Cystic Fibrosis is one of the most common genetic recessive diseases among Caucasians and is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene on chromosome 7. There are different classes of CFTR mutation, leading to differences in disease severity among patients. In addition to the CFTR genotype, secondary genetic factors, modifier genes, also influence CF phenotypes. Due to the dysfunction of CFTR protein and production of thickened mucus, bacterial infection in the lungs is favored and can lead to further clinical complications in CF patients. Pseudomonas aeruginosa is one of the most common bacteria detected among patients. The aim of this project was to investigate four candidate modifier genes, Factor B, Complement Factor 3, Toll-like Receptor 4 and Heme oxygenase-1, which might affect the status of Pseudomonas aeruginosa infection. A total of 22 single nucleotide polymorphisms (SNPs) were selected in these four genes and they were tested against five phenotypic traits, including age of diagnosis, FEV1% predicted andstandard deviation value, age of first Pseudomonas aeruginosa infection and Pseudomonas aeruginosa infection status. Among the selected SNPs, both case-control studies and family-based analysis were performed in order to establish any correlation between the genotypes and the phenotypes. In addition, haplotype analysis was performed to determine whether there was interaction between SNPs or whether there were unidentified SNPs in the vicinity of the selected ones that might contribute to the observed phenotypic traits. Among the 22 chosen SNPs, 13 of them were found to be significantly linked to one or more of the tested phenotypes. The three most significant associations were BF_2557 with lung function, HMOX1_9531 with lung function and BF_7202 with age of diagnosis. Several haplotypes were significantly associated with one of the five phenotypes. There was no evidence for the presence of unidentified SNPs or interaction between SNPs. Most of haplotype associations were likely due to the presence of a single SNP which was found to be significantly linked to the phenotype. Conclusively, both SNPs and haplotype analyses suggest that the four candidate genes are modifiers of disease severity in CF. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

The oxidation of glucose by Pseudomonas aeruginosa is known to follow the sequence: glucose→ gluconic acid→2-ketogluconic acid→pyruvic acid and thence into the tricarboxylic acid cycle. The most striking aspect of this pathway is that the first two oxidative steps do not involve phosphorylated intermediates at the substrate level. In the present study radioactive phosphorus was used in an attempt to elucidate the carbohydrate metabolism of P. aeruginosa. Cell free preparations of P. aeruginosa, obtained by crushing a cell paste in the Hughes press, incubated with added cofactors, ADP and P³² resulted in the formation of labelled ADP and ATP. The presence of glucose or succinate in the reaction mixture greatly depressed the amount of ATP found. The cell free preparations were found to yield ATP as measured in the hexokinase trap, but the formation of ATP was not increased by the addition of glucose, gluconic acid, 2-ketogluconic acid or succinic acid. These results suggested that no net energy was gained by the extract by the oxidation glucose→ gluconic acid→ 2-ketogluconic acid. In manometric experiments it was found that the cell free preparation did not oxidize glucose-6-phosphate, ribose-5-phosphate, α-ketoglutarate, citrate and isocitrate. Glucose was oxidized with the uptake of two atoms of oxygen per mole of substrate. In the presence of ATP, glucose was oxidized with the uptake of only one atom of oxygen. Gluconic acid and gluconolactone were oxidized with the uptake of one atom of oxygen; ATP had no effect on these last two oxidations. From these data two reactions beyond 2-ketogluconate have been postulated. [Formulas omitted] / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

I have investigated the evolutionary response of Pseudomonas aeruginosa to salt (NaCl) stress, and the physiological mechanisms responsible for this adaptation. Populations of P. aeruginosa founded from the same ancestral genotype were selected at three different concentrations of NaCl, low, moderate and high for about 660 generations with four independent replicates for each concentration. Adaptation was measured as the fitness of the evolved populations relative to the ancestor assessed in direct, head-to-head competition experiments conducted in the same environment in which they were selected (direct response) as well as in all alternative environments (correlated response). Results suggest that selection in each salt environment led to adaptation to that environment and a modest degree of specialization that evolved because correlated responses to selection were smaller than direct responses. In order to identify the physiological mechanisms contributing to the populations' adaptation in high NaCl concentration, I chose a sample of evolved lines that showed the strongest evidence for specialization to salt and competed them against the common ancestor in KCl and sucrose. Results suggested that increased Na+ /H+ antiporter activity is probably the primary mechanism behind adaptation to high NaCl concentration, however alternative mechanisms cannot be excluded. Tolerance curves, which measure the performance of a genotype across a gradient of salt concentrations, suggested no change in the high salt group’s ability to tolerate extreme concentrations of NaCl. We conclude that high salt evolved population showed improvements to its ionic/osmotic stress resistance strategies mainly to Na+ efflux strategies but with no changes to salt niche.

