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Pseudomonas aeruginosa es un importante patógeno oportunista del ser humano y tiene amplia versatilidad metabólica por lo que se puede adaptar a vivir en ambientes ubicuos, pudiendo persistir en ciertas superficies como tejidos formando comunidades especializadas llamadas biopelículas, además de elaborar otros factores de virulencia, como el pili tipo IV (T4P) vinculado no sólo a la motilidad llamada “contracción” sino también pareciera estar relacionada con las etapas tempranas en la formación de las biopelículas. La presente tesis tuvo como principal objetivo determinar la presencia de los diferentes genotipos del T4P así como los factores de virulencia asociados en cepas clínicas de diferentes fuentes, encontrando que la distribución de genotipos fue 25,9 % para el Grupo II, 19 % para el Grupo I, 24,1 % para el Grupo III, 10,3 % el Grupo IV y 20,7 % para el Grupo V, de tal manera que en nuestro medio existe distribución homogénea de los diversos genotipos. Palabras clave: Pseudomonas aeruginosa, Genotipos de pilA, Motilidad

Los avances en la ciencia y la tecnología desde la revolución industrial, han aumentado la capacidad del ser humano para explotar los recursos naturales causando un constante aumento de perturbaciones en los diversos ecosistemas. En este contexto, el uso de los biosurfactantes que solubilizan fracciones de hidrocarburos contaminantes, representa una prometedora alternativa de aplicación para cualquier proceso tecnológico de remediación de ambientes naturales. El objetivo del presente trabajo se basó en la evaluación de la actividad emulsificante y la capacidad de remoción de metales pesados de un biosurfactante de naturaleza ramnolipídica producido empleando glicerol como única fuente de carbono por una cepa de Pseudomonas aeruginosa PB25. Con los resultados obtenidos se logró determinar una velocidad de crecimiento específica (µ) de 0,0351 h^-1 y un tiempo generacional (tg) de 19,75 h; registrándose a su vez una concentración máxima de ramnolípidos de 2,47 g/L en la fase estacionaria de crecimiento, con valores de rendimiento de 0.13 gramos de ramnolípido por gramo de glicerol y de productividad de 0.082 g/L-h. De igual forma se logró conseguir un valor máximo de actividad emulsificante frente a crudo de petróleo de 5.257 UAE/mL e Índices de Emulsificación E24 de 53, 64, 62 y 84 % para crudo de petróleo, petróleo diesel 2, gasolina y kerosene respectivamente. Asimismo, se obtuvieron valores máximos de remoción del 98% y 99% para plomo y cadmio respectivamente presentes en soluciones acuosas a pH 11. Por lo tanto, es posible afirmar que este tipo de biosurfactante puede ser empleado en futuros procesos biotecnológicos de remediación de ambientes naturales contaminados por la presencia de metales pesados y/o crudo de petróleo y sus derivados. Palabras clave: Remediación, biosurfactantes, ramnolípidos, Pseudomonas aeruginosa, actividad emulsificante, hidrocarburos, metales pesados. / --- Since the industrial revolution, advances in science and technology have increased the human capacity to exploit natural resources causing ever-increasing disturbances in ecosystems. In this context, the use of biosurfactants represents a promising alternative application for any technological process of remediation of natural environments. The objective of this work was based on the evaluation of the emulsifying activity and the ability to remove heavy metals with a rhamnolipidic biosurfactant produced by Pseudomonas aeruginosa strain PB25, using glycerol as sole carbon source. In this media this strain had a specific growth rate (μ) of 0,0351 h^-1 and a generational time (td) of 19,75 h. At the same time it produced maximum concentration of 2.47 g/L rhamnolipid, with yields (Y) of 0.13 g/g and productivity of 0.082 g/L-h. Similarly, the rhamnolipid is managed to achieve a maximum value of emulsifying activity compared to crude oil from 5.257 UAE/mL and E24 emulsification index of 53, 64, 62 and 84% for crude oil, diesel oil 2, gasoline and kerosene respectively. In addition, the rhamnolipid got to remove 98% of lead and 99% of cadmium in aqueous solutions at pH 11. In conclusion, this type of biosurfactant can be used in future biotechnological processes for the remediation of natural environments contaminated by the presence of heavy metals and/or crude oil and its derivatives. Keywords: Remediation, biosurfactant, rhamnolipid, Pseudomonas aeruginosa, emulsifying activity, hydrocarbons, heavy metals.

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

The role of microorganisms in the degradation of xenobiotics in the environment is well established. Bacteria from the genus Pseudomonas are particularly well adapted to the degradation of hydrocarbons, aromatics, and numerous other natural and introduced substrates. We have isolated a strain of Pseudomonas putida, designated PC2P15, that uses toluene, phenol, benzene, and a number of other substrates as its sole sources of carbon and energy.

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.

Graduation date: 1997

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 101-116). Also available on the World Wide Web.