Publicación a texto completo no autorizado por el autor. / Los ramnolípidos son biosurfactantes producidos por una gran cantidad de microorganismos, estos presentan gran diversidad de aplicaciones, siendo una de sus más importantes aplicaciones es en procesos de biorremediación en áreas contaminadas con hidrocarburos y metales pesados. El presente estudio está enfocado en investigar algunos de los parámetros fermentativos críticos para la producción de ramnolípidos por Pseudomonas aeruginosa 6k-11 usando dos fuentes de nitrógeno orgánica e inorgánica y dos diseños experimentales. El proceso para la producción de ramnolípidos fue optimizado usando un diseño central compuesto y Box Behnken como diseños experimentales y metodología de superficie de respuesta. Los factores tomados en cuenta para su estudio fueron el pH, temperatura, velocidad de agitación y porcentaje de inóculo. La máxima producción para la fuente de nitrógeno orgánica (triptona y urea) fue 23,2 g/L de ramnolípidos después de 168 horas de fermentación con las variables: 170 rpm de velocidad de agitación, 31,9°C de temperatura, 6,8 de pH, 6,8 (v/v) % de inóculo. La máxima producción teniendo como fuente de nitrógeno inorgánico (nitrato de sodio) fue 25,7 g/L de ramnolípidos luego de 168 horas de fermentación con las variables de fermentación: 185,8 rpm de velocidad de agitación, 31,3 °C de temperatura, 6,8 de pH, 7,3% de inóculo. Experimentalmente se confirmó 24,01 ±1,5 g/L y 28,01 ± 2,5 g/L de ramnolípidos para el medio I y II respectivamente. Los resultados de la presente investigación sugieren que la aplicación de la modelización matemática es muy eficaz para determinar las condiciones óptimas de reacción y mejorar el rendimiento de bioprocesos microbianos. / Tesis

Reductive dechlorination of chlorinated ethenes is dependent upon suitable substrates promoting microbial activity and creating anaerobic conditions. At the periphery of active reductive dechlorinating zones combinations of lesser chlorinated ethenes should exist along with end products of the anaerobic metabolism that is driving reductive dechlorination. Potential end-products of anaerobic metabolism were investigated for their ability to stimulate oxidative cometabolism of dichloro ethenes (DCEs) by the butane-degrading bacterium, Pseudomonas butanovora. Organic acids that supported butane-monooxygenase (BMO) activity were acetate, propionate, lactate, and butyrate. Lactate consistently supported and sustained greater rates of cooxidation than did the other organic acids. When propane replaced butane as the growth substrate, lactate remained the superior electron donor, while the ability of butyrate and acetate to support BMO activity decreased. In contrast, propionate-supported cooxidation was only observed in propane-grown cells. Lactate supported the degradation of 1,2-trans dichloroethylene (1,2-trans DCE), 1,2-cis dichloroethylene (1,2-cis DCE) and 1,1-dichloroethylene (1,1-DCE) in butane-grown P. butanovora. 78 nmoles (25 μM) of 1,2-cis DCE were completely degraded by butane-grown P. butanovora. In contrast, smaller amounts of 1,1-DCE and 1,2-trans DCE were degraded over the twenty minute time course. Decreasing rates of cooxidation over time were observed for of all three DCEs, and 50% of BMO activity was irreversibly lost after 15 min, 6 min, and 0.5 min exposures to 1,2-cis DCE, 1,2 trans-DCE, and 1,1-DCE respectively. Cell viability decreased by over 90, 95, and 99.95% during the transformation of 25 nmoles/mg protein of 1,2-cis DCE, 1,2-trans DCE and 1,1-DCE. These results indicate that cellular viability was more sensitive to cooxidation of 1,2-cis DCE and 1,2-trans DCE than was BMO. 1,2-cis DCE and 1,2-trans DCE induced BMO activity to 25 and 45% of the butane control, respectively. Induction by 1,2-trans DCE was observed at a threshold of about 20 μM and higher concentrations did not increase BMO activity. Fusion of lacZ to the BMO catabolic promoter, with consequent knock out of BMO activity, provided the opportunity to assess substrate induction without the confounding effects of enzyme inactivation and product induction. While BMO substrates, butane, 1,2-cis DCE, and ethylene, were unable to induce lacZ activity the BMO products, 1-butanol, and ethylene oxide, effectively induced lacZ activity. 1,2-trans DCE was unique among the BMO substrates tested in it's ability to induce expression of lacZ, 2-fold above background, in the reporter strain. A wide range of concentrations induced lacZ activities (10 to 100 μM), and low levels of 1,2-trans DCE achieved high levels of induction after 4 hrs. However, lacZ activities were limited to an induction of about four-fold above background and this limit allowed lower concentrations of 1,2-trans DCE to eventually produce equal levels of beta-galactosidease. These data provide proof-of- concept that BMO-dependent cometabolism can occur independently of butane as an inducer and electron donor for BMO gene expression and activity. / Graduation date: 2004

