Several cellular and subcellular fractions were prepared from Pseudomonas aeruginosa strain PA-7. Those found to be immunogenic in rabbits included a heat-stable lipopolysaccharide, a protein-lipopolysaccharide complex, a cell wall preparation arid a formalin-killed whole cell vaccine. However, a lipopolysaccharide preparation extracted with phenol and water was found to be a poor immunogen in rabbits. The cell wall fraction proved to be the most effective immunogen in terms of the amount of antibody evoked, and of the duration of the serum antibody response. Hyperimmune sera produced against all four antigens were found to contain a mixed population of 2-mercaptoethanol sensitive and 2-mercaptdethanol resistant antibodies. Gel filtration and ion exchange chromatography studies established the presence of both IgM and IgG immunoglobulins in all four types of hyperimmune serum. Whole immune serum, as well as the IgM and IgG serum fractions, afforded passive protection to mice challenged with twenty or more LD₅₀ of viable organisms. There was an indication that the IgG fraction of two of the four serum types provided better protection than did the IgM fraction, but precipitation studies indicated that this may have been due to greater numbers of IgG immunoglobulins. In addition serum containing a high proportion of 2-mercaptoethanol resistant antibody-was found to promote faster clearance of injected bacteria than did serum taken earlier in the response. Immunodiffusion studies indicated that all four antigenic preparations contained at least one common immunogen; moreover, all serum types were able to react with sheep red blood cells coated with the heat-stable lipopolysaccharide preparation in passive hemagglutination and hemolysin tests. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The oprF gene encoding porin protein F from Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed in large amounts in E. coli and retained its heat- and reduction-modifiable and immunological characteristics. The cloned oprF gene product was purified from E. coli and characterized with respect to pore-forming ability in black lipid bilayers. Small channels, with an average single channel conductance of approximately 0.4 nS, were observed. A similar small channel size was observed for native protein F. The oprF sequences were used as a DNA-DNA hybridization probe with chromosomal DNA from the 17 IATS (International Antigen Typing Scheme) strains of P. aeruginosa, 52 clinical isolates and the non-aeruginosa Pseudomonads. Conservation of oprF sequences was observed among all the P. aeruginosa strains and to a lesser extent among the non-aeruginosa strains of the P. fluorescens rRNA homology group. Insertion mutations in the oprF gene were created in vivo by Tn1mutagenesis of the cloned gene in E. coli and in vitro by insertion of the streptomycin-encoding Ω fragment into the cloned gene, followed by transfer of the mutated protein F gene back into P. aeruginosa and homologous recombination with the chromosome. The oprF mutants were characterized by gel electrophoresis and immunoblotting, and it was shown that the mutants had lost protein F. The P. aeruginosa oprF mutants were characterized with respect to growth rates, antibiotic permeability and cell surface hydrophobicity. The results of these studies indicated that major alterations in the cell surface had occurred and that the cells were unable to grow in a non-defined liquid medium without added electrolytes. Marginal differences were observed in MICs (minimum inhibitory concentrations) of hydrophilic antibiotics for the oprF mutants compared with their protein F-sufficient parents. The putative roles of protein F in antibiotic permeability and general outer membrane permeability are discussed. Evidence for extensive homologies between protein F, the OmpA protein of E. coli and PHIII of Neisseria gonorrhoeae are presented. A role for protein F in prophylactic anti-Pseudomonas therapy, as a target for vaccine development, is proposed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Cyanide utilization in Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated by an enzyme referred to as cyanide oxygenase (CNO), previously shown to require components in both high (H) (>30 kDa) and low (L) (<10 kDa) molecular weight cell fractions. In this study, tetrahydrobiopterin (H4biopterin) was identified as a cofactor in fraction L, thus making CNO appear as a pterin- dependent hydroxylase. CNO was purified 150-fold (specific activity 0.9 U/mg) and quantitatively converted cyanide to formate and ammonia as reaction products. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted. CNO was found to be an aggregate of known enzymes that included NADH oxidase (Nox), NADH peroxidase (Npx), cyanide dihydratase (CynD) and carbonic anhydrase (CA). A complex multi-step reaction mechanism is proposed in which Nox generates hydrogen peroxide which in turn is utilized by Npx to catalyze the oxygenation of cyanide to formamide accompanied by the consumption of one and two molar equivalents of oxygen and NADH, respectively. The further hydrolysis of formamide to ammonia and formate is thought to be mediated by CynD. The role of H4biopterin and of the enzyme CA in the proposed process remains unclear, but the involvement of each in reactive oxygen and radical chemistry is consistent with the proposed formation of such species in the catalytic process. H4biopterin may additionally serve as a protein stabilizing agent along with a protein co-purifying with CynD identified as elongation factor Tu, a known chaperone. At least two of the CNO components (Nox and CynD) are complex oligomeric proteins whose apparent association with Npx and CA appears to be favored in bacterial cells induced with cyanide allowing their purification in toto as a multiprotein enzyme complex.

