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

An aerobic, Gram-negative, rod shaped and polarlagellated bacterium, designated strain MS586, was isolated from soybean rhizosphere in Mississippi. The taxonomic position of MS586 was determined using a polyphasic approach. Analysis of the housekeeping genes supported the novel position of MS586. The results were also supported by average nucleotide identity (ANI) values. Based on these data, it is proposed that strain MS586 represents a novel species, Pseudomonas mississippiensis, within the genus Pseudomonas. The type strain is MS586. Strain MS586 showed a broad-spectrum of antimicrobial activity against plant pathogenic bacteria and fungi that are economically important in agriculture. Preliminary studies using transposon-based mutagenesis showed that the gltB gene was associated with production of antifungal activity against the indicator fungus Geotrichum candidum. The research findings of strain MS586 have provided insights into its potential use as a biocontrol agent in plant disease management.

Pseudomonas aeruginosa is a Gram-negative pathogen that forms biofilms, which increase tolerance to antibiotics. Biofilms are dense, surfaceassociated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentration (sub-MIC) levels of antibiotics induce the formation of biofilms, indicating a potential role in response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unknown. We show that treatment with sub-MIC levels of cefixime (cephalosporin), carbenicillin (β-lactam), tobramycin (aminoglycoside), chloramphenicol (chloramphenicol), thiostrepton (thiopeptide), novobiocin (aminocoumarin), ciprofloxacin (fluoroquinolone), or trimethoprim (antifolate) induces biofilm formation, with maximal induction at ~ ¼ to ½ MIC. We demonstrate that addition of exogenous eDNA or cell lysate does not stimulate biofilm formation to the same extent as antibiotics, suggesting that the release of common goods by antibiotic action does not solely drive the biofilm response. We show that increased biofilm formation upon antibiotic exposure requires the outer membrane porin OprF and the extracytoplasmic function sigma factor SigX. Through transposon mutant screening and deletion studies, we found that OprF is important for biofilm induction, as mutants lacking this protein did not form increased biofilm when exposed to sub-MIC antibiotics. OprF expression is v controlled by SigX, and its loss increases SigX activity. Loss of SigX also prevents biofilm induction by sub-MIC antibiotics. Together, these results show that antibiotic-induced biofilm formation may constitute a type of stress response. This response may be useful to screen for new antibiotics due to its ability to reveal antibiotic activity at concentrations below the MIC. Further study of this response may also provide targets for adjuvant therapies that reduce biofilm formation in P. aeruginosa infections and increase the efficacy of current antibiotics. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a bacterium that causes illness in patients with compromised immune systems, like patients with cystic fibrosis. This bacterium forms biofilms, which are dense groups that stick to surfaces within a protective slime that contains proteins, sugars, and DNA. Biofilms make the bacteria harder to treat with antibiotics. If the bacteria are treated with low levels of antibiotics, they respond by forming more biofilm but how this happens is unknown. We showed that adding DNA does not increase biofilm formation, while adding dead cell debris only causes a small increase. By testing a library of mutant bacteria, we found that they need two genes, oprF and sigX, to form more biofilm when they are treated with low levels of antibiotic. By studying how bacteria respond to low levels of antibiotics, we can find ways to identify new antibiotics and to make our current antibiotics work better.

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