Thesis (M.S.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Thomas S. Livinghouse. Includes bibliographical references (leaves 76-82).

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

La mucoviscidose reste une maladie génétique grave sans traitement curatif. L’amélioration de la prise en charge de la maladie s’est accompagnée d’une augmentation importante de l’espérance de vie qui était de moins de un an dans les années 1950 et dépasse en moyenne 40 ans pour les enfants nés aujourd’hui. La mucoviscidose se caractérise au niveau pulmonaire par un mucus anormalement épais qui est difficilement évacué. Le mucus s’accumule alors dans les voies respiratoires et les bactéries y persistent et s’y multiplient. 80 à 95% des patients décèdent des suites de l’insuffisance respiratoire causée par les infections chroniques bactériennes concomitantes aux inflammations des voies aériennes. Les patients souffrent particulièrement d’infections à P. aeruginosa qui est la bactérie la plus fréquemment rencontrée dans la mucoviscidose. Lors des premières infections à P. aeruginosa les bactéries sont de phénotype non mucoïde et sont relativement sensibles aux antibiotiques. Les bactéries finissent par s’adapter dans les voies aériennes des patients atteints de mucoviscidose et forment des biofilms multiresistants aux antibiotiques. Les patients sont alors colonisés par P. aeruginosa de façon permanente. Les infections chroniques à P. aeruginosa sont responsables du déclin de la fonction respiratoire, d’exacerbations pulmonaires nécessitant une hospitalisation et de la mortalité des patients atteints de mucoviscidose. Il est donc urgent d’améliorer les traitements symptomatiques existants dans le but d’augmenter la qualité et l’espérance de vie des patients en attendant de nouveaux traitements curatifs visant à corriger l’anomalie génétique. Ce travail s’est donc orienté vers l’étude de l’efficacité in vitro de différentes combinaisons d’antibiotiques vis-à-vis des biofilms bactériens. Parmi l’ensemble des associations d’antibiotiques testées une synergie d’activité a été observée entre la tobramycine et la clarithromycine sur des biofilms âgés de 24 heures mais aussi de 12 jours. Notamment parmi les 26 souches testées une synergie d’action est observée chez 11 souches après une coadministration pendant 28 jours de tobramycine et clarithromycine en doses répétées sur un biofilm « mûr » âgé de 12 jours. Parmi ces 11 souches, 7 biofilms sont entièrement détruits à la fin du traitement. Sur l’ensemble des souches étudiées le traitement avec l’association est soit supérieur ou équivalent à celui de la tobramycine seule et a permis d’obtenir une action dans 53.8% des cas pour la combinaison versus 30.8% pour la tobramycine. Cependant aucune corrélation n’est trouvée entre le profil génétique, le phénotype mucoïde ou non des souches et la présence de synergie d’action ou la destruction entière du biofilm. Cette dernière étude s’inspire du traitement actuel de référence TOBI® qui s’administre par cycle de 28 jours, 2 fois par jour. Les différents travaux de cette thèse ont permis tout d’abord de développer un modèle in vitro proche des conditions rencontrées in vivo chez les patients atteints de mucoviscidose. Ce modèle pourra également être utilisé pour une série d’autres études et mises au point de nouveaux médicaments sous forme de molécule isolée ou en combinaison. Ce travail a également permis d’envisager le développement d’un nouveau médicament dans le traitement d’infections chroniques à P. aeruginosa chez les patients atteints de mucoviscidose. Cependant des études futures in vivo sur souris mais aussi toxicologiques et cliniques seront indispensables pour confirmer le profil d’efficacité et de sécurité de cette association.

