Evalúa la eficacia de dos soluciones multipropósito comerciales (Renu Fresh ® y Multi 3 Max®) aplicando un método analítico cuantitativo. Utiliza el microorganismo Pseudomonas aeruginosa (Cepa clínica aislada) debido a su capacidad formadora de biofilm. Se elabora una curva de crecimiento utilizando lentes de contacto de Etalficon A, demostrando la acción de las soluciones multipropósito (SMP) a las concentraciones de 75 y 100%, frente al crecimiento de P. aeruginosa vegetativa. Para probar el desempeño se inocula la cepa en placas con agar tripticasa de soya, luego es llevado a una solución normalizada de caldo tripticasa de soya en la cual se ponen los lentes de contacto y las soluciones multipropósito (SMP) a concentraciones definidas. / Tesis

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

Pseudomonas aeruginosa is an opportunistic bacterial pathogen associated with severe nosocomial infections in immunocompromised hosts and patients with cystic fibrosis (CF). During infection the bacteria secrete proteins that are essential to the infection process. Several of these virulence-associated proteins have been identified using genetic methods. The aim of this research, using a proteomic approach, was to identify novel extracellular proteins that are secreted by P. aeruginosa during infection of a CF patient. Extracellular proteins from P. aeruginosa strain PAO1 grown in vitro were separated by two-dimensional gel electrophoresis (2-DE). The humoral response of chronically infected CF patients to the separated proteins was elucidated using western blotting. Growth phase, cell population and iron limitation were identified as important regulators of the extracellular proteome. The number of extracellular proteins significantly increased upon entry into stationary phase, as did the number of proteins detected by CF patient sera. The detection of several known quorum-controlled proteins by patient sera indicated the importance of this regulatory mechanism during infection. In iron-limiting medium, the proportion of proteins detected by CF patient sera significantly increased compared to extracellular proteins from cells grown in iron-replete conditions. Proteomic analysis of a PAO1 pvdS mutant strain showed that PvdS (an iron-regulated alternative sigma factor) directs production of many extracellular proteins made during infection of a CF patient. Examination of extracellular proteins from a second strain, PA4, indicated it had a shared set of extracellular proteins. The identities of selected proteins were determined and these included well-characterised extracellular virulence factors such as elastase (LasB). Also identified were proteins with a potential virulence role such as azurin (a copper containing redox protein), PA2939 (a likely aminopeptidase) and proteins with unknown functions. This study provides the first evidence for the production of these proteins during infection. In summary, the proteomics methodology developed here facilitated the rapid identification and enumeration of proteins secreted by P. aeruginosa during infection.

Cet ouvrage présente la synthèse d'analogues du L-fucose comme inhibiteur de l'adhésion de la bactérie Pseudomonas aeruginosa (PA), dont la colonisation des poumons est mortelle chez les patients atteints de fibrose kystique. Ces dérivés fucosides devaient être reconnus de la PA-IlL, une lectine produite par la bactérie, ce qui leur permettra d'empêcher l'adhésion de P. aeruginosa aux poumons des patients atteints de la maladie. La préparation de O-, C-, N- et S-α-L-fucosides a été effectuée principalement à partir des réactions de O-glycosylation, de 'Click Chemistry' et de cycloadditions 1,3-dipolaires. La synthèse des dérivés α-L-fucosides a été réalisée en peu d'étapes, avec de bons rendements et avec un contrôle sur la configuration anomérique, ce qui a permis le développement de nouvelles méthodologies de synthèse sur des glycomimétiques. Le design rationnel de nos analogues fucosides se base sur des images cristallographiques présentant les interactions entre la PA-IlL et son ligand naturel le L-fucose. Une image cristallographique du complexe formé par un fucoside synthétique (composé 57) et la PA-IlL, a même pu être obtenue nous fournissant ainsi des informations supplémentaires. C'est à partir de ces nouvelles données qu'une exploration du pharmacophore de ce fucoside a été effectuée. L'insertion de pharmacophores en position C-6 du L-fucose a également été effectuée. La synthèse du 6,7,8-trideoxy-L-galacto-oct-7-enose a été réalisée avec un bon rendement global par le Dr. Karine Marotte. La fonction allyle en position C-6 a ainsi pu être fonctionnalisée par les réactions de métathèse croisée et de dihydroxylation asymétrique. Un homologue du L-galactose a également été préparé en deux étapes. Des résultats préliminaires en inhibition compétitive (tests ELLA) ont démontré que la lectine PA-IlL pouvait avoir une affinité 14 fois plus élevée pour certains de nos analogues, qu'elle en avait pour le fucose présent chez le trisaccharide Lewis a, le ligand naturel présumé de la bactérie. Les trois composés ayant démontré le meilleur potentiel d'inhibition sont des fucosides possédant des pharmacophores de type triazole et isoxazole. De tels agents anti-adhésion pourraient représenter une thérapie alternative ou un complémentaire aux antibiotiques dans le traitement d'infections mortelles.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

