No description available.

No description available.

When Pseudomonas aeruginosa ATCC 9027 var. RCII, a chloramphenicol-tolerant substrain, is grown in a phosphate limited, complex medium, along with the drug (150 (mu)g/ml), it accumulates high intracellular levels of inorganic phosphate and fails to synthesize normal levels of alkaline phosphatase and pyocyanine. Glucose transport is additionally hindered, and, accordingly, extracellular glucose is mainly oxidized to 2-ketogluconate. The preference of NAD(H)-linked enzymatic activities suggests the absence of transhydrogenase activity. The cytochrome content and intracellular ATP pool of this substrain are also greater. The ATP pool is further augmented when chloramphenicol is omitted from the medium. H('+)/O analysis confirmed that the substrain gained one additional ATP conservation site. Drug tolerance in P. aeruginosa ATCC 9027 is clearly accompanied by greater energy production. Slower growth arises since more energy is delegated towards maintenance and survival in the presence of the drug.

Intrinsic antimicrobial resistance of the opportunistic human pathogen Pseudomonas aeruginosa has mainly been attributed to the presence of several chromosomally-encoded multidrug efflux systems. The MexAB-OprM system exports the largest range of structurally unrelated antimicrobial agents and its expression is modulated by multiple regulatory controls. To develop a better understanding of mexAB-oprM overexpression in nalC mutants, which characteristically produce the effector protein PA3719 that binds and disrupts MexR transcriptional repression of mexAB-oprM, the PA3719-MexR interaction domains were investigated. Using a bacterial two-hybrid system, the C-terminus of PA3719 was found to be sufficient to mediate MexR-binding, and the binding region was found to be distinct from the MexR DNA-binding motif. The two-hybrid system was also used in an attempt to understand the role of PA3720, a protein of unknown function that is also overexpressed in nalC mutants. Results from this study confirm that PA3720 does not function to bind and alleviate NalC transcriptional repression of the PA3720-PA3719 operon. This study also attempted to identify the signals involved in overexpressing PA3720-PA3719, in the hopes to elucidate the natural function of MexAB-OprM. Random transposon mutagenesis using a PA3720-PA3719 promoter-lacZ fusion containing P. aeruginosa strain was conducted, but failed to clearly identify any disrupted genes associated with PA3720-PA3719 overexpression. Using the same PA3720-PA3719 promoter-lacZ fusion, expression of these genes was assessed as a function of growth in both wildtype and nalC mutant P. aeruginosa strains. Interestingly, PA3720-PA3719 expression was found to be growth-regulated, with an increased amount of expression occurring in late log/early stationary phase, even in the absence of nalC. This suggests that another regulator(s) is/are involved in modulating PA3720-PA3719 levels in late log/early stationary phase. Since PA3719 ultimately influences mexAB-oprM expression, its involvement in mediating growth-phase mexAB-oprM expression was assessed by examining mexA expression in both wildtype and PA3719 deletion P. aeruginosa strains. PA3719 was found to be involved in some, but not all, of the growth phase control of mexAB-oprM. These results suggest that mexAB-oprM growth-phase regulation is complex, as both MexR-dependent and MexR-independent regulatory pathways seem to exist. Overall, this study has produced a better understanding of mexAB-oprM regulation in nalC mutant P. aeruginosa strains. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-09-25 19:00:42.929

