This thesis is an investigation of two common skin conditions of pigs: exudative epidermitis (EE) and ear necrosis (EN). The cause of exudative epidermitis and risk factors are well understood, however the study was prompted because of reports of treatment failure. A survey of veterinary practitioners (n=15) and pork producers (n=58) was conducted to determine which treatments are commonly used. Amongst farmer respondents topical treatments were often used and in serious cases injectable penicillin G was administered. Thirty farms with a history of EE were visited and skin samples taken from affected pigs. The antimicrobial resistance pattern for isolates of Staphylococcus hyicus and Staphylococcus aureus revealed that almost all isolates were resistant to penicillin G and ampicillin. In addition, certain isolates of S. hyicus as well as S. aureus were shown to possess the mecA gene which is associated with resistance to methicillin. The presence of widespread resistance to penicillin G among staphylococci isolates suggests a reason for poor treatment response. The presence of the mecA gene in staphylococci other than S. aureus recovered from pigs has not been reported before and is of interest from a public health standpoint. A second study investigated EN. The causative agent(s) and the associated risk factors are not well understood. Eleven case farms were visited and skin biopsies and oral swabs taken from pigs in early, mid and late stages of the disease. Bacteriological culturing was performed for staphylococci and spirochetes as well as histological examination of the biopsy samples. Farm-level risk factors were assessed on 14 case farms and 9 control farms. Staphylococci were generally recovered in abundance from the majority of samples but spirochetes were not cultured and only identified microscopically in a small number of tissue samples. Histology revealed that the disease appeared to occur first as a lesion on the epidermal surface that caused tissue damage and led to subsequent invasion of the dermis. This pathogenesis was consistent with the hypothesis that staphylococci colonize the skin surface and produce exfoliating toxins. Ear biting was noted to be commonly present and may be an important contributing factor. / Ontario Pork Animal Health Strategic Initiative Fund Ontario Ministry of Agriculture, Food and Rural Affairs(OMAFRA) Ontario Veterinary College, University of Guelph

Advantages and disadvantages of administration of resuscitation fluids in burns patients have been discussed at length. However, the effect of resuscitation fluids on tissue physiological endpoints and tissue antibiotic distribution is scarcely reported, yet clinically crucial. The preliminary studies of this thesis involved evaluation of the literature and the development of a non - recovery anaesthetized rat model of burn injury suitable for the study of plasma and tissue physiological changes and antibiotic pharmacokinetics (PK). Therefore, the first series of the studies for this thesis was designed to examine the relative effects of a range of crystalloid and colloid-containing resuscitation fluids on tissue pH following burn injury in a rat model. The secondary aims were to examine the effects of these fluids on tissue blood flow, plasma protein extravasation (PPE) and evaporative water loss (EWL). In these studies we confirmed that the burn injury and fluid resuscitation were accompanied by a tissue acidosis. Administration of Lactated Ringers’ Albumin (LRA) and Lactated Ringers’ Dextran (LRD) effectively attenuated the degree of tissue acidosis in the thermally injured and non injured sites for 180 minutes post burn and the transepidermal water loss (TEWL) on the non injured sites during the first 60 minutes of the acute phase of burn injury. The second phase of the work was designed to assess the changes in antibiotic distribution with the administration of these different fluids in plasma as well as in interstitial tissues in the burn and the non burn sites. This study showed that for cephalothin (4g/kg body weight, administered intravenously (IV)), Lactated Ringers solution (LR) and Hypertonic Saline (HS) showed similar plasma PK with Time > Minimum inhibitory concentration (MIC) (> 180 minutes) in plasma. However, the antibiotic tissue distribution was more skewed towards lower levels for HS when compared with LR. For piperacillin (18g/kg body weight, administered IV), Time > MIC was considerably low comparatively, being only 55 min for both LR and HS. Antibiotic concentrations did not reach the MIC with LRA resuscitation. When considering the interstitial tissues, Time > MIC for cephalothin was lower than HS with LR on both the burn and the non burn sites. T > MIC for piperacillin was zero for all fluids in both burn and non burn sites. The major finding of this study was that with LRA resuscitation, antibiotic distribution was significantly lower than seen with LR and HS for both antibiotics studied in the interstitial tissue fluid space in both the burn and non burn sites. The final phase of the work was designed to study the apparent permeability co efficient of Keratinocytes (KC) to antibiotics in the presence of simulated pH changes observed in burn tissue in thermal injury using colloids and crystalloids. This study found that there was no significant difference between the basolateral and apical concentrations of antibiotics observed neither with the different pH values nor with time. However, there was definitely a significant difference in the apparent permeability of the cells with LR vs LRA and that the permeability was higher with LR than LRA. This study confirmed that the presence of LR allows greater permeation of the antibiotic into the KC, and also that with LRA resuscitation, the antibiotic tends to stay at higher concentrations in the interstitial compartment. These studies demonstrate that choice of resuscitation fluid following burn injury can affect both changes in tissue physiology and antibiotic distribution, warranting further study in both animal models and patient populations.

