MexCD-OprJ is an RND-type multidrug efflux pump present in P. aeruginosa and is capable of exporting, and as such providing resistance to, several clinically important antimicrobials including fluoroquinolones, cephems, macrolides, and several biocides including chlorhexidine (CHX). Expression of mexCD-oprJ is negatively regulated by NfxB, a LacI-type repressor. The promoter region of mexCD-oprJ was identified and included two inverted repeat operator sites, B1 and B2, both of which are required in order for NfxB to bind, thereby repressing mexCD-oprJ. NfxB oligomerizes into a tetramer in solution and likely functions as a dimer of NfxB homodimers. In addition to being derepressed by loss of NfxB, MexCD-OprJ is inducible by a variety of non-antibiotic membrane-damaging agents (MDAs) such as CHX. A homologue of NfxB, PA4596, was found to be induced in response to CHX-promoted envelope stress in an AlgU-dependent manner and is directly repressed by NfxB. Loss of PA4596 resulted in increased resistance to the biocide CHX, shown to be a result of increased CHX-dependent expression of mexCD-oprJ. Susceptibility to CHX was restored upon expression of PA4596 from the plasmid pAK1900 as was decreased expression of mexCD-oprJ in the presence of CHX, indicating that PA4596 contributes to mexCD-oprJ repression in the presence of CHX. PA4596 was found to form oligomers in solution, likely dimers and tetramers. In the absence of NfxB, PA4596 is unable to contribute to repression of mexCD-oprJ. However, NfxB and PA4596 interact and together form a repressor capable of regulating mexCD-oprJ expression. Screening of transposon mutants for increased resistance to erythromycin potentially indicative of increased mexCD-oprJ expression lead to the identification of several novel genes including PA0479, cupA3, faoA, PA3259, mucD, and clpA whose loss generated a multidrug resistance profile consistent with increased production of MexCD-OprJ. However, further studies are required to determine how each of these genes may be affecting expression of mexCD-oprJ. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-06-12 12:07:28.67

La presente tesis tuvo como objetivo principal evaluar la influencia del meropenem en la formación de piocianina y alginato por cepas de Pseudomonas aeruginosa formadoras de biopelículas. Se detectó un 75% (60/80) de cepas formadoras de biopelículas por el método de O'Toole - Kolter de 80 aislados provenientes de pacientes de los Hospitales Nacionales Edgardo Rebagliati Martins y Guillermo Almenara Irigoyen, de las cuales el 30.0% (18/60) evidenciaron producción de piocianina en una concentración de 6.0 ug/mL a 6.5 ug/mL y el 36.6% (22/60) se evidenció producción de alginato en concentraciones de 6 ug/mL a 10 ug/mL expresado como ácido urónico en presencia de meropenem a la concentración de 10 μg . Sin embargo en ausencia de meropenem se obtuvo una menor producción de piocianina y alginato, por lo que se comprobó que el pigmento de piocianina y la producción de alginato son mecanismos de virulencia que favorecen la supervivencia de Pseudomonas aeruginosa en presencia del antibiótico.

The growth of environmentally persistent biofilms in cooling towers causes several associated problems, including microbiologically-induced corrosion (MIC) and biofouling. Current chemical control methods are not only ineffective against biofilms and costly to procure, they also have downstream environmental impacts when released untreated, or incur additional treatment costs. Bacteriophages are alternative biofilm control agents that have the potential to be more effective, cheaper to produce and yet have a more benign effect on the environment. In this study, biofilms grown under conditions simulating seawater fed cooling towers were characterized and the differences in growth and community make-up across time and different substrates were assessed. An MIC associated bacterium common in cooling tower water, P. aeruginosa, was chosen. Seven bacteriophage strains found to be effective against the chosen bacterium were isolated from wastewater influent. The relative effectiveness of these strains was measured against P. aeruginosa across different salinities. Separate biofilms fed with P. aeruginosa enriched seawater were characterized and the effectiveness of the isolated strains, singly and in cocktails, against the enriched biofilms was measured.

This investigation was concerned with the relationship of the slime layer material of Pseudomonas aeruginosa, Verder and Evans strain 1369, to the presumably somatic "O" type of antigen used by these authors as the base for their serological schema.

The purpose of this study was to extend our understanding of the ecological relationships of P. aeruginosa by investigating the differences or similarities between the strains of this organism found in sewage and those found as pathogens in human infections. This research was approached by comparing the serological types of P. aeruginosa isolated from sewage contaminated waters in Argentina (South America) to those isolated from sewage contaminated waters in Texas. They were typed with sera obtained using P. aeruginosa isolated from human infections. The data obtained revealed that bacteria isolated from sewage in Texas and from soil and water in Argentina are antigenically similar to those isolated from human infections.

