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.

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.

No description available.

No description available.

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 fragi CRDA 037 was used as source of intracellular esterase, which remained attached to the cell membrane, and characterized. Several mechanical methods of disruption were used including glass beads (MSK), sonication, French press and combinations of these methods. The cellular debris were also treated with detergents such as CHAPS and Triton X-100 in the presence of chelating agent ethylenediaminetetra acetic acid (EDTA). The esterase activity remained in the cellular debris which was therefore use as a source of enzyme for kinetic studies. In the case of glass beads homogenization, the activity was found to decrease as a function of time of disruption. The results of chemical treatment showed that the esterase was characterized in terms of detergent and EDA action as well as substrate specificity. Triton X-100 and EDTA had no effect on the esterase activity and did not denature the enzyme. The substrate specificity with cells and cellular debris were carried out. The valeric acid was the best in term of esterase activity among fatty acids used in the study. (Abstract shortened by UMI.)

Pseudomonas fragi CRDA 037 was used to produce both intracellular and extracellular lipases. The crude lipase fractions were fractionated using ammonium sulfate to obtain partially purified intracellular and extracellular lipases; the fraction precipitated at 20-60% of saturation of the intracellular proteins was retained as the source of the endolipase, whereas the culture medium precipitated at 20-40% of saturation, was used as the source of exolipase. Native gel electrophoresis (PAGE) suggested that the partially purified extracellular lipase has a molecular weight of 25,500 Da. Three major electrophoretic bands were present in the intracellular lipase fraction at 70,000, 49,000, and 35,500 Da. The partially purified lipases were characterized with respect to their optimum pH and temperature for lipase activity, and well as for their kinetics, specificities, and reactions toward inhibitors. The optimum pH for the activity of the endolipase was found to be 9.0 whereas that of the exolipase was 8.75. With respect to optimum temperature, 30$ sp circ$C was determined to be the best for the endolipase while 35$ sp circ$C was the optimal value for the exolipase. Enzyme specificity was carried out using triacetin, tributyrin, trimyristin, and triolein as substrates. The results for the exolipase indicated that the lowest K$ sb{m}$ was obtained with trimyristin and the highest K$ sb{m}$ was obtained with triolein whereas for the endolipase, the highest K$ sb{m}$ was obtained with tributyrin and the lowest was with trimyristin. Experiments carried out with inhibitors indicated that both lipases are serine lipases, sulfydryl enzymes, and that tryptophan is essential for maintaining the conformation of the proteins. The most potent inhibitor was ferrous chloride, and sodium deoxycholate was a weak inhibitor for both lipases, however, it was an activator at low concentrations for the exolipase.

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.