Pseudomonas is the most common cause of chronic lung infections leading to death in cystic fibrosis patients. While chronic infection is extremely difficult to eradicate, the initial bacterial-host interactions prior to biofilm formation and establishment of chronic infections represents an attractive therapeutic target. It is clear that interaction between pathogens and the host is a very complex process and successful adaptation requires tight control of virulence factor expression. The aim of this project was to look for early changes in P. aeruginosa global gene expression in response to attachment to epithelial cells. P. aeruginosa PA01 was incubated with polarized HBE cells at a MOI of 100 for 4 hours and bacteria attached to epithelial cells (interacting) were collected separately from those in the supernatant (non-interacting). To minimize media effects observed by others, iron and phosphate were supplemented at appropriate levels to avoid expression changes due to limitation of these nutrients, as confirmed in our microarray experiments. Analysis of 3 independent experiments demonstrated that 766 genes were up or down regulated by more than 1.5 fold during attachment. Among these, 371 genes, including ion, oprC, as well as 3 genes in quorum-sensing systems and 9 genes involved in the pmrAB and phoPQ two-component regulatory systems were found to be induced in the interacting bacteria. On the other hand, 395 genes, including oprG outer membrane porin and pscP involved in type III secretion system were down regulated. To understand the roles of these differentially expressed genes, a cytotoxicity (LDH release) assay was performed and demonstrated that oprG and ion mutants were less capable than the wild type of killing HBE epithelial cells. These findings suggest that, under these interaction assay conditions, regulation of the expression of certain virulence factors provides a potential advantage for successful adaptation. In addition, a mutant lacking a filamentous hemagglutinin like protein was found to be less cytotoxic to HBE cells and also deficient in A549 epithelial cell binding, indicating that this probable non-pilin adhesin has multiple functions in P. aeruginosa.

A pathogenicity locus of Pseudomonas syringae pv. syringae identified by Tn5 mutagenesis was investigated. The mutant strain PS9024 is attenuated for disease expression in its host, Phaseolus vulgaris, but produces the hypersensitive reaction (HR) in the nonhost, tobacco (Nicotina tabacum). A cosmid clone carrying 16 kilobases (kb) of contiguous genomic DNA partially complements this mutant. Altered growth of the mutant in planta was also partially restored. Marker exchange mutagenesis with Tn3- HoHo1 at two other sites within this locus results in mutants with attenuated and severely reduced pathogenicity. The locus is complex and contains repetitive DNA sequences. Northern analysis reveals that this locus is expressed in planta, but is not expressed in a rich growth medium, and the transcript is larger than 10 kb, suggesting that the locus is transcribed as a polycistronic mRNA. Comparison of total cellular protein profiles of R32 and PS9024 using SDS-PAGE analysis further reveals that at least nine protein bands ranging from approximately 100 kD or above in size are present in the wild type strain R32, but absent from the mutant. Additionally, a protein of approximately 45 kD is absent from the mutant. The site of Tn5 insertion has been partially sequenced. The initial search of the data banks suggested a gene or genes related to the ornithine biosynthetic pathway map to this locus. Further study strongly suggest a gene that encodes a membranceassociated protein and under the control of a promoter identical to appA gene promoter maps at this site and it is involved in the process of pathogenesis. / Graduation date: 1991

Pseudomonas aeruginosa, es uno de los más importantes patógenos humanos oportunistas, que puede colonizar no solo superficies abióticas, sino también superficies bióticas. Una vez colonizadas estas superficies, dicha bacteria produce exopolisacárido originando la formación de biopelículas, en consecuencia, estas bacterias son mil veces más resistentes, no sólo a antibióticos, sino también a biocidas y desinfectantes, constituyendo un problema de salud pública. Para este trabajo de investigación se recolectaron 62 cepas aisladas de líquidos biológicos y secreciones de pacientes procedentes del Hospital Nacional Edgardo Rebagliati Martins durante el periodo de Enero a Setiembre del 2006, identificándose el 100% de ellas como cepas de Pseudomonas aeruginosa. Sólo se escogió el 81% de las cepas formadoras de biopelículas para investigar la influencia del mucolítico NAC sobre las biopelículas de Pseudomonas aeruginosa, ya que éstas presentaron mayor producción durante la cuantificación realizada por el Método de O’Toole and Kolter. / Pseudomonas aeruginosa, is one of the most important human pathogens opportunistic, that can colonizes not only abiotics surfaces, also biotics surfaces. Once colonized these surfaces produce extracellular polysaccharides originating the biofilms formation, in consequence, these bacterias are thousand times more resistant to antibiotics, biocides and desinfectants, constituting a problem of public health. For this study, were collected 62 strains isolated from biological fluids and secretions of patients from the Edgardo Rebagliati Martins National Hospital during the period from January to September 2006, indentifying 100% of them like Pseudomonas aeruginosa strains. The determination of biofilm production we preferred to use Congo Red Agar Method (ARC), for its sensibility and reproducibility which showed that 42% were biofilm producers, whereas 48% were biofilm non developed. Pseudomonas aeruginosa ATCC 9027 (non producer) were used as negative control. Only the 81% of them was chosen to investigate the influence of mucolitic N-acetylcysteine (NAC) on Pseudomonas aeruginosa biofilms, by the biggest biofim production during the quantification by O’Toole and Kolter Method.