Antibiotic resistance is one of the most serious medical problems in the world. To counteract the increase in antibiotic resistance, new rapid and effective analytical methods are needed. To effectively treat infections in critically ill patients, optimal antibiotic dosages are required. DrugLog® is an instrument that uses a spectrophotometric method to analyze antibiotics in plasma in the wavelength range 200-800 nm. The aim of this study was to do a method validation of the instrument DrugLog®.     The study material that was used was whole blood from healthy donor and routine citrate plasma samples from the laboratory. The precision of the method and stability of plasma, the best way to filtrate lipids from plasma and four antibiotics (meropenem, cefotaxime, vancomycin, piperacillin/tazobactam) were investigated.     The precision of the method, measured as CV% was less than 0.62 and stability plasma showed a CV% of 135.74 after 24 h in room temperature. The stability for the different antibiotics after 24 h in room temperature showed a CV% of 8.11 for meropenem, 40.80 for vancomycin, 16.55 for cefotaxime and 2.92 for the combination antibiotic piperacillin/tazobactam. It was also determined that bacterial filter was the best way to remove lipids from plasma.     In conclusion DrugLog® is a suitable instrument to analyze concentration of antibiotics in patients during antibiotic treatment, however further validations are needed.

Orientadores: Telma Teixeira Franco, Adrianus Johannes Straanhof, Lucas Antonius Maria van der Wielen / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-05T06:02:03Z (GMT). No. of bitstreams: 1 Ferreira_JulianadeSouza_D.pdf: 5552134 bytes, checksum: f2234304e4f228a88bb1b83073350bfa (MD5) Previous issue date: 2004 / Resumo: Este trabalho estuda a hidrólise enzimática da penicilina G (PenG) em ácido 6-aminopenicilânico (6-APA) e ácido fenil acético (PAA). Em um reator contra-corrente multi-estágio e em baixos valores de pH, a reação enzimática ocorre na fase aquosa e os produtos são separados entre a fase aquosa e a fase orgânica (acetato de butila). Além disso, em pH baixo, a cristalização do 6-APA ocorre quando concentrações de PenG são altas. Ambos fenômenos deslocam o equilíbrio no sentido de conversão do substrato, promovendo alta produtividade. A primeira etapa deste trabalho refere-se à avaliação da atividade e estabilidade da penicilina amidase a baixo pH e na presença de acetato de butila (BuAc). A enzima apresentou máxima atividade na faixa de pH 8,0 - 9,0 e permaneceu estável mesmo em pHs baixos (3,0 - 6,0) num período de incubação de até 32 dias. Embora a atividade enzimática sofra um decréscimo de aproximadamente 80%, isto não representa empecilho para sua utilização no emprego da hidrólise de PenG em processo contínuo e bifásico (água e BuAc) em pH baixo. O efeito de PenG, PAA e BuAc na cristalização do 6-APA e os parâmetros cinéticos de cristalização também foram avaliados. Os resultados mostraram que as impurezas não exerceram efeito sobre a cristalização de 6-APA, na faixa de pH entre 4 e 5 e nas concentrações de impurezas de 0,55 mM - 3,0 mM. A avaliação da cinética de cristalização possibilitou o uso de um modelo que pode predizer as taxas de cristalização de 6-APA. Um modelo quantitativo foi desenvolvido para o cálculo do pH e das concentrações do substrato e dos produtos nos estágios do reator contra-corrente. Os dados fornecidos pelo modelo podem ser utilizados para otimizar as condições de operação como: estágio de alimentação, vazão volumétrica das fases e concentração inicial do substrato. Na última etapa deste trabalho foi feita uma revisão bibliográfica sobre biorreatores extrativos em que são apresentadas as vantagens de cada configuração e as restrições dos processos biocatalíticos. Através desta revisão, foi verificado que o uso de um sistema, formado por agitadores acoplados a hidrocic1ones em série, pode representar uma opção adequada de reatores multi-estágio contra-corrente para a hidrólise de PenG em escala de laboratório / Abstract: This work studies the enzymatic hydrolysis of penicillin G (PenG) into 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). In a multi-stage countercurrent reactor and at low pH, the enzymatic reaction