published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Focus of this study was on the development of one of the faster, simpler, cost effective and environmentally friendly sample pre-treatment techniques which employs a supported liquid membrane, in this case a Hollow-fiber supported liquid membrane (HF-SLM) for determination of seven (7) quinolone antibiotics (enrofloxacin, ciprofloxacin, danofloxacin, difloxacin, norfloxacin, nalidixic acid and sarafloxacin) in bovine kidney samples followed by LC-MS/MS analysis. The key parameters of the method were optimized and the method was validated following the 2002/657 EC guidelines. The optimum HF-SLM conditions were therefore; NaH2PO4 as a donor phase at pH 7, 0.1% formic acid at pH 3 as acceptor phase. Triethylamine was the optimized liquid membrane and the stirring time was optimized at 1 hour. Separation of the 7 quinolones including 3 internal standards (enrofloxacin-d5, norfloxacin-d5 and difloxacin-d3) was carried out on a Phenomenex Kinetex 2.6 μm XB-C18, 100 mm x 4.6 mm, 100Å column. Validation parameters such as Correlation coefficients (r2) ranging from 0.9714-0.9975 were obtained, while limit of detection (LOD) ranged between 3-39 ug kg-1 and limit of quantification (LOQ) ranged between 10-130 ug kg-1. The obtained limits at which it can be concluded with an error probability of α = 95% that a sample is non-compliant (CCα) ranged from 28 – 422 ug kg-1 while CCβ; the smallest content of the substance that may be detected, identified or quantified in a sample with an error probability of β = 95%, ranged from 29 – 454 ug kg-1. The method was found to be reproducible with CVs ≤ 23 %. The tested samples from Botswana local abattoirs showed no presence of quinolone antibiotics when the method was applied to real bovine kidney samples. Hollow-fiber supported liquid membrane can therefore be used for extraction of biological samples since it is a “greener technique” which uses less solvent which are less harmful to the environment when disposed as compared to dispersive Solid Phase Extraction (dSPE). / Chemistry / M. Sc. (Chemistry)

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Lee, Ching Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-103). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Quinolone Antimicrobial Agents --- p.1 / Chapter 1.1.1. --- Development --- p.1 / Chapter 1.1.2. --- Mode of action --- p.3 / Chapter 1.1.3. --- Mechanisms of resistance to quinolones --- p.4 / Chapter 1.1.3.1. --- Target genes mutations --- p.4 / Chapter 1.1.3.2. --- Decreased intracellular quinolone accumulation --- p.5 / Chapter 1.1.3.3. --- Plasmid-mediated quinolone resistance --- p.6 / Chapter 1.2. --- Plasmid-mediated Quinolone Resistance Genes (qnr) --- p.8 / Chapter 1.2.1. --- Discovery of qnrA genes --- p.8 / Chapter 1.2.2. --- Discovery of qnrS genes --- p.9 / Chapter 1.2.3. --- Discovery of qnrB genes --- p.10 / Chapter 1.2.4. --- Discovery of qnrC genes --- p.11 / Chapter 1.2.5. --- Discovery of qnrD genes --- p.12 / Chapter 1.2.6. --- Origins of qnr genes --- p.12 / Chapter 1.2.7. --- Qnr proteins and mode of action --- p.14 / Chapter 1.2.8. --- Epidemiology and quinolones resistance activity of qnr genes --- p.16 / Chapter 1.2.9. --- Epidemiology of fluoroquinolone-resistant Enterobacteriaceae --- p.17 / Chapter 1.2.10 --- Multidrug-resistant in extended-spectrum-B-lactamase- and AmpC-producing Enterobacteriaceae --- p.19 / Chapter 1.3. --- Background of Study --- p.20 / Chapter 1.4. --- Objectives of Study --- p.21 / Chapter Chapter 2 --- Materials & Methods / Chapter 2.1. --- Study Design --- p.22 / Chapter 2.2. --- Antimicrobial Susceptibility Testing --- p.24 / Chapter 2.2.1 --- Bacterial isolates --- p.24 / Chapter 2.2.2. --- Screening for ESBL and AmpC production by disk diffusion test --- p.24 / Chapter 2.2.3. --- Determination of minimal inhibitory concentrations (MICs) --- p.25 / Chapter 2.3. --- "Detection of qnrA, qnrB and qnrS Genes by Multiplex PCR" --- p.27 / Chapter 2.3.1. --- Total DNA preparation --- p.27 / Chapter 