Pseudomonas aeruginosa is a common cause of chronic respiratory infection in cystic fibrosis (CF). Infection is established within the lung epithelial mucus layer, through adhesion to mucins. Terminal residues on mucin oligosaccharide chains are highly sulphated and sialylated, which increases their resistance to degradation by bacterial enzymes. However, a number of microbes display mucin sulphatase activity, including P. aeruginosa. Using ion chromatography, the levels of sulphation on different respiratory mucins and the availability of inorganic sulphate in CF sputum were quantified, and the ability of clinical P. aeruginosa isolates to desulphate mucin was tested by providing mucin as a sole sulphur source for growth. All tested P. aeruginosa strains isolated from the CF lung were able to use human respiratory mucin as source of sulphur for growth, whereas other non-clinical Pseudomonas species were not. However, measured levels of inorganic sulphate in CF sputum suggest that bacteria resident in the lung have sufficient inorganic sulphate for growth and are unlikely to require access to mucin-sulphur as a sulphur source during chronic infection. This was confirmed when expression of sulphate-repressed P. aeruginosa genes, atsK and msuE, were found by quantitative PCR to be repressed in CF sputum. These results indicate that sulphate-starvation is unlikely to occur in pathogens residing in CF sputum and, therefore, mucin desulphation may have an alternative purpose in the association between P. aeruginosa and CF airways.Transcriptomic studies showed enhanced expression of 5 main islands on the P. aeruginosa genome in the presence of mucin as a sulphur source, when compared to sulphate. These islands include general sulphur-starvation response gene clusters, encoding desulphurizing enzymes AtsA, SsuD and MsuD, plus the locus PA2083-PA2094. This locus has not been characterised but encodes a putative sulphonatase, an extracellular-function (ECF) type sigma factor, with associated TonB-dependent transducer, and Major Facilitator Superfamily transporters. Transcriptional studies of this locus in response to various sulphur sources revealed that the locus comprises two transcriptional units under sulphate-limited conditions, and putative σ70-type promoters were identified using 5’-RACE and sequence alignment. Transcriptional regulation of the locus is contributed to by the encoded ECF-type σ factor and anti-σ factor, as a mutant carrying only a disrupted copy of these genes displayed a lack of transcriptional downregulation of the locus in the presence of sulphate. The influence of mucin on transcription levels of the locus was also investigated by RT-qPCR, showing that for maximum transcriptional levels both sulphate-limitation and the presence of mucin are required. However, despite repression of P. aeruginosa sulphate-regulated genes in CF sputum, the level of expression of the locus PA2083-PA2094 in CF sputum was comparable to that of P. aeruginosa culture grown in sulphate-limited conditions. The influence of the lung environment may, therefore, have a greater impact on expression levels of the locus than seen in in vitro studies with mucin. To further investigate the role of the locus, mutants were constructed and screened for changes in their ability to utilise a range of sulphur sources, including mucin, for growth. However, none of the mutants showed significant change in their growth patterns in response to any of the other sulphur sources tested, suggesting that the locus may be involved in desulphurization of a compound not tested in this study or may be functionally replaced by other organosulphur utilisation pathways in its absence. With the aim of identifying genes involved in mucin desulphurization, a P. aeruginosa transposon library was generated, combining the high-throughput nature of a random library with the variable expression reporter capabilities afforded by a promoterless GFP insert. The GFP reporter transposon produced varying fluorescence levels over time during growth of individual mutants, based on the activity of the promoter upstream of the transposon insertion site. A preliminary method was devised using fluorescence-activated cell-sorting to isolate mutants displaying altered GFP expression levels in response to sulphate availability and to mucin. Overall, this work explores the prevalence and importance of mucin desulphurization by P. aeruginosa, with relation to cystic fibrosis lungs, and provides some insight into the transcriptional patterns of the P. aeruginosa locus PA2083 to PA2094.

Intact bovine Longissimus dovsi muscle was subjected to a mild washing procedure in order to reduce the concentration of the sarcoplasmic fluid. Intact and washed muscle samples were inoculated with Pseudomonas fragi to evaluate the effect of a sarcoplasmic reduction on bacterial growth and subsequent spoilage during storage at 4°C for 12 days. Aseptic controls were stored under similar conditions. Alterations in the water-soluble, salt-soluble, urea-soluble and urea-insoluble protein fractions, as well as the total carbohydrate, pH and bacterial numbers, were monitored in both intact and washed inoculated muscle samples. Scanning and transmission electron microscopy were employed to monitor ultrastructural changes on the muscle surface as a consequence of the growth of P. fragi. Analysis of water-soluble components (non-protein nitrogen, water-soluble proteins and carbohydrates) indicated that the washing procedure effectively removed the majority of these components. Increases in the extractability of the water-soluble and salt-soluble protein fractions were observed in the intact inoculated muscle sample. Alterations in the SDS-gel electrophoretic pattern of the water-soluble, salt-soluble, urea-soluble and urea-insoluble proteins were evident. Total carbohydrate decreased as a result of growth of P. fragi. An increase in pH of the intact muscle occurred as bacterial numbers increased. Significantly (P < 0.01) higher growth rates were observed on the intact muscle tissue than the washed muscle tissue. Relatively little change in the non-protein nitrogen, water-soluble and salt-soluble protein content was observed in the washed inoculated muscle tissue. A slight decrease in total carbohydrate was seen. Minor changes in the SDS-gel electrophoretograms of the salt-soluble proteins were apparent. Little change in pH of the washed inoculated sample occurred due to the growth of P. fragi. Scanning electron micrographs indicated that surface degradation of both intact and washed inoculated muscle samples were apparent only in areas of localized colonization. Glycocalyx appeared to mediate not only cell to cell attachment, but also cell to muscle surface adhesion. Bacteria were observed growing between muscle fibers. Transmission electron micrographs of intact inoculated muscle tissue confirmed the mediation of glycocalyx in bacterial adhesion. Cellular evaginations were present on the surface of the bacteria. Autolysis was minimal in both intact and washed aseptic muscle controls. / Land and Food Systems, Faculty of / Graduate