takes place in the aqueous phase and the products are separated between the aqueous phase and organic phase (butyl acetate - BuAc). Furthermore, 6-APA crystallization occurs at low pH when PenG concentrations are high. Both phenomena shift the equilibrium towards conversion of substrate, favoring high productivity. The first step of this work, concerns the evaluation of activity and stability of penicillin amidase at low pH and in the presence of butyl acetate (BuAc). The enzyme presented the maximum activity in the pH 8.0 - 9.0 and remained stable at low pHs (3.06.0) during at least 32 days. Although the enzyme activity decreased by 80%, this does not represent a drawback in the application of a biphasic (water and BuAc) and continuous PenG hydrolysis at low pH. The effect of PenG, PAA and BuAc in APA crystallization and the kinetic parameters were also analyzed. The results showed that impurities have no effect on APA crystallization, in the pH range 4 - 5 and in the impurity concentrations of 0.55 mM - 3.0 mM. The evaluation of crystallization kinetics allowed the use of a mo del that predicts the APA crystallization rates. A quantitative model was developed in order to calculate the pH and substrates and products concentrations in the countercurrent reactor. The data provided by the model can be used to optimize the operation conditions: stage of feed, flow rate of phases and initial substrate concentration. In the last step of this work, a literature review concerning extractive bioreactor was made. This review presents the advantages of each configuration and the restrictions of the biocatalytic processes. Through this review, a integrated system of mixers and hydrocydone was suggested as an appropriate option of multi-stage countercurrent reactor for PenG hydrolysis in laboratory scale / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.

No description available.

Infectious diseases cause substantial morbidity and mortality, exacerbated by increasing antibiotic resistance. In critically ill patients, recent studies indicate a substantial variability in β-lactam antibiotic levels when standardized dosing is applied. New methods for characterizing nosocomial outbreaks of bacterial infections are needed to limit transmission. The goals of this thesis were to investigate new strategies towards individualized treatment and control of bacterial infections.  In Paper I we confirmed high variability in β-lactam antibiotic levels among intensive care unit (ICU) patients from southeastern Sweden, where 45 % failed to reach treatment targets (100 % fT>MIC). Augmented renal clearance and establishing the minimum inhibitory concentration of the bacteria were important for evaluating the risk of not attaining adequate drug levels. In Paper II a rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 11 commonly used antibiotics was developed and tested in clinical samples. Performance goals (CV<15%) were reached. A microbiological method for quantification of β-lactam antibiotics in serum was developed in Paper III. The method could be important for hospitals without access to an LC-MS method. Paper IV and Paper V investigated ligation-mediated qPCR with high resolution melt analysis (LMqPCR HRMA), for transmission investigation of extended spectrum β-lactamase (ESBL)-producing E. coli and other common bacterial pathogens. Results comparable to the reference method (PFGE) could be achieved within one day in a closed system and confirmed a nosocomial outbreak in Kalmar County. In Paper VI whole genome sequencing followed by bioinformatic analysis resolved transmission links within a nosocomial outbreak due to improved discriminatory power compared to LMqPCR HRMA. The high proportion of ICU patients with insufficient β-lactam drug levels emphasizes the need for individualized treatment by therapeutic drug monitoring (TDM). TDM is enabled by a highly sensitive method, such as UPLC-MS/MS, but if unavailable, also by a microbial method. Molecular typing methods used for transmission investigation can detect nosocomial outbreaks. LMqPCR HRMA can be used for screening purposes. For enhanced resolution, whole genome sequencing should be used, but always together with a rigorous epidemiological investigation.

