A lysozyme from Bacteriophage 7v was purified 7.7 fold over the original lysates of the bacteriophage 7v and Pseudomonas aeruginosa PS-7. This purification process includes ultracentrifugation, ammonium sulfate precipitation, dialysis, and fractionation in a Sephadex G-150 column. The phage lysozyme exhibits a greater specificity when assayed with P. aeruginosa cells as a substrate, but still is capable of acting on the standard lysozyme Micrococcus lysodeikticus substrate. The pH optimum, heat inactivation range, and action on other bacteria is described. The molecular weight was found to be 14,300. The values of this 7v phage lysozyme are in close agreement with values found with other phage lysozymes. A possible treatment for burn wounds infected with Pseudomonas aeruginosa is also described.

The present study was undertaken to define the standard conditions for growth of bacteriophage 7V on its host Pseudomonas aeruginosa strain PS-7 and to determine the factors which affect the quantity and quality of plaques in the plaque count assay. Observations from the single-step growth experiment and single-burst experiment are also included. Plaque count assays were performed under various environmental conditions. Conditions were selected as “standard” if they yielded: 1) relative maximum number of infective centers per ml of stock 7V phage, 2) clear, haloed plaques at least 2.0 mm in diameter, and 3) reproducible assays limited only by the sampling error. These conditions are: 1. fresh NBYE or NBYE agar for the growth medium 2. NYBE or buffered salts solution for diluents 3. Physiologically young cells in the log phase between 1-5 X 10⁸ bacteria/ml 4. Stock and diluted stock suspensions stored at refrigerator temperatures. Adsorption rate experiments which measured both unadsorbed phage and infective centers were performed in minimal media, minimal media supplemented with organic and and ionic cofactors, and complete media. Although overnight lysates of PS-7 in minimal media produced a high titer of phage, the rate of adsorption of phage 7V in PS-7 was extremely slow in minimal media. Addition of tryptophan caused a decrease in free phage without a corresponding increase in infective centers. Casamino acids plus tryptophan caused an increase in the velocity of the adsorption reaction which was less than the rate of adsorption of phage 7V to its host PS-7 in NBYE. In NBYE 90 percent of the initial phage were adsorbed in 5 minutes, but the recovery of phage as free phase of infective centers was not equal to the input of phage. These results suggest that this system requires a cofactor, organic, ionic, or both, in order that adsorption of phage 7V to its host PS-7 proceed at a maximum rate. And it further suggests that the incidence of abortive infection in this system is high. In this particular system under standard conditions it appears that the size of the plaques is controlled mainly by environmental factors, while the relative number of plaques is a characteristic of the system.

Pseudomonas aeruginosa, es uno de los más importantes patógenos humanos oportunistas, que puede colonizar no solo superficies abióticas, sino también superficies bióticas. Una vez colonizadas estas superficies, dicha bacteria produce exopolisacárido originando la formación de biopelículas, en consecuencia, estas bacterias son mil veces más resistentes, no sólo a antibióticos, sino también a biocidas y desinfectantes, constituyendo un problema de salud pública. Para este trabajo de investigación se recolectaron 62 cepas aisladas de líquidos biológicos y secreciones de pacientes procedentes del Hospital Nacional Edgardo Rebagliati Martins durante el periodo de Enero a Setiembre del 2006, identificándose el 100% de ellas como cepas de Pseudomonas aeruginosa. Sólo se escogió el 81% de las cepas formadoras de biopelículas para investigar la influencia del mucolítico NAC sobre las biopelículas de Pseudomonas aeruginosa, ya que éstas presentaron mayor producción durante la cuantificación realizada por el Método de O’Toole and Kolter. / Pseudomonas aeruginosa, is one of the most important human pathogens opportunistic, that can colonizes not only abiotics surfaces, also biotics surfaces. Once colonized these surfaces produce extracellular polysaccharides originating the biofilms formation, in consequence, these bacterias are thousand times more resistant to antibiotics, biocides and desinfectants, constituting a problem of public health. For this study, were collected 62 strains isolated from biological fluids and secretions of patients from the Edgardo Rebagliati Martins National Hospital during the period from January to September 2006, indentifying 100% of them like Pseudomonas aeruginosa strains. The determination of biofilm production we preferred to use Congo Red Agar Method (ARC), for its sensibility and reproducibility which showed that 42% were biofilm producers, whereas 48% were biofilm non developed. Pseudomonas aeruginosa ATCC 9027 (non producer) were used as negative control. Only the 81% of them was chosen to investigate the influence of mucolitic N-acetylcysteine (NAC) on Pseudomonas aeruginosa biofilms, by the biggest biofim production during the quantification by O’Toole and Kolter Method. / Tesis

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, we decided to test the ability of the DHOase mutant strain to produce other virulence factors as well. Here we report that a block in the conversion of carbamoyl aspartate (CAA) to dihydroorotate significantly impairs the ability of P. aeruginosa to affect virulence. We believe that the accumulation of CAA in the cell is the root cause of this observed defect. This research demonstrates a potential role for pyrimidine intermediates in the virulence response of P. aeruginosa and may lead to novel targets for chemotherapy against P. aeruginosa infections.

Nucleotide pools were extracted and quantified from Pyr^+ and Pyr^- strains of P. aerucjinosa. Strains were grown in succinate minimal medium with and without pyrimidines, and nucleotides were extracted using trichloracetic acid (TCA; 6% w/v). The pyrimidine requirement was satisfied by uracil, uridine, cytosine or cytidine. Pyr^- mutants were starved for pyrimidines for two hours before nucleotide levels were measured. This starvation depleted the nucleotide pools which were restored to wild type levels by the addition of pyrimidines to the medium. When the pyrimidine analogue, 6-azauracil, known to inhibit OMP decarboxylase, was added to cultures of the wild type strain, the uridine and cytidine nucleotides were depleted to near zero. Thus, the nucleotide pool levels of Pseudomonas strains can be manipulated.