Pseudomonas aeruginosa, a ubiquitous environmental bacterium and an opportunistic human pathogen, is one of the most and best studied biofilm-forming organisms and has emerged as a model organism in the study of surface- and biofilm-induced gene expression. P. aeruginosa forms biofilms through a series of interactions between the cells and adherence to surfaces, which is mediated by surface appendages such as flagella and type IV pili. A gene cluster, designated htpABCDEFGI, which appears to encode protein products with homology to those encoded by recently described novel pilus biogenesis and assembly systems, has been identified in P. aeruginosa PAO1. Since the pili produced by these systems, designated Flp, are associated with the ability of the bacteria to bind non-specifically to inert surfaces, the aims of this study were to characterize the transcriptional organization of the putative P. aeruginosa PAO1 htp gene cluster and to determine the functional importance of the htp gene cluster in the ability of P. Aeruginosa PAO1 to adhere to surfaces. In silico evidence has suggested that the pilin subunit gene flp is not part of the P. Aeruginosa htp gene cluster thought to encode proteins involved in the synthesis, assembly and export of these pili. To determine the transcriptional organization of this gene cluster, total RNA from P. aeruginosa PAO1 was analyzed by reverse transcriptase-polymerase chain reaction (RTPCR). Primers designed to amplify regions spanning gene junctions yielded amplicons at each individual gene junction from htpA to htpI, as well as an amplicon for flp. Moreover, corresponding sigma 70 (σ70) consensus sequences were identified in the intergenic region between the htpA and flp genes and promoter function of the flp and htpA upstream region was subsequently confirmed using lacZ reporter gene constructs transformed into P.aeruginosa PAO1. The results therefore indicated that the htp gene cluster is an operon transcribed as a polycistronic message, whilst the flp gene is transcribed independently as a monocistronic message. To determine the functional importance of thehtp gene cluster in P. aeruginosa PAO1, the htpD gene, encoding a putative NTPase, was inactivated by in vivo homologous recombination with an appropriately constructed allelic exchange vector to generate the isogenic mutant strain PAOHtpD. Comparative analysis of the wild-type P. aeruginosa PAO1 and mutant PAOHtpD strain revealed that the mutant strain was impaired in its ability to attach to a glass wool substratum and also in its ability to grow as a biofilm. Since the mutant PAOHtpD strain was not growth-impaired, these results indicate that the htp gene cluster plays a role in P. aeruginosa PAO1 biofilm development under the culturing conditions used in this study. Thus, it can be proposed that the flp and htp gene cluster of P. aeruginosa PAO1 may play a role in its ability to successfully colonize abiotic surfaces. / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Publicación a texto completo no autorizada por el autor / El documento digital no refiere asesor / Determina los factores de riesgo asociados a la adquisición de PARC en los pacientes hospitalizados en el Hospital Nacional Arzobispo Loayza. Estudio tipo casos y controles, de carácter retrospectivo y descriptivo cuya población fue las interconsultas de pacientes con al menos un aislamiento para pseudomonas aeruginosa en cultivo, entre enero 2012 a diciembre 2013 con una muestra de 108 pacientes. En el análisis univariado se identificaron como factores de riesgo para la adquisición de PARC, procedencia de áreas críticas (Unidad de cuidados intermedios, Unidad de cuidados intensivos), antecedente de hospitalizaciones previas y estancia en Unidad de cuidados intensivos, hemodiálisis, ventilación mecánica, dispositivos