2.3.2. --- "Multiplex PCR assay for qnrA, qnrB and qnrS genes detection" --- p.27 / Chapter 2.3.3. --- Agarose gel electrophoresis --- p.29 / Chapter 2.4. --- "Detection of TEM-, SHV-, CTX- and PMAmpC Type B-Lactamase Genes by PCR" --- p.30 / Chapter 2.5. --- PCR Assays for Further Genotypic Characterization Purpose --- p.32 / Chapter 2.5.1. --- PCR assay to amplify qnrB genes --- p.32 / Chapter 2.5.2. --- PCR assay to amplify qnrS genes --- p.33 / Chapter 2.5.3. --- "PCR assays for genotypic characterizations of the co-existed blaTEM, blaSHV, blaCTX-M and PMAmpC genes of all qnr-positive isolates" --- p.33 / Chapter 2.5.3.1. --- Genotypic characterizations of the co-existed bla-TEM and genes --- p.33 / Chapter 2.5.3.2. --- PCR assays to amplify the co-existed blaCTX_M genes --- p.33 / Chapter 2.5.3.3. --- PCR assay to amplify the co-existed PMAmpC genes --- p.34 / Chapter 2.5.4. --- Sequencing reaction --- p.36 / Chapter 2.5.4.1. --- Purification of PCR product and sequence determination --- p.36 / Chapter 2.5.4.2. --- Sequence analysis --- p.37 / Chapter 2.6. --- Collection of Clinical Data --- p.38 / Chapter 2.6.1. --- Demographics and clinical data --- p.38 / Chapter 2.6.2. --- Definitions --- p.38 / Chapter 2.6.3. --- Data analysis --- p.40 / Chapter Chapter 3 --- Results / Chapter 3.1. --- Bacterial Isolates --- p.41 / Chapter 3.2. --- "Demographics, Medical History, Clinical Features and Clinical Outcomes of Patients" --- p.42 / Chapter 3.3. --- Antimicrobial Susceptibility Testing --- p.44 / Chapter 3.4. --- Detection of qnr Genes --- p.48 / Chapter 3.4.1. --- "Detection of qnrA, qnrB and qnrS genes by multiplex PCR" --- p.48 / Chapter 3.5. --- Detection of ESBLs --- p.49 / Chapter 3.5.1. --- Detection of TEM- and SHV-type ESBLs --- p.49 / Chapter 3.5.2. --- Detection of CTX-M- type ESBLs --- p.51 / Chapter 3.6. --- Detection of PMAmpC Genes --- p.52 / Chapter 3.6.1. --- Detection of PMAmpC genes --- p.52 / Chapter 3.7. --- "The Distribution of qnr and bla Genes for TEM, SHV, CTX-M and PMAmpC" --- p.53 / Chapter 3.8. --- The Characteristics of qnr Isolates --- p.54 / Chapter 3.8.1. --- Genotypes of qnrB and qnrS --- p.54 / Chapter 3.8.2. --- Antimicrobial susceptibility of qnr isolates --- p.58 / Chapter 3.9. --- "The Associations of qnr Genes with Other Bacterial Resistance Genotypes, and the Clinical Characteristics and Outcomes of Patients" --- p.62 / Chapter 3.9.1. --- "Univariate analysis of the associations of qnr genes with other bacterial resistance genotypes, and the clinical characteristics and outcomes of patients" --- p.62 / Chapter 3.9.2. --- "Multivariate analysis of the associations of qnr genes with other bacterial resistance genotypes, and the clinical characteristics and outcomes of patients" --- p.65 / Chapter 3.9.2.1. --- "Multivariate analysis of the associations of qnr genes with other bacterial resistance genotypes, and the clinical characteristics of patients" --- p.65 / Chapter 3.9.2.2. --- "Multivariate analysis of the associations of mortality with qnr genes, bacterial resistance genotypes and other clinical characteristics of patients" --- p.66 / Chapter Chapter 4 --- Discussion / Chapter 4.1. --- Prevalences and Susceptibility of ESBL and PMAmpC in Bacteraemic Enterobacteriaceae Isolates --- p.67 / Chapter 4.2. --- Epidemiology of Plasmid-mediated Quinolone Resistance (qnr) Genes --- p.69 / Chapter 4.3. --- Genotypes of qnr-positive Isolates --- p.72 / Chapter 4.4. --- Antimicrobial Susceptibility of qnr-positive Isolates --- p.75 / Chapter 4.5. --- "The Associations of qnr Genes with Other Bacterial Resistance Genotypes, and the Clinical Characteristics of Patients" --- p.79 / Chapter 4.6. --- "The Associations of Mortality with qnr Genes, Bacterial Resistance Genotypes and Other Clinical Characteristics of Patients" --- p.80 / Chapter 4.7. --- Clinical Importance and Clinical Implications of qnr Genes --- p.82 / Chapter 4.8. --- Limitations of the Current Study --- p.85 / Chapter 4.9. --- Future Studies --- p.87 / Chapter 4.10. --- Conclusions --- p.89 / References --- p.90