Pseudomonas aeruginosa is an opportunistic pathogen found in soil and aquatic environments that possesses a broad range of intrinsic antibiotic resistance mechanisms, including a highly impermeable outer membrane and several RND-type efflux pumps that export a number of clinically relevant antibiotic classes. Chronic P. aeruginosa infections in cystic fibrosis (CF) patients gradually develop high levels of resistance to antimicrobial therapy due to conditions that favour the acquisition and selection of numerous chromosomal mutations, the nature of which are poorly understood. To identify chromosomal contributors to aminoglycoside resistance a P. aeruginosa transposon mutant library was screened for increases in aminoglycoside susceptibility. Six genes of interest (pstB, lptA, faoA, amgR, PA0392, and PA2798) were identified, the deletion of which meaningfully decreased aminoglycoside minimum inhibitory concentrations in wild-type P. aeruginosa. Combinations of gene deletions were constructed to determine if any of these genes contributed to aminoglycoside resistance via a common mechanism or whether they operated independently to promote intrinsic aminoglycoside resistance. In all cases, double deletion had an additive impact on aminoglycoside susceptibility, suggesting that each gene of interest contributes to resistance through an independent mechanism. Deletions in pstB, lptA, faoA, amgR, PA0392, and PA2798 were introduced into pan-aminoglycoside-resistant CF-lung isolates where they dramatically compromised aminoglycoside resistance, indicating that these genes also contribute to acquired aminoglycoside resistance in chronic P. aeruginosa infections. A fluorimetric assay was developed to measure aminoglycoside-induced membrane depolarization using the voltage sensitive probe DIBAC4(3). Gentamicin-induced membrane depolarization was found to be substantially increased in the amgR, pstB, and PA0392 mutant strains when compared to wild-type P. aeruginosa. These increases in depolarization paralleled declines in cell viability as measured by a gentamicin killing assay, suggesting that the cytoplasmic membranes of these mutant strains are more sensitive to the membrane perturbing effects of aminoglycoside-induced mistranslated proteins, and supporting a role for the disruption of the selective barrier of the cytoplasmic membrane in the bactericidal activity of the aminoglycosides. This study describes novel contributors to intrinsic and acquired aminoglycoside resistance in P. aeruginosa, and highlights the importance of membrane functions in resisting these activities. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2012-09-21 21:27:23.303

Meat Juice Medium (MJM), an aqueous extract of meat, was inoculated with Pseudomonas aeruginosa and incubated for 7 d at 4$ sp circ$C under shaking conditions (100 rev.min$ sp{-1}$). Two predominant compounds produced during spoilage of MJM were detected using HPLC. These compounds with retention times (RT) of 21.48 and 32.04 min were tentatively identified as acetic and butyric acids, respectively. These compounds were also produced when MJM was replaced with Brain Heart Infusion Broth medium. In later experiments, the effect of glucose supplementation on the rate of MJM spoilage was examined. Glucose 0.5% (wt/vol) was added to the MJM, inoculated with P. aeruginosa and incubated at 30$ sp circ$C under shaking conditions (100 rev.min$ sp{-1}$). HPLC of samples after 1d of incubation indicated the presence of 8 predominant compounds including acetic and butyric acids. Their concentrations were, in general, higher in control samples of MJM without added glucose. Using HPLC, TLC, Pyrolysis/GC/MS, FTIR and GC-MS methodologies, the compounds with RT of 8.91, 9.67, 11.96, 13.33, 17.74, 21.48, 26.07 and 32.04 min were tentatively identified as cadaverine, 2-keto gluconic acid, fructose, lactic acid, acetic acid, methanol and butyric acid. In contrast to the results of previous researchers, cadaverine was produced in large amounts while no putrescine was produced by P. aeruginosa. During the spoilage period, the levels of glucose, fructose and total carbohydrate were monitored. Addition of glucose to MJM delayed slime production by 4 days and increase to maximum pH of 8.3 by 7 days. Results suggest that addition of glucose to MJM delays spoilage by P. aeruginosa.

Pseudomonas aeruginosa grew in the presence of 10 $ mu$g/ml tetracycline (TC) or chlorotetracycline (CTC) in a minimal medium containing Mg$ sp{++}$. Growth is inhibited, with a six-fold increase in length of the lag phase. Cells revert to sensitivity when returned to antibiotic-free medium. Substitution of Mg$ sp{++}$ in the growth medium of CTC-resistant strains with Ca$ sp{++}$ and Sr$ sp{++}$ resulted in dramatic changes in growth and cell mass of cultures. Exposure of CTC-grown cells to EDTA did not result in cell lysis. SDS-PAGE of outer membrane proteins of resistant cells revealed loss of a protein band of molecular weight 73,500 D and the appearance of a 54,000 D protein band. Growth of cells resistant to CTC was hampered by subsequent exposure to penicillin G. Chelation of divalent cations from the outer membrane of sensitive cells leading to cell disruption is postulated as the primary mode of action of this antibiotic against P. aeruginosa.