invasivos como catéter venoso central y catéter urinario, uso previo de antibióticos como Imipenem, Meropenem y Ceftazidima; no obstante al realizar el análisis multivariado, solo se constituyeron como factores de riesgo independientes el uso previo de Imipenem (OR: 31.25; IC95%: 0.004 – 0.256, p: 0.001), Meropenem (OR: 11.7; IC95%: 0.014 – 0.512, p: 0.007) y Ceftazidima (OR:5.7; IC95%: 0.042 – 0.711, p: 0.015). El uso previo de antibióticos Carbapenemicos, sobre todo Imipenem y Ceftazidima están relacionados de manera independiente con la adquisición de Pseudomonas aeruginosa resistente a Carbapenems (PARC). El aislamiento de PARC se relaciona más con una permanencia en hospitalización mayor a 30 días, en comparación con el aislamiento de Pseudomonas aeruginosa sensible a Carbapenems. El tratamiento correcto: antibiótico adecuado por el tiempo adecuado es indispensable para mejorar el pronóstico del paciente con infección por PARC. / Trabajo de investigación

ExsA is an AraC-family transcriptional regulator that controls expression of T3SS genes in P. aeruginosa. ExsA binds to DNA at T3SS promoters and activates transcription. In the work presented here I examine the stoichiometry, ligand-interaction properties, and transcriptional activation mechanism of ExsA. I determined that ExsA is largely monomeric in solution. ExsA binds T3SS promoter DNA with high affinity resulting in two ExsA-DNA complexes. Whereas the lower molecular weight complex represents a single molecule of ExsA bound to DNA, the higher molecular weight complex represents two molecules of ExsA bound to adjacent sites at T3SS promoters. I next analyzed the mechanism by which ExsD negatively effects ExsA function. Chromatin Immuno-Precipitation Assays (ChIP) demonstrate that ExsD inhibits the DNA-binding activity of ExsA in vivo. Finally, I characterized the mechanism of transcriptional activation by ExsA. ExsA-dependent promoters contain regions that resemble consensus σ70 -35 and -10 recognition hexamers. The spacing between these regions, however, is increased 4-5 bp compared to the σ70 consensus. Nevertheless, I demonstrate that T3SS promoters are dependent on σ70-RNA polymerase (RNAP). Using the abortive initiation assay I discovered that ExsA recruits RNA polymerase to the PexsC and PexsD promoters. Potassium permanganate footprints indicate that following recruitment, RNAP facilitates unwinding of DNA at the -10 hexamer of T3SS promoters. Transcriptional activators generally recruit RNAP by contacting the α or σ70 subunits (or both). I have found that ExsA recruits RNAP to the PexsC and PexsD promoters by contacting region 4.2 of σ70. Although I have established the role of the -10 hexamer, the function of a near-consensus, putative -35 remains puzzling. in vitro transcription assays with mutations in the PexsC -35 hexamer reveals that this region is dispensable for ExsA-independent transcription. This data may suggest that what was thought to be a -35 hexamer is really just an ExsA binding site. Consistent with this hypothesis, I provide evidence that suggests an extended -10 element at PexsC may function to compensate for the lack of a -35 hexamer.

The complete nucleotide sequence of the region encoding the DHCDH function of the pDK1 lower operon was determined. DNA analysis has shown the presence of two open reading frames, one gene consisting of 777 nucleotides encoding a polypeptide of 27.85 kDa and another gene of 303 nucleotides encoding a polypeptide of 11.13 kDa. The results of enzymatic expression studies suggest that DHCDH activity is associated only with xy1L. However although the addition of xy1T cell-free extracts to xy1L cell-free extracts does not produce an increase in DHCDH activity, subclones carrying both xy1L and xy1T exhibit 300- 400% more DHCDH activity than subclones carrying only xy1L.