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Pseudomonas aeruginosa ATCC 9027 grew in a chemically defined medium with n-hexadecane or glucose, 0.5% (v/v or w/v, respectively), as the sole carbon source, and the K$ sb2$HPO$ sb4$, concentration was either 0.6 mM or 2.3 mM with both carbon sources. Only cells grown on n-hexadecane and 0.6 mM K$ sb2$HPO$ sb4$ produced pyocyanine. The variable lag period associated with cells grown on n-hexadecane was regulated by the state of the inoculating culture, grown on glucose and 2.3 mM K$ sb2$HPO$ sb4$. It was found that organic phosphate was more prevalent in cells grown on both phosphate concentrations with glucose and the high phosphate concentration with n-hexadecane, than it was for the low phosphate, n-hexadecane grown cells. The inorganic phosphate levels remained low under all conditions, decreasing as the cell culture became older. The inorganic polyphosphate level remained stable for all conditions, except in the high phosphate, n-hexadecane grown cells, where there was an increase.

The MexCD-OprJ multidrug efflux pump of Pseudomonas aeruginosa confers resistance to a range of antimicrobials. Although not expressed under normal laboratory conditions, exposure to the membrane-active biocides, chlorhexidine or benzalkonium chloride, results in mexCD-oprJ expression. This suggests that membrane disruption provides the inducing signal. Consistent with this, increased mexCD-oprJ expression was demonstrated in the presence of additional membrane-damaging agents including polymyxin B, ethanol, SDS, EDTA, the organic solvents n-hexane and p-xylene, and the antimicrobial peptides melittin, V8 and V681. MexCD-OprJ expression was initially verified through increased resistance to known MexCD-OprJ antimicrobial substrates and subsequently using a mexC-lacZ transcriptional fusion and RT-PCR. Since the P. aeruginosa sigma factor AlgU is responsive to envelope stress, it was of interest to ascertain whether AlgU is capable of mediating this increased mexCD-oprJ expression. Thus, the impact of AlgU loss on mexCD-oprJ expression in response to membrane-damaging agents was assessed in a algU strain. In contrast with above, little or no mexCD-oprJ expression (assessed using resistance to MexCD-OprJ antimicrobial substrates, the mexC-lacZ transcriptional fusion and RT-PCR) occurred in response to membrane-damaging agents in the algU strain, consistent with AlgU playing a role in the envelope stress inducibility of mexCD-oprJ. Overall, envelope stress, and the ability to react to this stress through AlgU, appears to play an important role in mexCD-oprJ induction. This suggests an important role for MexCD-OprJ in alleviating envelope stress, independent of its ability to export and provide resistance to antimicrobials. A gene, PA4596, whose product shows substantial homology to the NfxB repressor of mexCD-oprJ expression, occurs downstream of mexCD-oprJ and shows AlgU-dependence and chlorhexidine inducibility, suggesting a role in the chlorhexidine-induced, AlgU-mediated expression of mexCD-oprJ. Thus, the impact of PA4596 loss on mexCD-oprJ expression was assessed. Paradoxically, the loss of PA4596 increases mexCD-oprJ expression in wild-type cells in response to chlorhexidine treatment (as assessed through RT-PCR), while its loss compromises mexCD-oprJ expression in an nfxB mutant. Nonetheless, this suggests that PA4596 is involved in the induction of mexCD-oprJ and that its ability to induce mexCD-oprJ differs depending on the state of nfxB. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-07-31 12:03:52.535