Although the importance of tricarboxylic acid cycle activity in the metabolism of aerobic bacteria is well established, detailed studies on the utilization of intermediates of the cycle designed to assess the nature, importance and control of the enzymes concerned have not been performed with pseudomonads. Results of this investigation have shown that Pseudomonas aeruginosa ATCC 9027 lacks NAD or NADP linked L-malic dehydrogenase. Studies with cell fractions have shown that an NAD and NADP independent, particulate L-malic dehydrogenase catalyses the oxidation of L-malic acid to oxalacetic acid. The labelling patterns of citrate obtained from succinate-1,4-¹⁴ C and succinate-2,3-¹⁴ C have demonstrated the involvement of the particulate malic dehydrogenase and have excluded any other possibility. Phosphofructokinase could not be detected in the cell-free extract preparations and thus accounting for the non-functional Embden-Meyerhof pathway in this organism. Cells grown in succinate medium either do not have or have extremely low levels of glucose metabolizing enzymes. The glucose effect on tricarboxylic acid cycle enzymes was not observed. Further, the addition of α-keto- glutarate and glutamate to the medium did not repress these enzymes. These observations suggest that tricarboxylic acid cycle activity is of special importance for growth and metabolism in pseudomonads. The data have also indicated that during growth in a succinate medium, pentose synthesis must occur by the action of transketolase upon compounds derived from tricarboxylic acid cycle intermediates. It has been shown that both the glucose permease and the glucose metabolizing enzymes were induced simultaneously on shift from succinate to glucose medium. The glucose permease was found to be very specific since glucose uptake was not inhibited even in the presence of 100-fold excess of α-CH glucoside, 2-deoxyglucose, galactose, fructose or mannose. Particulate malic dehydrogenase was inhibited by adenine nucleotides while it was activated by GTP and GDP. The mechanism of this regulation is not clear, however, it is obviously important in the control of tricarboxylic acid cycle activity. Addition of glutamate to the medium repressed the synthesis of glutamic dehydrogenase. High levels of malic enzyme were maintained on growth in glucose and succinate media, whereas the levels were low in acetate medium. These observations demonstrated the capacity of the organism to regulate the synthesis of enzymes in response to the particular environment. Tricarboxylic acid cycle intermediates and glyoxylate have been found to exert "fine control" over the activities of isocitrate dehydrogenase and isocitrate lyase in such a way that flow of isocitrate through the tricarboxylic acid cycle and the glyoxylate cycle is precisely regulated to suit the needs of the cell. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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 major outer membrane protein OprF from Pseudomonas aeruginosa was compared with OprF from the fluorescent phytopathogen Pseudomonas syringae. The P. syringae oprF gene was subcloned and sequenced and found to code for a sequence of 344 amino acids containing a 24 amino acid leader sequence. The mature protein, with a deduced molecular weight of 34,225, contained four cysteine residues and an alanine-proline rich area. Comparison of the P. syringae OprF amino acid sequence with the P. aeruginosa OprF and the E. coli OmpA sequences showed that the sequences were most similar at the carboxy-terrninal ends. Restriction enzyme site heterogeneity near the oprF gene from nine different P. syringae pathovars was determined. All pathovars had a conserved SalI site within the gene and conserved PstI. and BamHI sites near the ends of the gene. The location of the PstI and the SalI sites outside the gene was variable, although similar. Immunological relatedness between P. syringae OprF from the different pathovars and P. aeruginosa OprF was confirmed. Protein OprF from all the pathovars was shown to be 2-mercaptoethanol modifiable and more easily heat modifiable than was OprF from P. aeruginosa. / 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

The human opportunistic pathogen, Pseudomonas aeruginosa PAO1, has been shown to kill the nematode Caenorhabditis elegans. C. elegans has been a valuable model for the study of bacterial pathogenesis, and has reinforced the notion that common virulence and host defense mechanisms exist. Recently, the pyrimidine pathway was shown to regulate virulence levels. Therefore, mutations in the pyrimidine pathway of PAO1 showed decrease virulence in the nematode. When starving the nematode, bacterial resistance was also shown to increase. It was hypothesized that starvation induced the DAF pathway, which regulates the transcription of genes involved with the antibacterial defense mechanism. Further research will be conducted to test this theory by performing RNAi experiments for the genes functioning in the antibacterial defense mechanism.

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