Pseudomonas aeruginosa is a ubiquitous environmental microorganism that opportunistically colonizes immune-compromised hosts. P. aeruginosa is capable of establishing complex. matrix-encased biofilms during colonization of both environmental and living host surfaces. Biofilms formed by P. aeruginosa are physiologically very different from free-living P. aeruginosa cells, and exhibit increased resistance to environmental stresses, including antibiotic treatment. While the development and establishment of P aeruginosa biofilms has been extensively studied in vitro, several new behaviours of P. aeruginosa in biofilms have recently been observed that may greatly impact on the spread, recolonization and function of biofilms. These processes include bacteriophage mediated lysis and dispersal of P. aeruginosa biofilms, and the generation of phenotypic and genetic variation among bacterial cells that disperse from the biofilm. In this project, the role of bacteriophage activity and phenotypic variation in the development of P. aeruginosa biofilms has been investigated. Induction of a Pf1-like prophage of P aeruginosa (here named Pf4), during biofilm formation was characterized and was shown to increase over the progression of biofilm development. It was observed in this study that the activity of Pf4 caused the emergence of small colony variant (SCY) phenotypes in the effluent run-off from P. aeruginosa biofilms. Computational analysis of the genome ofPf4 resulted in the identification of a novel Toxin-Antitoxin (TA) gene pair, not previously identified within the genome of P. aeruginosa, of which the putative toxin gene product was determined here to play a role in growth-inhibition and the small colony phenotype of P. aeruginosa. TA gene pairs are proposed to induce stress responses in host cells and therefore play a role in survival during periods of environmental stresses such as oxidative or starvation stress. To study the effects of the Pf4 toxin and its possible role in the stress response of P aeruginosa, the Pf4 toxin gene was cloned and placed under the regulation of an inducible arabinose promoter. The proteome expression and biofilm formation as a result of toxin over expression were compared. The proteomic studies performed here indicated that P. acrllginosu biofilms do respond to expression of the toxin component of this putative TA element by increased expression of stress related proteins. Many stress-related groups of proteins were found to be over expressed during induction of the toxin indicating a possible role in stress survival of P. acrllginosa. Homology studies of the Pf4 toxin indicated a strong structural sequence relationship with the toxin ParE of the ParDE TA system. The mode of action of ParE toxin had previously been determined and showed the ParE toxin to be a strong gyrase inhibitor. The Pf4 antitoxin was, however, found to have homology to the Phd antitoxin of the Phd-Doe system of bacteriophage PI. The mode of action of the Doc protein remains to be clearly determined. To better understand the interaction between the Pf4 antitoxin and its cognate toxin protein interaction studies were performed. Peptide fragments of the Pf4 antitoxin were generated for an SPR binding assay and this study identified putative peptide sequences that are responsible for binding of the Pf4 antitoxin to its cognate toxin. Further investigation of a selected strong binding peptide showed that there were 3 key amino acids that were important in binding to the Pf4 toxin, namely His65, Ser67 and A 69 sp. Overall, this study has identified a key role for bacteriophage Pf4 in biofilm development and phenotypic variation in P. aeruginosu, and has provided initial insight into the molecular mechanisms by which this bacteriophage influences growth and gene expression in this organism.

Thesis (Ph. D.)--Colorado State University, 2004. / Includes bibliographical references.

Stuttgart, Universiẗat, Diss., 2008. / Aus: Appl. Environ. Microbiol. 2006 Jul;72(7):4819-28, FEMS Microbiol. Lett. 2006 Nov;264(2):220-5, Microbiology. 2008 Mar;154(Pt 3):789-96, FEMS Microbiol. Lett. 2005 May 1;246(1):25-31.