The opportunistic pathogen P. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis patients. During chronic infection of the cystic fibrosis lung, P. aeruginosa undergoes conversion to a mucoid phenotype, constitutively producing the exopolysaccharide alginate, composed of the uronic acids D-mannuronate and L-guluronate. This alginate production contributes significantly to virulence in the cystic fibrosis lung. Evidence suggests that the acetylation state of the mannuronate component of the alginate influences the ability of components of the immune system to phagocytose the organism. To garner new and relevant information regarding the mechanism of alginate acetylation in Pseudomonas aeruginosa, a variety of approaches were undertaken. Analysis of the alginate produced by algX, algG, and algK alginate biosynthesis mutants revealed that the small oligouronides they produced were unacetylated. This strongly supports the hypothesis that the mannuronates are acetylated in periplasm, and that a polymer of at least some specific size is required. While three alginate biosynthesis gene products (AlgI, AlgJ, and AlgF) have been shown to be involved in alginate acetylation, another gene in the cluster, algX, shares 30% identity with one of them and thus generates speculation as to its potential involvement in the process. To test this possibility, an algX mutant was complemented with a plasmid carrying a mutation at a conserved residue shown to be required for alginate acetylation in the homologous protein. Analysis of alginate from this construct suggested that AlgX is not involved in alginate acetylation. To determine if changes in levels of alginate acetylation are accomplished at the transcriptional level, transcript levels of several alginate biosynthesis genes in different media were determined by real-time PCR. As qRT-PCR had not been previously performed on any of the alginate biosynthesis genes, this yielded important information about the transcription of the operon. In addition, beta-galactosidase assays on upstream regions of several biosynthesis genes identified two previously unrecognized promoters, one upstream of algG and one upstream of algI. The remaining approach was to examine protein interactions of AlgF, the protein product of one of the three acetylation genes. 2-D redox SDS-PAGE gels indicated that disulfide bonding may be important for interactions with this protein. While mass spectrometry was unable to identify the binding partners of AlgF, efforts are ongoing to create a mutation in the P. aeruginosa genome that changes the cysteine residue in AlgF to a serine residue. This would be a definitive method for determining the importance of disulfide bonding in AlgF.

As Pseudomonas aeruginosa adapts to the Cystic Fibrosis (CF) and chronic obstructive pulmonary disease (COPD) lung environments, mucoid strains often appear as a result of alginate overproduction. Such mucoid conversion is associated with the establishment of a chronic pulmonary infection. Alginate confers resistance to phagocytosis and has other pathogenic properties. The regulation of alginate production is complex and involves an alternate sigma factor, anti-sigmas and several DNA-binding transcriptional regulators. Here we examined the possibility that the catabolic repression control (Crc) protein repressor may affect alginate gene expression. A putative Crc binding site was observed adjacent to the ribosome binding site of algD, the first gene in the 12-gene alginate biosynthetic operon. We hypothesized that Crc binding here would act as a repressor of algD expression. Taking a genetic approach, Gateway technology was used to construct crc::GmR (nonpolar) mutants of P. aeruginosa strain PAO1 and its mucoid (mucA) mutant derivative, PDO300. The crc mutation had the expected phenotypes with respect to pyocyanin production, biofilm formation and diauxic growth. When a PalgD-lacZ (translational) fusion was tested, the crc mutant showed increased algD expression as predicted for a mRNA-binding repressor. Another Ptrc-algD-lacZ (translational) construct, but missing the upstream promoter (PalgD) elements, also showed increased activity in crc mutants as predicted if Crc was acting directly as a repressor of algD transcriptional / translational expression. However, this was not consistent with the production of alginate. The crc mutant of mucoid PDO300 showed lower levels of alginate production than its parent strain. Under conditions were the algD operon was induced by ammonium metavanadate in the growth medium to produce alginate, the crc mutation again resulted in a lower level of alginate production than wild-type, which was again inconsistent with the algD-lacZ expression data. This suggests that crc mutation, which has global effects on carbon flow in the cell, could be affecting metabolic pathways that feed precursors into the alginate biosynthetic pathway. Future directions for this research are discussed.