Con el objetivo de caracterizar bioquímicamente la actividad lipolítica de Pseudoalteromonas atlantica PAR 2, aislada de la Bahía de Paracas (Ica), se procedió a cultivar la bacteria en medio LB (Luria-Bertani) a temperatura ambiente durante 24 horas. Para determinar la actividad lipolítica de la Pseudoalteromonas atlantica PAR 2, se utilizó agar SW 5 % con tributirina 1 % y se incubó a temperatura ambiente por 48 horas; la hidrólisis del sustrato se evidenció por la formación de un halo transparente. Así también, se cuantificó la actividad lipolítica utilizando como sustratos por un lado tween 80 y por otro, aceite de oliva al 1 %; los cuales no fueron degradados; esta respuesta evidenció que la enzima en estudio es una lipasa del grupo VI o esterasa. Por ese motivo, se utilizó como sustrato -nitrofenol acetato en buffer fosfato 25 mM pH 7, el producto liberado se midió por espectrofotometría a 405 m y se obtuvo una actividad enzimática de 79,32 mol/mL y una actividad específica de 661,00 mol/mg de proteína en el extracto crudo. Pseudoalteromonas atlantica PAR 2 produce una lipasa del grupo VI o esterasa, la cual además presenta actividad óptima a 20 ºC, pH 7 y concentración salina 5 %. -- Palabras clave: Pseudoalteromonas atlantica, actividad lipolítica, esterasa, -nitrofenol acetato. / -- In order to characterize biochemically the lipolytic activity of Pseudoalteromonas atlantica PAR 2, isolated from the Bay of Paracas (Ica), it was proceeded to grow the bacteria in LB medium (Luria-Bertani) at environment temperature during 24 hours. To determine lipolytic activity of Pseudoalteromonas atlantica PAR 2, SW 5 % agar with tributyrin 1 % was incubated at environment temperature for 48 hours, where substrate hydrolysis was evidenced by the formation of a transparent halo. Additionally, lipolytic activity was quantified using as substrates Tween 80 on one side and on the other, olive oil, 1%, which were not degraded, this response revealed that the enzyme under study is a lipase or esterase group VI. For that reason, was used as substrate -nitrophenol acetate in 25 mM phosphate buffer pH 7, the released product was measured by spectrophotometry at 405 m and was obtained 79,32 mol/mL as enzymatic activity, and 661,00 mol/mg protein as specific activity in the crude extract. Pseudoalteromonas atlantica PAR 2 produces a lipase group VI or esterase, which also has optimal activity at 20 °C, pH 7 and 5% salt concentration. -- Keywords: Pseudoalteromonas atlantica, lipolytic activity, esterase, -nitrophenol acetate / Tesis

Pseudomonas aeruginosa Ps-1C produces an extracellular proteolytic enzyme which from preliminary studies appears to be inducible, and responsible for corneal destruction in injured dyes. In the present study the nutritional requirements for this bacterium to produce the proteolytic enzyme(s) were investigated. Preliminary studies indicated that proteose peptone offered the required nutrients for good enzyme production. The separation of the components of proteose peptone by Sephadex C-10 and Sephadex G-75 descending column chromatography was undertaken to illucidate the nutritional requirements. It was also noted that casamino acids hydrolysate served as a good substrate for Pseudomonas aeruginosa to produce this enzyme. The separation of amino acid groups was undertaken using paper and Ceon electrophoresis and various types of thin layer chromatography. The three amino acids found to be required for good protease production were, phenylalanine, isoleucine, and valine in their respective concentrations of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml. Isoleucine was found to be inhibiting at higher concentrations. Dextrose also inhibited protease production, but not growth, at concentrations greater than 0.05%. Divalent metal ions in varying concentrations were tested as nutritional requirements for enzyme production. Magnesium ion provided very good enzymatic activity at a concentration of 0.01 M, whereas cobalt, copper, calcium and zinc ions did not allow appreciable enzyme activity and even in some cases were inhibitive.

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.

No description available.