Del mes de enero al mes de octubre del año 2008 en el Servicio de Microbiología del Hospital Nacional Guillermo Almenara Irigoyn – ESSALUD, se colectaron 186 cepas de Pseudomonas aeruginosa resistentes al Imipenem (IMP) y al Meropenem (MEM) o con resistencia Intermedia a los dos antibióticos o a uno de ellos. Utilizando el Método Fenotípico de Doble Difusión en Disco con Monodiscos de EDTA; se llegó a detectar 13 cepas de P. aeruginosa positivas para MBLs, correspondiente al 6.99 % de las cepas testadas; de las cuales, el 53.84 % de la sinergia (efecto de la metaloenzima) se manifestó en el disco de Meropenem, el 30.75 % se manifestó en ambos discos (Imipenem y Meropenem) y solo el 15.38 % se manifestó en el disco de Imipenem. El 100 % de cepas de P. aeruginosa en las que se detectó las metaloenzimas provinieron de pacientes hospitalizados, de estos aislados el 69.76 % correspondieron al sexo masculino; el 61. 53 % de casos corresponden a pacientes cuyas edades están entre 70 a 90 años, y sólo el 14.14% de casos fueron en menores de 1 año y jóvenes. Las muestras biológicas en las que aisló a P. aeruginosa productora de MBLs fueron: orina, aspirado bronquial, BAL, herida no operatoria, secreción otica, líquido de diálisis peritoneal y sangre; fue orina en la que se aisló el mayor número de casos: 46. 15 %; en el Servicio de Medicina‐1, se detecto más casos 30.76 %, seguido de Cardiología y UCI con 23.07 %. Se detectó la enzima MBLs en cepas peruanas de Pseudomonas aeruginosa y se estableció la incidencia en 6.99 %, la cual es inferior a las reportadas por otros países, por lo cual se debe hacer estudios multicentricos para establecer la incidencia real. / In the study, 186 samples of Pseudomonas aeruginosa strains resistant to the Imipenem (IMP) and the Meropenem (MEM) or with Intermediate resistance to both antibiotic and one of them were collected in the Service of Microbiology of the Guillemo Almenara Irigoyen National Hospital ‐ ESSALUD from the month of January to the month of October of the year 2008. Using the Phenotypical Method of Double Disc Diffusion with Monodiscs of EDTA; 13 positive strains of Pseudomonas aeruginosas for MBLS were detected (6.99 % of the sampled strains): of them, the 53.85% of the synergy (effect of metal‐enzyme) was present in the disc of Meropenem, the 30.75% was present in both discs (Imipenem and Meropenem) and only the 15.38% was present in the disc of Imipenem. The 100% of the P. aeruginosa strains in which metal‐enzyme was detected belonged to hospitalized patients and the 69.76% of them belonged to masculine sex. The 61.54 % of cases correspond to patients whose ages are between 70 to 90 years, and only the 15.38% of the cases were in minors of 1 year and young people. The biological samples, in which the producing P. aeruginosa of MBLs was isolated, were: urine, inhaled bronchial, BAL, non operating wound, ear secretion, liquid of dialysis peritoneal and blood; but it was in the urine in that the greater number of cases was isolated the 46. 15 %. In the Service of Medicine‐1 were detected mayor number of cases (23.07%) followed for Cardiology and UCI (23.07%). The MBLs enzyme was detected in Pseudomonas aeruginosa peruvian strains. The incident was of 6.99% that is lower tanning the incident reported in other countries. For which Multicentral studies must be done for establish the real incident.

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 27-37).