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.

Pseudomonas chlororaphis strain PA23 has demonstrated biocontrol of Sclerotinia sclerotiorum (Lib.) de Bary, a fungal pathogen of canola (Brassica napus L.). The objectives of this research were two-fold: to optimize PA23 phyllosphere biocontrol and to investigate PA23’s influence in the rhizosphere. PA23 demonstrated longevity when inoculated on B. napus under greenhouse conditions. Carbon source differentially effected growth rate and antifungal metabolite production of PA23 in culture. Carbon source did not have a significant effect on in vivo biocontrol. PA23 demonstrated biocontrol ability of the fungal root pathogens Rhizoctonia solani J.G. Kühn and Pythium ultimum Trow in radial diffusion assays. PA23’s ability to promote seedling root growth was demonstrated in sterile growth pouches, but in a soil system these results were reversed. This research is essential for developing PA23 into an effective biocontrol agent in the phyllosphere and it opens the door for use of PA23 as a rhizosphere seed treatment. / October 2016

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.

Pseudomonas aeruginosa secretes several proteases associated with pathogenesis, but the most abundant and active is elastase (M4 metalloendopeptidase). Elastase (lasB), is first synthesized as a preproenzyme, with a signal peptide, an 18-kDa N-terminal propeptide, and a 33-kDa mature domain. The propeptide functions as an intramolecular chaperone that is required for the folding and secretion of elastase, but ultimately is proteolytically removed and degraded. Previous research has identified the conserved residues in the propeptide of elastase as compared to other M4 protease precursors and showed some among them to be important for the production of active elastase. In this project, the ability of the propeptide alone to fold into a defined secondary structure was explored and a molecular model was created. Furthermore, the effects of substitutions on conserved residues in the propeptide of plasmid-encoded lasB pro alleles were assessed by expressing them in a lasB propeptide mutant. The kinetics of elastase activity in culture supernatants was quantitated using a fluorescent substrate, Abz-AGLA-p-Nitro-Benzyl-Amide, to provide an accurate assessment of the effects of mutant propeptides. In vitro refolding studies were also performed to determine the effects of specific substitutions on foldase activity of the propeptide. When wild-type propeptide and mature elastase were denatured as separate proteins in guanidine-HCl buffer and renatured together, restoration of activity of the refolded elastase was measured, which was propeptide-dependent. Several mutant propeptides have now been shown to have defects using this in vitro foldase assay. Additional mutants were near wild-type activity level suggesting their role in recognition by the secretion apparatus. Residue locations were determined on a molecular model of the complex and confirmed the role of the secretion mutants as residues on the exterior. Residues that had diminished ability to refold in the in vitro assay were found to be in the interior parts of the complex, confirming their ability to be critical residues at the interface of the proteins or important in the stability of the propeptide’s intrinsic structure. The goal was to perform a series of comprehensive analyses of the propeptide and its conserved residues in order to determine its role as an intramolecular chaperone.

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.

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.