(3R,5R)-Clavulanic acid is a clinically important inhibitor of Class A β-lactamases. Progress has been made in to establishing the steps of clavulanic acid biosynthesis leading to (3S,5S)-clavaminic acid. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde remains elusive. It is believed that the products of the later genes (orf10-orf18) of the clavulanic acid biosynthesis gene cluster are probably involved in this conversion. Part I of this thesis describes biochemical and structural studies carried out on OAT2, a member of N-terminal nucleophile (Ntn) hydrolase superfamily of enzymes. OAT2 has been characterised to be an ornithine acetyl transferase (OAT) and is involved in clavulanic acid biosynthesis. OAT2 catalyses the reversible transfer of the acetyl group between N-acetyl-L-ornithine and L-glutamate. It was found that this reaction is catalysed via the formation of an acyl-enzyme intermediate. Subsequent studies including mass spectrometry, 13C NMR spectroscopy, infrared spectroscopy, X-ray crystallography and molecular dynamics simulations, further confirmed the viability of the intermediate. This acyl-enzyme intermediate of OAT2 was found to be exceptionally stable at physiological pH, as compared to the acyl-enzyme intermediates involved in catalysis by hydrolytic enzymes including proteases, Ntn hydrolases and β-lactamses. The X-ray studies revealed possible reason for this unusual stability. The infrared studies revealed two conformations for the acyl-enzyme. Modeling (MDS) studies assigned one of these to the structure observed by X-ray and proposed the other one to result from a hydroxyl hydrogen 'flip' involving the oxyanion hole component Thr-111 resulting in a singly hydrogen bonded acyl-enzyme intermediate. α, β Subunit co-expression studies with OAT2 were used to investigate the autocatalytic cleavage step. In one case an interesting N-acyl enzyme species was observed. Part II of this thesis describes efforts carried out to characterise the ORF10 and ORF15 proteins of clavulanic acid biosynthesis. ORF10 was characterised to be an 'active' cytochrome P450 and ORF10 crystals were obtained in the presence spinach ferredoxin, highlighting the role of the ferredoxin interaction in assisting ORF10 crystallisation. ORF15 was shown to be a probable peptide transporter, which binds bradykinin as observed in the crystal structure.

This research developed validated QSPKR and PK-AS models for predicting human systemic PK properties of three, preselected, pharmacological classes of drugs, namely opioids, β-adrenergic receptor ligands (β-ARL) and β-lactam antibiotics (β-LAs) using pertinent human and animal systemic PK properties (fu,, CLtot, Vdss, fe) and their biologically relevant unbound counterparts from the published literature, followed by an assessment of the effect of different molecular descriptors on these PK properties and on the PK-AS slopes for CLtot and Vdss from two species (rat and dog). Lipophilicity (log (D)7.4) and molecular weight (MW) were found to be the most statistically significant and biologically plausible, molecular properties affecting the biologically relevant, systemic PK properties: For compounds with log (D)7.4 > -2.0 and MW < 350 D (e.g., most opioids and β-ARL), increased log (D)7.4 resulted in decreased fu and increased Vdssu, CLtotu and CLnonrenu, indicating the prevalence of hydrophobic interactions with biological membrane/proteins. As result, the final QSPKR models using log (D)7.4 provided acceptable predictions for fu, Vdssu, CLtotu and CLnonrenu. CLnonrenu and CLtotu. For both the datasets, inclusion of drugs undergoing extrahepatic clearance worsened the QSPKR predictions. For compounds with log (D)7.4 < -2.0 and MW > 350 D (e.g., β-LA), increased MW (leading to more hydrogen bond donors/acceptors) resulted in a decrease in fu, likely indicating hydrogen bonding interactions with plasma proteins. In general, it was more difficult to predict PK parameters for β-LAs, as their Vdssu approached plasma volume and CLrenu and CLnonrenu were low - as a result of their high hydrophilicity and large MW, requiring specific drug transporters for distribution and excretion. The PK-AS analysis showed that animal body size accounted for most of the observed variability (r2> 0.80) in systemic PK variables, with single species methods, particularly those using dog, gave the best predictions. The fu correction of PK variables improved goodness of fit and predictability of human PK. There were no apparent effects of molecular properties on the predictions. CLren, CLrenu, CLnonren, and CLnonrenu were the most difficult variables to predict, possibly due to the associated interspecies differences in the metabolism, renal and hepatobiliary drug transporters.