Magíster en Bioquímica área de Especialización en Bioquímica ambiental / Memoria para optar al Título de Bioquímico / Los problemas ambientales y la disminución de las reservas de petróleo han reiniciado la búsqueda de combustibles alternativos a los combustibles fósiles. El etanol se presenta como una alternativa sustentable para resolver esta problemática. En la naturaleza existen microorganismos silvestres productores de etanol, tales como la bacteria Zymomonas mobilis y la levadura Saccharomyces cerevisiae. Si bien estos dos microorganismos son altamente eficientes en la generación de etanol, sólo son capaces de utilizar azúcares de 6 carbonos, limitando la producción industrial de etanol solo a materias primas que contengan hexosas. En los residuos lignocelulósicos se encuentra una gran reserva de hidratos de carbono almacenada como polímeros de glucosa (celulosa) o azúcares de 5 carbonos como xilosa y arabinosa. Para el aprovechamiento de pentosas disponibles en esta materia prima se han modificado estos microorganismos, mediante la introducción de los genes de las enzimas necesarias para metabolizar arabinosa y xilosa o se han incorporado los genes de las enzimas de la ruta etanológica de Z. mobilis, piruvato descarboxilasa (pdc) y alcohol deshidrogenasa II (adhB) a bacterias capaces de utilizar pentosas, como Escherichia coli, Klebsiella oxycota, Lactobacillus plantarum, Lactococcus lactis y Bacillus subtilis. El género Pseudomonas, principalmente Pseudomonas aeruginosa, posee muchas de las características necesarias para la producción industrial de etanol, es una versátil bacteria Gram negativo, capaz de adaptarse y sobrevivir bajo amplio rango de condiciones ambientales y al igual que Z. mobilis asimila azúcares por la ruta de Entner-Doudoroff, además posee una toleracia a etanol mayor que bacterias E. coli recombinantes utilizadas en la producción de etanol. El objetivo general de este trabajo es la construcción de un operón productor de etanol para la expresión de los genes pdc y adhB de la ruta etanologénica de Z. mobilis en P. aeruginosa PAO1. El diseño del operón artificial incluyó los siguientes elementos genéticos: el promotor del operón inducible arabinosa (PBAD), sitio de unión a ribosoma (RBS), gen pdc, gen adhB y un terminador transcripcional para P. aeruginosa. Las primeras etapas para la construcción del operon pet, contemplaron la realización de 2 construcciones genéticas mediante PCR. La primera comprende la mínima región codificante del gen pdc y una región adaptadora de 33 pb (adp), la segunda corresponde a la unión de adp2 (secuencia que contiene una zona complementaria a adp y un sitio de unión a ribosoma) con la mínima región codificante del gen adhB y un terminador transcripcional específico para Pseudomonas. Los análisis de restricción, PCR y secuenciación mostraron la obtención de un fragmento 1740 pb correspondiente al producto pdc-adp, un fragmento de 1247 pb para el producto adp2- adhB-term. Para los productos de PCR de la fusión de las construcciones pdc-adp con adp2- adhB-term se obtuvieron amplicones inespecíficos de tamaños superiores e inferiores al tamaño teórico esperado (3 Kb). De manera alternativa para la obtención de la construcción que contenga los genes pdc y adhB con la secuencia adaptadora y de unión a ribosoma, se realizaron reacciones de ligación de ambos fragmentos utilizando la enzima T4 DNA ligasa, las cuales fueron clonadas en el vector pSC-B. De todos los clones analizados solo uno contenía la construcción esperada (plasmidio pCL27-B), pero los análisis de PCR revelaron que los genes no se encontraban en la orientación adecuada, por lo tanto no fue posible obtener la construcción del fragmento pdc-adp-RBS-adhB, construcción clave en el término de la construcción del operón pet. / Environmental problems and declining oil reserves have resumed the search for alternative fuels to fossil fuels. Ethanol is presented as a sustainable alternative to solve this problem. In nature there are wild ethanol producing microorganisms, such as bacteria Zymomonas mobilis and yeast Saccharomyces cerevisiae. While these two microorganisms are highly efficient in the generation of ethanol, are capable of using only 6 carbon sugars, limiting the industrial production of ethanol only to raw material containing hexoses. In lignocellulosic waste there is a large reservoir of stored carbohydrates like glucose polymers (cellulose) or 5-carbon sugars such as xylose and arabinose. For the utilization of available pentoses in this raw material, such microorganisms have been modified by the introduction of genes of the necessary enzymes to metabolize arabinose and xylose or enzymes genes have been incorporated of etanologic pathway from Z. mobilis, pyruvate decarboxylase (pdc) and alcohol deshidrogenasa II (adhB) to bacteria capable to use pentoses, such as Escherichia coli, Klebsiella oxycota, Lactobacillus plantarum, Lactococcus lactis and Bacillus subtilis. The genus Pseudomonas, mainly Pseudomonas aeruginosa, has many of the features needed for industrial production of ethanol, is a versatile Gram negative bacteria, able to adapt and survive under wide range of environmental conditions and like Z. mobilis assimilated sugars by the Entner-Doudoroff pathway, and in addition has ethanol higher tolerance than recombined E. coli bacteria used in the production of ethanol. The overall aim of this work is the construction of an ethanol producer operon for the expression of pdc and adhB genes from the Z. mobilis etanologic pathway on P. aeruginosa PAO1. The artificial operon design included the following genetic elements: the arabinose inducible operon promoter (PBAD), ribosome binding site (RBS), pdc gene, adhB gene and a transcriptional terminator to P. aeruginosa. The first step to construct the pet operon, contemplated 2 genetic constructs by PCR. The first involves a pdc gene minimum coding region and a adapter region of 33 bp (adp), the second corresponds to the union of adp2 (sequence that contains a complementary zone to adp and a ribosome binding site) with adhB gene minimal encoding region and a specific transcriptional terminator to Pseudomonas. The restriction analysis, PCR and sequencing showed the obtention of a 1740 pb fragment corresponding to the pdc-adp product, a 1247 pb fragment for the product adp2-adhB-term. For the PCR products of pdc-adp and adp2-adhB-term construction fusion, non specific amplicons were obtained in higher and lower sizes than the theoretical size expected (3 Kb). Alternatively for the obtention of the construction which have the pdc and adhB genes with the adapter sequence and ribosome binding, ligation reactions were performed of both fragments using T4 DNA ligase enzyme, which were cloned on the vector pSC- B. Of all the clones analyzed just one contained the expected construction (plasmid pCL27-B), but PCR analysis revealed that genes were not in the proper orientation, therefore it was not possible to obtain the construction of fragment pdc-adp-RBS-adhB, key construction in the completion of the pet operon construction.

