The multidrug resistance associated proteins (MRP(human)Mrp (rat) are ATP-dependent transporters responsible for the efflux of a wide range of substrates, including endogenous compounds e.g. bilirubin, drug metabolites e.g. paracetamol glucuronide and fluorescent dyes e.g. 5 (and 6)-carboxy-2’,7’-dichlorofluorescein (CDF). Mrp1-6 (<i>abccl-6)</i> are expressed in rat liver, with Mrp2 being expressed at the highest level. Isolation of hepatocytes by <i>in situ</i> collagenase perfusion causes bile canalicular disruption and internalisation of Mrp2. Cells cultured in a sandwich configuration of Matrigel or collagen (Type 1) showed bile canalicular reformation at different days in cell culture, depending on the extracellular matrix and time of overlay. We have developed a method for quantifying Mrp-mediated efflux in hepatocytes cultured in a sandwich configuration of collagen (Type 1). This method is unique in its ability to distinguish between sinusoidal efflux, canalicular efflux and diffusion in intact hepatocytes. Alternative <i>in vitro</i> models of Mrp2-mediated efflux include the vesicular (direct and indirect methods) and the ATPase assays. We have used these assays to identify atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin as substrates of human and rat MRP2/Mrp2. A close correlation was seen between the kinetic parameters of transport of the Mrp2 substrate; CDF determined in sandwich cultured rat hepatocytes using the method above (Km = 3.5 – 9.9 μM), vesicle preparations (Km = 37.9 μM) and membrane preparations (Km = 18.7 μM). We also present data to implicate the nuclear receptors, PXR, CAR and FXR in the regulation of <i>abcc2</i> and <i>abcc3</i> and PXR and CAR in <i>abcc1</i> gene regulation. <i>Abcc2 </i>and abcc5<i> </i>are also up-regulated in response to a toxic insult to the cell, probably via Nrf2 activation, suggesting a role in cell defence.

The problem in this investigation consists of, first, the procurement of several strains of Micrococcus pyogenes var. aureus; second, the comparison of the degree and rate of development of resistance of these organisms to terramycin; and, third, to study the morphological and physiological changes which occur during the development of resistance.

No description available.

Wong, Ah Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (p. 125-130). / Abstracts in English and Chinese. / ABSTRACT --- p.V / 摘要 --- p.VIII / TABLE OF CONTENTS --- p.X / ACKNOWLEDGEMENTS --- p.XII / INTRODUCTION --- p.13 / Adaptive Mutation versus Spontaneous Mutation --- p.13 / Fluctuation Test --- p.13 / Adaptive Reversion of lacI - lacZ Fusion Mutant --- p.17 / Putative Models of Adaptive Mutation Mechanisms --- p.18 / Point Mutagenesis --- p.18 / Hypermutation --- p.19 / Gene Amplification --- p.21 / Controversy over the Mechanism of Adaptive Mutation --- p.21 / Induced Mutagenesis under Other Stresses --- p.23 / The General Stress Response --- p.23 / The SOS Response --- p.24 / Reduced Mismatch Repair --- p.24 / Adaptive Mutation in Other Micro-organisms --- p.25 / Mutation Generation under Antibiotic Stress --- p.26 / Fluoroquinolones --- p.26 / Beta-Lactams --- p.27 / Aminoglycosides --- p.27 / Justification and Objectives of this Study --- p.28 / MATERIAL AND METHODS --- p.30 / Bacterial Strains --- p.30 / Culture Media --- p.30 / Antibiotics --- p.30 / Resistance Induction Assay --- p.31 / Rationale of Experimental Design --- p.31 / Agar Selection Method --- p.32 / Broth Selection Method --- p.34 / Isolation of Organisms which Exhibited Reduced Drug Susceptibility and Determination the Minimal Inhibitory Concentration (MIC) --- p.36 / Indole Test --- p.37 / DNA Extraction --- p.37 / Polymerase Chain Reaction (PCR) on the gyrA and rpoB Genes --- p.37 / PCR Product Purification and Nucleotide Sequencing --- p.39 / RESULTS --- p.40 / Solid Agar Selection Approach --- p.41 / Broth Selection Approach --- p.48 / Strain MG1655 --- p.49 / Strain BW25113 --- p.53 / recA Deletion Mutant --- p.54 / mutS Deletion Mutant --- p.56 / DISCUSSION --- p.59 / Development of Resistance Induction Assay --- p.59 / Background Resistance to Gentamicin and Rifampicin --- p.62 / Resistance Induction Effect of Ciprofloxacin --- p.64 / Relative Effects of recA and mutS Deletion --- p.66 / Putative Origins of Antibiotic Resistance Gene Mutations --- p.69 / TABLES AND FIGURES --- p.71 / REFERENCES --- p.125

Leung, Siu Sing. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 92-97). / Abstracts also in Chinese. / Title from PDF title page (viewed on 25, October, 2016).