Background: The incidence of Pseudomonas aeruginosa bacteremia has not been defined in a population-based investigation. Methods: We performed a retrospective, population-based incidence study using resources of the Rochester Epidemiology Project of Olmsted County, Minnesota. We identified all Olmsted County residents with P. aeruginosa bacteremia between January 1, 1997, and December 31, 2006, by microbiology records in the only 2 laboratories in the county. Medical records were reviewed to confirm diagnosis, residency status, and clinical characteristics. Results: Age-adjusted incidence per 100,000 person-years was 10.8 (95% confidence interval [CI], 7.5-14.0) in men and 3.7 (95% CI, 2.2-5.2) in women for total P. aeruginosa bacteremia, and 8.4 (95% CI, 5.5-11.2) in men and 2.5 (95% CI, 1.3-3.8) in women for monomicrobial P. aeruginosa bacteremia. There was no significant change in incidence of total P. aeruginosa bacteremia during the past decade (P = .418). Incidence increased exponentially with age, with a greater magnitude of increase in men compared with women for total and monomicrobial P. aeruginosa bacteremia (P = .007 and P = .015, respectively). In patients with monomicrobial P. aeruginosa bacteremia, the median age was 69 years, and 78.4% of cases were either nosocomial or health care associated. Most patients had multiple comorbid conditions. The urinary tract was the most common primary source of infection. The 28-day all-cause mortality of monomicrobial P. aeruginosa bacteremia was 25.5%. In vitro susceptibility to ciprofloxacin was 95.3%. Conclusion: To our knowledge, this is the first population-based incidence study of P. aeruginosa bacteremia. The incidence of P. aeruginosa bacteremia has remained stable during the past decade. Fluoroquinolone susceptibility is high among local P. aeruginosa bacteremia isolates.

The identification of antimicrobial compounds that inhibit multidrug-resistant (MDR) bacteria continues to be a significant area of research to combat the public health threat posed by MDRs. Pseudomonas aeruginosa (PA) is a Gram-negative, MDR bacterium found within both the environment and healthcare settings. Our laboratory has observed another Gram-negative bacterium, Proteus species, exerts an interesting polymicrobial interaction with Pseudomonas aeruginosa by compromising the quorum sensing (QS) factor, pyocyanin. Production of pyocyanin by Pseudomonas is a main method the bacterium uses for communication and coordination of virulence. In this study, we examined Proteus mirabilis (PM) and Proteus vulgaris (PV13) effectiveness to compromise pyocyanin production in Pseudomonas aeruginosa. Through the use of pyocyanin isolation and extraction techniques, data was gathered for Pseudomonas aeruginosa’ s molecular interaction with the two Proteus bacteria described. Further observations were made on microbial interaction between Pseudomonas aeruginosa and Proteus mirabilis through measuring the rate of metabolic activity, twitching motility rate, and observing differences in biofilm formation. Using the data obtained from this research, we hope to identify new methods of controlling Pseudomonas virulence and infection by inhibiting its ability to communicate and coordinate in polymicrobial infections.

East Fork Poplar Creek (EFPC) in Oak Ridge, TN was highly contaminated with elemental mercury in the 1950 and 1960. The area is still experiencing the effects of mercury contamination, and researchers are searching for ways to remediate the EFPC. One possible mechanism for bioremediation is the use of biogenic Mn oxides to remove heavy metals from water systems. Native Pseudomonas bacteria species were isolated from EFPC in order to examine biogenic Mn oxides production and bioremediation of Oak Ridge slurries. Pseudomonas isolates did produce Mn oxides which bound to mercury, and mercury bound to organic matter significantly decreased. However, after a significant decrease of dissolved mercury, dissolved mercury was cycled back into the water system on day 10. Given a longer experimental timeline, biogenic Mn oxides have the potential to decrease mercury cycling.