Pseudomonas aeruginosa ist ein gramnegatives Bakterium, welches insbesondere bei abgeschwächter Immunabwehr schwere Infektionen auslösen kann. Es besitzt eine hohe intrinsische Resistenz gegenüber Antibiotika, so dass nur eine begrenzte Anzahl von Antimikrobiotika wie beispielsweise Aminoglykoside für die Behandlung zur Verfügung steht. Unter Antibiotikatherapie wird zudem eine schnelle Resistenzentwicklung beobachtet, daher ist die Weiterentwicklung und Optimierung der Therapieoptionen von großer Bedeutung. Die vorliegende Arbeit untersucht den Einfluss von Colistin auf die Aminoglykosidresistenz bei Pseudomonas aeruginosa. 25 Bakterienstämme, die zu Beginn gegenüber Amikacin, Tobramycin und Gentamicin resistent waren, wurden Colistin ausgesetzt. Mithilfe des Epsilometertests wurde die minimale Hemmkonzentration (MHK) der Antibiotika für die zu untersuchenden Bakterienstämme vor und nach Colistineinfluss bestimmt. Es konnte ein signifikanter Rückgang der minimalen Hemmkonzentration nach Colistineinwirkung dokumentiert werden. Zu den Hauptresistenzmechanismen von Pseudomonas aeruginosa gehören die Effluxpumpen, welche die Antibiotika aus dem Bakterium ausschleusen. Für den Transport von Aminoglykosiden ist die MexXY-Pumpe verantwortlich, die in dieser Arbeit weiteruntersucht wurde. Durch eine quantitative Echtzeit-PCR unter Nutzung des Fluoreszenz-Resonanz-Energie-Transfers (FRET) wurde die Expression der Pumpe vor und nach Colistin verglichen. Es konnte nachgewiesen werden, dass durch Colistin die Expression reduziert wurde. Ein linearer Zusammenhang zwischen MHK-Veränderung und mexXY-Expressionslevel wurde anhand der Untersuchungsergebnisse nicht ermittelt. Es ist dementsprechend davon auszugehen, dass andere Resistenzmechanismen ebenfalls durch Colistin beeinflusst werden und so die MHK-Reduktion erklären können.

Identifica la prevalencia de genes que codifican carbapenemasas de tipo metalo β-lactamasas (MBL) en aislados clínicos de P. aeruginosa. Analiza 76 aislados clínicos de P. aeruginosa resistentes a Ceftazidima y “no sensibles” (intermedio o resistentes) a Imipinem y/o Meropem colectados en el Hospital Militar Central de enero a setiembre del 2016. Muestras de secreciones respiratorias, heridas, orinas y hemocultivos de pacientes hospitalizados son procesadas en el Laboratorio de Microbiología. Determina la sensibilidad antimicrobiana por el método de disco de difusión según los criterios del Clinical and Laboratory Standards Institute. Realiza la detección fenotípica de MBL por el test de sinergia de doble disco con imipenem, meropenem y EDTA. La detección genotípica se realiza amplificando por PCR multiplex, los genes blaIMP y blaVIM, mientras que para el gen blaNDM se hizo PCR convencional. Obtiene fenotípicamente 25 de 76 pruebas positivas para MBL, 24(31.58%) de las cuales se confirmaron genéticamente, encontrando el gen blaIMP (23/24, 95.83%) y el gen blaVIM (1/24, 4.17%). La sensibilidad de la prueba fenotípica es del 100% y la especificidad del 98%. Concluye que el 31.58% de los aislamientos clínicos de P. aeruginosa recuperados de pacientes hospitalizados en el HMC, presentan MBL, siendo el gen blaIMP el más prevalente. / Tesis