鮑曼不動桿菌(Acinetobacter baumannii)是一種威脅生命的醫院獲得性病菌。該細菌有很强的環境適應能力。它能夠在重症監護室被分離出來并有很高的幾率感染免疫系統受損的病人。鮑曼不動桿菌有很高的傾向獲得多重抗藥性。目前在亞洲和歐洲有多株泛抗藥性菌株被發現。一些基因組比對研究著重報告了鮑曼不動桿菌的抗藥基因片段和與抗藥性相關的基因突變。然而，抗藥基因的轉錄調控和該細菌在抗生素治療過程中引發的反應并未得到很好的研究。因此，我們運用鏈特異性轉錄組測序技術（RNA-seq）對一些抗藥菌株和非抗藥菌株在不同環境下生長的樣本進行測序，來研究該細菌，尤其是在抗生素治療中抗藥菌株的基因轉錄調控。 / 本研究運用轉錄組測序技術（RNA-seq）系統分析了十二株鮑曼不動桿菌在培養液生長狀況下的轉錄組。本次研究共收集了九株多重抗藥性菌株和三株敏感菌株，其中包括了一些快速生長的菌株和慢速生長的菌株。在快速生長的菌株中，氨基酸代謝途徑、甘油脂代謝途徑和钳铁化合物生物合成途徑被向上調控並扮演着重要角色。多重抗藥性菌株擁有更多與轉位酶（transposase）相關的抗生素抗性基因，但除此之外，在對數期的生長過程中多重抗藥性菌株與敏感菌株並未在許多其他代謝途徑中表現差異性控制。 / 三株擁有相同脉冲场凝胶电泳（PFGE）樣式但是表現出不同抗藥性的菌株分別生長於含有阿米卡星(Amikacin)、亞胺培南（Imipenem）或美羅培南（Meropenem）的培養液中，然後它們的轉錄組也被進行了研究。菌株生長在含有抗生素的培養液中時，與能量製造相關的的途徑和核醣體合成途徑被向上調控。作用機制不同的抗生素對細菌有不同的影響，阿米卡星誘發更多基因被向上調控，例如與蛋白質折疊相關的基因；碳青霉烯类抗生素誘發更多的基因被向下調控，例如甘油脂代謝途徑。然而，許多在抗生素治療過程中被緊密調控的基因功能仍舊未知。在抗生素環境生長的條件下基因調控和抗藥機制可能會更複雜。 / 最後，本研究找到一些新的與抗藥性相關的基因和单核苷酸变异（SNVs）。其中，源自於同一操縱子的大环内酯二位轉磷酸酶（macrolide2’ phosphotransferase）同源體Mph和大环内酯外排泵蛋白同源體Mel只存在並一同表達於鮑曼不動桿菌的阿米卡星抗藥株中。這兩個基因或對阿米卡星的抗藥性有一定貢獻作用。總而言之，這些成果爲將來的深度研究提供了重要依據。 / Acinetobacter baumannii is a life-threatening nosocomial pathogen, which has versatile adaptability to the environment. It can be isolated from intensive care unit (ICU) and causes high prevalence of infection among immunocompromised patients. A. baumannii has high tendency to develop multidrug resistance. Currently, pan-drug resistant strains have been reported in Asia and Europe. Several comparative genomic studies revealed the structures of drug resistant islands and antibiotic-related mutations in A. baumannii. However, the transcriptional regulation of drug resistant genes, and the multidrug resistant response of A. baumannii under the treatment of antibiotics are not well studied. By applying strand-specific RNA-sequencing on sensitive and multidrug resistant strains growing in various conditions, we aimed to study the transcriptional responses and gene regulation of A. baumannii, specifically under the antibiotic treatment. / The transcriptome of twelve A. baumannii strains, including nine multidrug resistant strains and three sensitive strains, were systematically analyzed in planktonic state by RNA-seq. Among the multidrug resistant strains there are both fast-and slow-growing strains. Amino acid metabolic pathways, glycerol lipid metabolic pathways and siderophore biosynthetic process are found to be key pathways that are up-regulated in fast-growing strains. Except that multidrug resistant strains possess more transposase-associated antibiotic resistant genes, intriguingly, only a few pathways are differentially regulated between multidrug resistant and sensitive strains during fast growth in antibiotic-free medium. / Three strains of the same PFGE pattern but with different antibiotic resistance patterns were treated by amikacin, imipenem, and meropenem, and their transcriptomes were analyzed. The energy generation-related pathways and ribosome synthesis pathway were commonly up-regulated when the strains were grown in antibiotic-treated media. Amikacin triggers more genes up-regulated, including genes responsible for protein folding, while carbapenems trigger more genes down-regulated, including glycerol lipid metabolic process, revealing the different actions of antibiotics. However, many tightly-regulated genes during antibiotic treatment were functionally unknown, suggesting that gene regulation during antibiotic response and the actual mechanisms involved could be far more complex. / Finally, this study also identified several novel genes and single nucleotide variations (SNVs) which were correlatedto antibiotic-specific resistance. A macrolide 2’ phosphotransferase homolog Mph and a macrolide efflux protein homolog Mel, which commonly exist only in A. baumannii amikacin resistant strains and are co-expressed in the same operon, may contribute to amikacin resistance. In summary, the results presented in this thesis have opened the venue for future investigations. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qin, Hao. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 107-114). / Abstracts also in Chinese.

Chan, Lap Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 131-147). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xv / LIST OF ABBREVIATIONS --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- Prevalence of antimicrobial reisitance in gonococcal strains --- p.4 / Chapter 1.2.1 --- Prevalence of penicillin resistant gonococcal strains --- p.5 / Chapter 1.2.2 --- Prevalence of tetracycline resistant gonococcal strains --- p.6 / Chapter 1.2.3 --- Prevalence of quinolone resistant gonococcal strains. --- p.7 / Chapter 1.2.4 --- Emergence of generation cephalosporin reduced susceptible gonococcal strains --- p.8 / Chapter 1.2.5 --- Monitoring the prevalence of gonorrhea --- p.9 / Chapter 1.3 --- Gonococcal Antimicrobial Surveillance Program --- p.10 / Chapter 1.4 --- Antimicrobial Resistance Mechanisms in N. gonorrhoeae --- p.11 / Chapter 1.4.1 --- "Innate Resistance Mechanisms in N, gonorrhoeae" --- p.11 / Chapter 1.4.1.1 --- Natural mechanisms in N. gonorrhoeae against toxic substance --- p.11 / Chapter 1.4.1.2 --- Efflux pump inhibitors --- p.13 / Chapter 1.4.2 --- Development of acquired antimicrobial resistance in N. gonorrhoeae --- p.14 / Chapter 1.4.2.1 --- Penicillin --- p.14 / Chapter 1.4.2.1.1 --- Chromosomal-mediated resistance --- p.14 / Chapter 1.4.2.1.2 --- Plasmid-mediated resistance --- p.16 / Chapter 1.4.2.2 --- Tetracycline --- p.16 / Chapter 1.4.2.2.1 --- Plasmid-mediated resistance --- p.17 / Chapter 1.4.2.2.2 --- Chromosomal mediated resistance --- p.18 / Chapter 1.4.2.3 --- Fluroquinolone --- p.18 / Chapter 1.4.2.3.1 --- Resistant mechanism in quinolone resistant gonococcal strains --- p.19 / Chapter 1.4.2.3.1.1 --- gyrA andparC --- p.19 / Chapter 1.4.2.3.1.2 --- NorM efflux system --- p.21 / Chapter 1.4.2.4 --- 3rd generation cephalosporins --- p.22 / Chapter 1.4.2.4.1 --- Mosaic penA structure in reduced susceptible gonococcal strains --- p.22 / Chapter 1.4.2.4.2 --- Other mechanisms related to reduced susceptibility in gonococcal strains --- p.24 / Chapter 1.5 --- Application of molecular typing methods to study the epidemiology of N. gonorrhoeae --- p.24 / Chapter 1.5.1 --- Opa typing --- p.25 / Chapter 1.5.2 --- K gonorrhoeae Multi-Antigen Sequence Typing --- p.25 / Chapter 1.6 --- Project Objectives --- p.27 / Chapter CHAPTER 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Collecting gonococcal strains --- p.29 / Chapter 2.2 --- Culturing of N. gonorrhoeae --- p.29 / Chapter 2.3 --- Identification --- p.29 / Chapter 2.3.1 --- Gram staining test --- p.30 / Chapter 2.3.2 --- Oxidase test --- p.31 / Chapter 2.3.3 --- Cabohydrate utilization test --- p.31 / Chapter 2.4 --- In-vitro antimicrobial susceptibility testing --- p.32 / Chapter 2.4.1 --- Preparation of cell cultures for MIC tests --- p.32 / Chapter 2.4.2 --- Preparation of antimicrobial agents for MIC tests --- p.32 / Chapter 2.4.3 --- Inoculum preparation and delivering --- p.33 / Chapter 2.5 --- Preparation of genomic DNA for detection of mutations --- p.34 / Chapter 2.6 --- Study of Resistant Mechanism against fluoroquinolone --- p.34 / Chapter 2.6.1 --- PGR detection of mutations in gyrA and parC genes --- p.35 / Chapter 2.6.2 --- Optimization of gyrA and parC genes PGR --- p.35 / Chapter 2.6.3 --- Detection of PGR products for gyrA and parC genes --- p.37 / Chapter 2.6.4 --- Purification of Amplified DNA products --- p.37 / Chapter 2.7 --- Tests of efflux inhibitor on N. gonorrhoeae --- p.38 / Chapter 2.7.1 --- Effect ofCCCP --- p.39 / Chapter 2.7.2 --- Effect of Reserpine --- p.39 / Chapter 2.8 --- Study of Resistant mechanism against β-lactams --- p.40 / Chapter 2.8.1. --- Detection for the presence of β-lactamase --- p.40 / Chapter 2.8.2 --- Mosaic penA patterns --- p.40 / Chapter 2.8.2.1 --- Detection of mutations in penA gene --- p.40 / Chapter 2.8.2.2 --- Optimization of penA gene PGR --- p.41 / Chapter 2.8.2.3 --- Detection of PGR products --- p.43 / Chapter 2.8.2.4 --- Purification of Amplified DNA products --- p.44 / Chapter 2.9 --- Detection of the presence of tetM determinant --- p.45 / Chapter 2.9.1 --- Optimization of tetM determinant PCRs --- p.46 / Chapter 2.9.2 --- Detection of PGR products --- p.47 / Chapter 2.10 --- Detection of different allele types in tbpB andpor genes --- p.48 / Chapter 2.10.1 --- Optimization of PGR for NG-MAST --- p.48 / Chapter 2.10.2 --- Detection of PCR products --- p.49 / Chapter 2.10.3 --- PCR product purification --- p.50 / Chapter 2.11 --- Sequencing of the PCR products --- p.51 / Chapter 2.12 --- Data Analysis --- p.52 / Chapter CHAPTER 3. --- RESULTS / Chapter 3.1 --- Gonococcal strains collected --- p.55 / Chapter 3.2 --- Identification of gonococcal strains --- p.55 / Chapter 3.3 --- MIC of Antimicrobial agents --- p.56 / Chapter 3.3.1 --- Interpretive Criteria --- p.56 / Chapter 3.3.2 --- Ciprofloxacin --- p.56 / Chapter 3.3.3 --- Penicillin --- p.57 / Chapter 3.3.4 --- Tetracycline --- p.57 / Chapter 3.3.5 --- Ceftriaxone --- p.58 / Chapter 3.3.6 --- Cefixime --- p.58 / Chapter 3.3.7 --- Cefotaxime --- p.58 / Chapter 3.3.8 --- Spectinomycin --- p.58 / Chapter 3.3.9 --- Levofloxacin --- p.59 / Chapter 3.3.10 --- Ceftibuten --- p.59 / Chapter 3.4 --- Result of PGR --- p.60 / Chapter 3.4.1 --- gyrA andparC genes --- p.60 / Chapter 3.4.2 --- penA gene --- p.61 / Chapter 3.4.3 --- tbpB and por genes --- p.62 / Chapter 3.4.4 --- tetM determinant --- p.62 / Chapter 3.5 --- β-lactamase --- p.63 / Chapter 3.6 --- Efflux pump inhibitor --- p.63 / Chapter 3.6.1 --- CCCP --- p.64 / Chapter 3.6.2 --- Reserpine --- p.64 / Chapter 3.7 --- Detection of Mutations --- p.66 / Chapter 3.7.1 --- gyrA and parC genes --- p.66 / Chapter 3.7.2 --- penA gene --- p.68 / Chapter 3.8 --- NG-MAST --- p.70 / Chapter 3.8.1 --- tbpB and por gene --- p.71 / Chapter 3.9 --- Porin mutation --- p.72 / Chapter CHAPTER 4. --- DISCUSSION / Chapter 4.1 --- Sampling --- p.74 / Chapter 4.2 --- Methodology --- p.75 / Chapter 4.3 --- MIC distribution of different antimicrobial agents --- p.76 / Chapter 4.4 --- Mechanisms of quinolone resistance --- p.78 / Chapter 4.4.1 --- Mutations at QRDRs --- p.78 / Chapter 4.4.2 --- Association of the number of mutations at parC gene with MIC levels against fluroquinolones --- p.80 / Chapter 4.5 --- Penicillin and Tetracycline Resistant Mechanisms --- p.81 / Chapter 4.6 --- Efflux system --- p.85 / Chapter 4.7 --- NG-MAST --- p.88 / Chapter 4.8 --- Mosaic penA pattern --- p.89 / Chapter 4.9 --- Management of gonorrhea --- p.90 / Chapter CHAPTER 5. --- CONCLUSIONS / REFERENCES

