.35 / Chapter 1.7 --- Objectives --- p.37 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.39 / Chapter 2.1 --- Bacterial strains and isolates / Chapter 2.1.1 --- Isolates for studying blaIMP-4 --- p.39 / Chapter 2.1.2 --- Isolates for studying adeB --- p.39 / Chapter 2.1.3 --- Isolates for investigation of other efflux pump(s)in Acinetobacter GDG3 --- p.40 / Chapter 2.1.4 --- Isolates for studying the distribution of efflux pumps --- p.40 / Chapter 2.1.5 --- Reference strains --- p.41 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Sources of materials --- p.42 / Chapter 2.2.2 --- Buffers and solutions --- p.45 / Chapter 2.3 --- Instruments and software --- p.46 / Chapter 2.4 --- General bacteriological techniques / Chapter 2.4.1 --- Bacteriological dientification --- p.47 / Chapter 2.4.2 --- Stock isolates --- p.48 / Chapter 2.4.3 --- Retrieval of isolates --- p.48 / Chapter 2.5 --- General molecular biology techniques / Chapter 2.5.1 --- Agarose gel electrophoresis --- p.49 / Chapter 2.5.2 --- Polymerase chain reaction (PCR) --- p.50 / Chapter 2.5.3 --- Amplified Ribosomal Restriction DNA Analysis (ARDRA) --- p.51 / Chapter 2.5.4 --- Pulsed field gel electrophoresis (PFGE) --- p.53 / Chapter 2.5.5 --- Minimal inhibitory concentration (MIC) --- p.55 / Chapter 2.5.6 --- Antibiotic sensitivity test - disc diffusion test --- p.56 / Chapter 2.5.7 --- Detection of the presence of the common resistance mechanisms --- p.57 / Chapter 2.5.8 --- TA Cloning --- p.60 / Chapter 2.5.9 --- DNA Sequencing --- p.62 / Chapter 2.5.10 --- Sequence analysis --- p.64 / Chapter 2.5.11 --- CYBR Green Assay --- p.65 / Chapter 2.5.12 --- Complementary DNA (cDNA) preparation --- p.66 / Chapter 2.5.13 --- Real time RT-PCR --- p.67 / Chapter 2.5.14 --- Construction of Genome Walker Libraries --- p.69 / Chapter 2.6 --- "Selection of acinetobacters from ICU, blood culture and other clinical isolates" / Chapter 2.6.1 --- Isolates from existing stock cultures --- p.71 / Chapter 2.6.2 --- New isolates obtained for this study --- p.71 / Chapter 2.7 --- Study of expression level of the blaIMP-4 gene / Chapter 2.7.1a --- Verification of the specificity of primers for blaIMP-4 --- p.72 / Chapter 2.7.1b --- Verfication of the specificity of primers for 16S rRNA gene --- p.73 / Chapter 2.7.1c --- Construction of standard curve --- p.76 / Chapter 2.7.2 --- Expression levels of blaIMP-4 and meropenem MICin blaIMP-4+ blood culture isolates --- p.77 / Chapter 2.7.3 --- Intra-assay reproducibility --- p.11 / Chapter 2.7.4 --- Detection of the production of metallo-β-lactamase --- p.77 / Chapter 2.8 --- Study of adeABC expression / Chapter 2.8.1 --- Determination of the presence of the adeB gene --- p.78 / Chapter 2.8.2 --- Entirety of the adeABC operon --- p.79 / Chapter 2.8.3 --- Expression level of the adeB gene --- p.82 / Chapter 2.8.4 --- Expression levels of adeB in sets of serial isolates --- p.84 / Chapter 2.8.5 --- Intra-assay reproducibility --- p.84 / Chapter 2.8.6 --- Inter-assay reproducibility --- p.84 / Chapter 2.9 --- Investigation of other efflux pumps in acinetobacter genomic DNA group3 / Chapter 2.9.1 --- Detection of adeB homologue in a genomic DNA group 3isolate --- p.85 / Chapter 2.9.2 --- Chromosome walking of the adeB-like genes --- p.87 / Chapter 2.9.3 --- Sequences of AdeE and AdeY and their comparison --- p.105 / Chapter 2.9.4 --- Topology prediction of AdeE and AdeY --- p.105 / Chapter 2.9.5 --- The role of the putative pump AdeE --- p.106 / Chapter 2.10 --- Distribution of AdeB and the putative efflux pumps AdeE and AdeY in acinetobacters from different bacterial collections / Chapter 2.10.1 --- Distribution of adeB and the putative pumps (adeE and adeY) in blood cultures (1997-2000) --- p.113 / Chapter 2.10.2 --- Confirmation of the identity of the amplification products of adeE and ade Y in blood culture isolate (1997-2000) --- p.115 / Chapter 2.10.3 --- The presence of adeE in GDG 3 acinetobacters from different sources --- p.116 / Chapter 2.10.4 --- "The presence of adeB, adeE and adeY in antibiotic susceptibility" --- p.116 / Chapter 2.10.5 --- "adeB, adeE and adeY and the clonally and epidemiologically related sets of isolates" --- p.116 / Chapter 2.10.6 --- "adeB, adeE and adeY and the blaIMP-4+ isolates" --- p.116 / Chapter CHAPTER 3 --- "SELECTION OF ACINETOBACTERS FROM ICU, BLOOD CULTURE AND OTHER CLINICAL ISOLATES" --- p.119 / Chapter 3.1 --- Results / Chapter 3.1.1 --- Isolates from existing stock cultures --- p.119 / Chapter 3.1.2 --- New isolates obtained for this study --- p.127 / Chapter 3.2 --- Discussion / Chapter 3.2.1 --- Identification of clonally related isolates by PFGE --- p.129 / Chapter 3.2.2 --- Correlation between the presence of common resistance mechanisms and the changes in antimicrobial susceptibility --- p.129 / Chapter 3.2.3 --- Development of resistance in serial isolates --- p.131 / Chapter CHAPTER 4 --- STUDY OF blaIMP-4 EXPRESSION --- p.133 / Chapter 4.1 --- Results / Chapter 4.1.1 --- Study of expression level of the blaIMP-4 gene --- p.134 / Chapter 4.1.2 --- Expression levels of blaIMP-4 and meropenem MIC in blaIMP-4+ blood culture isolates --- p.136 / Chapter 4.1.3 --- Intra-assay reproducibility \ --- p.37 / Chapter 4.1.4 --- Detection of the production of metallo-β-lactamase --- p.140 / Chapter 4.2 --- Discussion / Chapter 4.2.1 --- Dissociation curve --- p.140 / Chapter 4.2.2 --- Reproducibility of real time RT-PCR --- p.140 / Chapter 4.2.3 --- Relationship between mRNA level of blaIMP-4 and the meropenem MIC --- p.142 / Chapter CHAPTER 5 --- STUDY OF adeABC EXPRESSION --- p.145 / Chapter 5.1 --- Results / Chapter 5.1.1 --- Determination of the presence of the adeB gene --- p.145 / Chapter 5.1.2 --- Entirety of the adeABC operon --- p.146 / Chapter 5.1.3 --- Expression level of the adeB gene --- p.148 / Chapter 5.1.4 --- Expression levels of adeB in sets of serial isolates --- p.151 / Chapter 5.1.5 --- Intra-assay reproducibility --- p.154 / Chapter 5.1.6 --- Inter-assay reproducibility --- p.154 / Chapter 5.2 --- Discussion / Chapter 5.2.1 --- Detection of adeB --- p.156 / Chapter 5.2.2 --- Entirety of the adeABC operon --- p.156 / Chapter 5.2.3 --- Reproducibility of real time RT-PCR --- p.157 / Chapter 5.2.4 --- Relationship between adeB-mRNA level and antimicrobial susceptibility --- p.157 / Chapter CHAPTER 6 --- INVESTIGATION OF OTHER EFFLUX PUMPS IN ACINETOBACTER GENOMIC DNA GROUP3 --- p.159 / Chapter 6.1 --- Results / Chapter 6.1.1 --- Detection of adeB homologue in a genomic DNA group3 isolate --- p.159 / Chapter 6.1.2 --- Chromosome walking of the adeB-like genes --- p.162 / Chapter 6.1.3 --- Sequences of AdeE and AdeY and their comparison --- p.173 / Chapter 6.1.4 --- Topology prediction of AdeE and AdeY --- p.175 / Chapter 6.1.5 --- The role of the putative pump AdeE --- p.177 / Chapter 6.2 --- Discussion / Chapter 6.2.1 --- The AdeE RND transporter --- p.181 / Chapter 6.2.2 --- The theoretical AdeY protein --- p.183 / Chapter CHAPTER 7 --- DISTRIBUTION OF AdeB AND THE PUTATIVE EFFLUX PUMPS AdeE and AdeY IN ACINETOBACTERS FROM DIFFERENT BACTERIAL COLLECTIONS --- p.184 / Chapter 7.1 --- Results / Chapter 7.1.1 --- Distribution of adeB and the putative pumps (adeE and ade Y) in blood cultures (1997-2000) --- p.184 / Chapter 7.1.2 --- Confirmation of the identity of the amplification products of adeE and adeY in blood culture isolates (1997-2000) --- p.187 / Chapter 7.1.3 --- The presence of adeE in GDG 3 acinetobacters from different sources --- p.195 / Chapter 7.1.4 --- "The presence of adeB, adeE and ade Y in antibiotic susceptibility" --- p.196 / Chapter 7.1.5 --- "adeB, adeE and adeY and the clonally and epidemiologically related sets of isolates" --- p.202 / Chapter 7.1.6 --- "adeB, adeE and adeY and the blaIMP-4+ isolates" --- p.202 / Chapter 7.2 --- Discussion / Chapter 7.2.1 --- PCR-RFLP typing --- p.205 / Chapter 7.2.2 --- Distribution of adeB --- p.205 / Chapter 7.2.3 --- Distribution of adeE --- p.206 / Chapter 7.2.4 --- Distribution of adeY --- p.207 / Chapter 7.2.5 --- Distribution of adeE and adeY in GDG 3 isolates --- p.207 / Chapter CHAPTER 8 --- GENERAL DISCUSSION --- p.209 / Chapter 8.1 --- Significance of adeB and the putative pumps (adeE and adeY) --- p.211 / Chapter CHAPTER 9 --- CONCLUSION --- p.218 / Chapter 9.1 --- Conclusion --- p.218 / Chapter 9.2 --- Future Plan --- p.219 / REFERENCES --- p.221 / APPENDIX --- p.246 / Appendix1 --- p.246 / Appendix2 --- p.247 / Appendix3 --- p.252 / Appendix4 --- p.253 / Appendix5 --- p.259
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324473 |
| Date | January 2003 |
| Contributors | Chau, Sze-lok., Chinese University of Hong Kong Graduate School. Division of Microbiology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xxi, 263 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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