Yip, Yuen Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 128-146). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vi / List of Figures --- p.xi / List of Tables --- p.xii / List of Appendix --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1 --- Pseudomonas aeruginosa --- p.1 / Chapter 1.1 --- Microbiology --- p.1 / Chapter 1.1.1 --- Morphology --- p.1 / Chapter 1.1.2 --- Identification --- p.1 / Chapter 1.1.3 --- Pathogenesis and virulence --- p.2 / Chapter 1.1.4 --- Host defenses --- p.2 / Chapter 1.1.5 --- Epidemiology --- p.2 / Chapter 1.1.6 --- Clinical manifestations --- p.3 / Chapter 1.1.7 --- Treatment --- p.3 / Chapter 2 --- β-Lactams --- p.4 / Chapter 2.1 --- Mode of action of β-lactams --- p.6 / Chapter 2.2 --- β-Lactams resistance --- p.7 / Chapter 2.3 --- Resistance mechanisms --- p.7 / Chapter 2.3.1 --- Changes in PBPs --- p.7 / Chapter 2.3.2 --- Impermeability --- p.8 / Chapter 2.3.3 --- β-Lactamases --- p.8 / Chapter 2.3.3.1 --- Extended spectrum β-lactamases --- p.10 / Chapter 2.3.3.2 --- Carbapenemases --- p.11 / Chapter 2.3.4 --- Efflux pump systems --- p.14 / Chapter 2.4 --- Mechanisms of imipenem resistance in P. aeruginosa --- p.16 / Chapter 2.4.1 --- Prevalence of imipenem resistant P. aeruginosa isolates --- p.18 / Chapter 3 --- Integrons --- p.20 / Chapter 3.1 --- Structure and classification --- p.20 / Chapter 3.1.1 --- Class 1 integrons --- p.21 / Chapter 3.1.2 --- Other class of integrons --- p.22 / Chapter 3.2 --- Integrons in imipenem-resistant P. aeruginosa --- p.23 / Chapter 4 --- Objectives --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 1 --- Materials --- p.25 / Chapter 1.1 --- Bacterial strains --- p.25 / Chapter 1.1.1 --- Bacterial strains used in this study --- p.25 / Chapter 1.1.2 --- Reference strains --- p.25 / Chapter 2 --- Methods --- p.26 / Chapter 2.1 --- Subculture of isolates --- p.26 / Chapter 2.2 --- Identification --- p.26 / Chapter 2.3 --- Antibiotic susceptibility testing --- p.26 / Chapter 2.3.1 --- Preparation of antibiotic plates --- p.27 / Chapter 2.3.2 --- Inoculation of antibiotic plates --- p.27 / Chapter 2.3.3 --- Determination of minimum inhibitory concentration (MIC) --- p.28 / Chapter 2.4 --- Phenotypic detection of metallo-beta-lactamase (MBL) production --- p.28 / Chapter 2.4.1 --- Preparation of inoculum --- p.28 / Chapter 2.4.2 --- Imipenem-EDTA disk test --- p.28 / Chapter 2.4.3 --- Determination of MBL strains --- p.29 / Chapter 2.5 --- Extraction of crude β-lactamase --- p.29 / Chapter 2.5.1 --- Detection of β-lactamase production --- p.29 / Chapter 2.6 --- Isoelectric focusing (IEF) --- p.30 / Chapter 2.6.1 --- Set up of electrophoresis equipment --- p.30 / Chapter 2.6.2 --- Sample application and instrument preparation --- p.30 / Chapter 2.6.3 --- Running conditions --- p.30 / Chapter 2.6.4 --- Detection of β-lactamase --- p.31 / Chapter 2.6.5 --- Determination of isoelectric point (pi) --- p.31 / Chapter 2.7 --- Bioassay of imipenem hydrolysis --- p.31 / Chapter 2.7.1 --- Preparation of inoculum and plate --- p.31 / Chapter 2.7.2 --- Preparation and incubation of sample mixtures --- p.32 / Chapter 2.7.3 --- Application of sample mixtures --- p.32 / Chapter 2.7.4 --- Determination of imipenem hydrolysis --- p.32 / Chapter 2.8 --- Detection of β-lactamase genes --- p.33 / Chapter 2.8.1 --- Polymerase chain reaction (PCR) --- p.33 / Chapter 2.8.2 --- Preparation of DNA template --- p.33 / Chapter 2.8.3 --- Preparation of PCR master mix --- p.33 / Chapter 2.8.4 --- PCR running conditions --- p.34 / Chapter 2.8.5 --- Agarose gel electrophoresis --- p.34 / Chapter 2.8.6 --- DNA sequencing --- p.35 / Chapter 2.9 --- Detection and characterization of integrons --- p.35 / Chapter 2.9.1 --- PCR --- p.35 / Chapter 2.9.2 --- DNA sequencing --- p.36 / Chapter 2.10 --- Detection and characterization of gene cassettes --- p.36 / Chapter 2.10.1 --- PCR --- p.36 / Chapter 2.10.2 --- DNA sequencing --- p.37 / Chapter 2.11 --- Investigation of membrane permeability --- p.37 / Chapter 2.11.1 --- Extraction of outer membrane proteins (OMP) --- p.37 / Chapter 2.11.2 --- Quantification of OMP --- p.38 / Chapter 2.11.3 --- Preparation of the albumin standards and working reagents --- p.38 / Chapter 2.11.4 --- Determination of protein concentration --- p.39 / Chapter 2.12 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.39 / Chapter 2.12.1 --- Sample preparation --- p.39 / Chapter 2.12.2 --- Gel preparation and sample application --- p.39 / Chapter 2.12.3 --- Staining and destaining of the gel --- p.40 / Chapter 2.13 --- Expression of the oprD gene --- p.40 / Chapter 2.13.1 --- Extraction of RNA --- p.40 / Chapter 2.13.1.1 --- Inhibition of RNase degradation --- p.41 / Chapter 2.13.1.2 --- Removal of DNA --- p.41 / Chapter 2.13.1.3 --- Quantification of RNA samples --- p.42 / Chapter 2.13.2 --- Real-time RT-PCR --- p.42 / Chapter 2.13.2.1 --- Preparation of real-time RT-PCR mixtures --- p.42 / Chapter 2.13.2.2 --- Real-time RT-PCR running conditions --- p.43 / Chapter 2.13.2.3 --- Construction of relative standard curves --- p.43 / Chapter 2.13.3 --- Analysis of real-time RT-PCR results --- p.43 / Chapter 2.14 --- Characterization of outer membrane protein regulator mexT --- p.44 / Chapter 2.14.1 --- PCR --- p.44 / Chapter 2.14.2 --- DNA sequencing --- p.44 / Chapter Chapter 3 --- Results / Chapter 1 --- Prevalence of imipenem-resistant P. aeruginosa isolated from patients in hospitals of the New Territories East Cluster (NTEC) from 2001 to 2005 --- p.46 / Chapter 1.1 --- Age and sex distribution of patients --- p.46 / Chapter 1.2 --- Antimicrobial susceptibilities --- p.46 / Chapter 1.2.1 --- Susceptibility to carbapenems --- p.46 / Chapter 1.2.2 --- Susceptibility to other β-lactams --- p.47 / Chapter 1.2.3 --- Susceptibility to aminoglycosides and fluoroquinolones --- p.47 / Chapter 1.2.4 --- Resistance patterns --- p.48 / Chapter 2 --- Phenotypic detection of metallo-beta-lactamase (MBL) producing strains --- p.48 / Chapter 2.1 --- Characterization of β-lactamases --- p.49 / Chapter 2.1.1 --- Production of β-lactamases --- p.49 / Chapter 2.1.2 --- Determination of isoelectric points of β-lactamases --- p.49 / Chapter 2.2 --- Imipenem hydrolysis by β-lactamases --- p.50 / Chapter 2.3 --- Detection of β-lactamase genes --- p.50 / Chapter 2.3.1 --- DNA sequence determination --- p.51 / Chapter 3 --- Detection and characterization of integrons --- p.51 / Chapter 3.1 --- Antibiotic susceptibility and resistance patterns of isolates harboring integrons --- p.51 / Chapter 4 --- Detection of gene cassettes --- p.52 / Chapter 5 --- Outer membrane permeability --- p.52 / Chapter 5.1 --- Outer membrane protein profiles --- p.52 / Chapter 5.2 --- mRNA expression of the oprD gene --- p.53 / Chapter 6 --- Regulatory gene studies --- p.53 / Chapter Chapter 4 --- Discussion / Chapter 1 --- Epidemiological characteristics of imipenem-resistant P. aeruginosa --- p.55 / Chapter 1.1 --- Prevalence of P. aeruginosa --- p.55 / Chapter 2 --- Antibiotic susceptibilities of imipenem-resistant P. aeruginosa --- p.56 / Chapter 3 --- Mechanisms of imipenem resistance in P. aeruginosa --- p.59 / Chapter 3.1 --- Production of β-lactamases --- p.59 / Chapter 3.2 --- Outer membrane permeability --- p.63 / Chapter 3.3 --- Effects of regulatory gene mutations --- p.64 / Chapter 4 --- Integrons in imipenem-resistant P. aeruginosa --- p.66 / Chapter 5 --- Conclusions --- p.67 / Chapter 6 --- Areas for further study --- p.67 / Figures --- p.69 / Tables --- p.82 / Appendix --- p.121 / References --- p.128