No description available.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

An epidemic of staphylococcal infections occurred in New Zealand hospitals and communities from 1955-1963. The 'H', or 'Hospital Bug', a strain of Staphylococcus aureus characteristic of the epidemic, was resistant to the most commonly used antibiotics. Post-operative patients, the frail elderly and mothers and babies were particularly vulnerable to staphylococcal colonization and infection. This thesis places the H-Bug epidemic in its historical context, discussing the ways in which the government and health professionals responded to the rising incidence of staphylococcal infection, and the major effects of the epidemic on medical and hospital practice. It also examines the impact of persistent staphylococcal infection on women and families in the community. Primary sources provided the basis for this thesis. The H-Bug epidemic has gone largely unrecorded except in contemporary documents. Health Department files and Auckland Hospital Board records as well as newspaper clippings were important sources. The New Zealand epidemic was clearly linked to the global pandemic of antibiotic resistant staphylococcal infection, 1946-1966, through medical literature and archival documents. International medical journals, including the New Zealand Medical Journal, published numerous articles on the epidemiology of antibiotic-resistant staphylococcal infection, providing an excellent record of research, case studies, current opinion, and recommended practice. The most valuable contribution to an understanding of the impact and experience of the H-Bug epidemic was, however, provided by the nineteen people who agreed to be interviewed for the study. Interviewees included a wide variety of health professionals and women and their children, all of whom had personal experience or association with the epidemic. In this thesis it is argued that the main focus of the medical response was the prevention and control of hospital cross-infection, both to protect patients and to preserve the public perception of the hospital as a safe venue for care. Although the emergence of resistant strains of staphylococci was widely attributed to the misuse of antibiotics, this thesis contends that the Health Department was reluctant to impose restrictions on medical prescribing and that Health Department official and senior clinicians chose instead to modify hospital environments and clinical practice. Rooming-in was widely introduced to counter the epidemic despite the fact that a trial in 1959, at National Women's Hospital, did not demonstrate a reduction in infection rates among neonates. The concept endured, however, as it held strong appeal for hospital administrators hard pressed to keep wards adequately staffed with trained personnel. It was also supported by women and health professionals who were convinced of the benefits of a close mother-baby relationship from birth. The H-Bug epidemic was eventually resolved by the introduction of the methicillin antibiotics in the early 1960s. As a consequence, confidence in a pharmaceutical solution to infectious disease remained intact until the emergence of multiple antibiotic resistant organisms in the 1980s. The lessons of the H-Bug epidemic had been largely forgotten in the intervening years, ignored until New Zealand clinicians were reminded once again that antimicrobial resistance would inevitably accompany the indiscriminate use of antibiotics and inadequate attention to infection prevention and control.