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Therapy of Experimental Pseudomonas Infections with a Nonreplicating Genetically Modified Phage. Steven Hagens, 2004.Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens . A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia . Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental Pseudomonas aeruginosa infection . An export protein gene of the P . aeruginosa filamentous phage Pf3 was replaced with a restriction endonuclease gene . This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell . The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum . Treatment of P . aeruginosa infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3 . However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5 . This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage . Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release . Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy . Functions Required for Extracellular Quinolone Signaling by Pseudomonas aeruginosa. Larry A. Gallagher, 2002.A set of 30 mutants exhibiting reduced production of the phenazine poison pyocyanin were isolated following transposon mutagenesis of Pseudomonas aeruginosa PAO1 . The mutants could be subdivided into those with defects in the primary phenazine biosynthetic pathway and those with more pleiotropic defects . The largest set of pleiotropic mutations blocked the production of the extracellular Pseudomonas quinolone signal (PQS), a molecule required for the synthesis of secondary metabolites and extracellular enzymes . Most of these pqs mutations affected genes which appear to encode PQS biosynthetic functions, although a transcriptional regulator and an apparent response effector were also represented . Two of the genes required for PQS synthesis (phnA and phnB) had previously been assumed to encode phenazine biosynthetic functions . The transcription of one of the genes required for PQS synthesis (PA2587/pqsH) was regulated by the LasI/R quorum-sensing system, thereby linking quorum sensing and PQS regulation . Others of the pleiotropic phenazine-minus mutations appear to inactivate novel components of the quorum-sensing regulatory network, including one regulator (np20) previously shown to be required for virulence in neutropenic mice . Development of a Plating Medium for Selection of Helicobacter pylori from Water Samples. A. J. Degnan, 2003.The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori . Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated . Eight strains selected as common waterborne organisms (Acinetobacter, Aeromonas, Bacillus, Escherichia coli, Enterobacter, Enterococcus, Helicobacter pylori, and Pseudomonas strains) were individually plated onto each of these media . Three organisms (Acinetobacter, E . coli, and H . pylori) were able to grow on all five media . This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days . Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed . This medium also included a phenol red color indicator for urease production . Aliquots of nonsterile well water that contained native flora (Flavobacterium, Serratia, Citrobacter, Pasteurella, Ochrobactrum, Rahnella, and unidentified molds) and were further adulterated with the eight strains listed above (106 CFU of each strain per 100 ml) were spiked with H . pylori and were plated . In spite of the heavy mixed microbial load, only H . pylori colonies grew during 7 days of incubation at 37°C . The color indicator system allowed presumptive identification of H . pylori colonies sooner (12 to 20 h) than the conventional media tested allowed . The HP formulation developed in this study provides a medium with superior selectivity for H . pylori from mixed microbial populations in water and reduces the time required to complete the assay . Purification and Characterization of a Novel Bacteriocin Produced by Enterococcus faecalis Strain RJ-11. Yukio Yamamoto, 2003.Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran . One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria . A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form . The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium .
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