Effect of chronic intermittent administration of inhaled tobramycin on respiratory microbial flora in patients with cystic fibrosis.
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Pseudomonas aeruginosa endobronchial infection causes significant morbidity and mortality among cystic fibrosis patients. Microbiology results from two multicenter, double-blind, placebo-controlled trials of inhaled tobramycin in cystic fibrosis were monitored for longitudinal changes in sputum microbial flora, antibiotic susceptibility, and selection of P. aeruginosa isolates with decreased tobramycin susceptibility. Clinical response was examined to determine whether current susceptibility standards are applicable to aerosolized administration. Treatment with inhaled tobramycin did not increase isolation of Burkholderia cepacia, Stenotrophomonas maltophilia, or Alcaligenes xylosoxidans; however, isolation of Candida albicans and Aspergillus species did increase. Although P. aeruginosa tobramycin susceptibility decreased in the tobramycin group compared with that in the placebo group, there was no evidence of selection for the most resistant isolates to become most prevalent. The definition of resistance for parenteral administration does not apply to inhaled tobramycin: too few patients had P. aeruginosa with a tobramycin MIC >/=16 microgram/mL to define a new break point on the basis of clinical response. (+info)
Gas supersaturation in the cecal wall of mice due to bacterial CO2 production.
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PCO2 in the lumen and serosa of cecum and jejunum was measured in mice. The anesthetic used was a fentanyl-fluanisone-midazolam mixture. PCO2 was recorded in vivo and postmortem. PCO2 was 409 +/- 32 Torr (55 +/- 4 kPa) in the cecal lumen and 199 +/- 22 Torr (27 +/- 3 kPa) on the serosa in normal mice. Irrigation of the cecum resulted in serosal and luminal PCO2 levels of 65-75 Torr. Cecal PCO2 was significantly lower in germ-free mice (65 +/- 5 Torr). Cecal PCO2 increased significantly after introduction of normal bacterial flora into germ-free mice. Introduction of bacterial monocultures into germ-free mice had no effect. After the deaths of the mice, cecal PCO2 increased rapidly in normal mice. The intestinal bacteria produced the majority of the cecal PCO2, and the use of tonometry in intestinal segments with a high bacterial activity should be interpreted with caution. We propose that serosal PCO2 levels >150-190 Torr (20-25 kPa) in the cecum of mice with a normal circulation may represent a state of gas supersaturation in the cecal wall. (+info)
Superiority of yeast over bacterial cytosine deaminase for enzyme/prodrug gene therapy in colon cancer xenografts.
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The enzyme/prodrug strategy using bacterial cytosine deaminase (bCD) and 5-fluorocytosine (5-FC) is currently under investigation for cancer gene therapy. A major limitation for the use of bCD is that it is inefficient in the conversion of 5-FC into 5-fluorouracil. In the present study, we show that the K(m) of yeast cytosine deaminase (yCD) for 5-FC was 22-fold lower when compared with that of bCD. HT29 human colon cancer cells transduced with yCD (HT29/yCD) were significantly more sensitive to 5-FC in vitro than HT29 cells transduced with bCD (HT29/bCD). In tumor-bearing nude mice, complete tumor regression was observed in 6 of 13 HT29/yCD tumors in response to 5-FC treatment (500 mg/kg i.p. daily, 5 days a week for 2 weeks), whereas 0 of 10 HT29/bCD tumors were cured. Our study demonstrates an improved efficacy of the CD/5-FC treatment strategy when yCD was used. This enzyme has, therefore, a high potential to increase the therapeutic outcome of the enzyme/prodrug strategy in cancer patients. (+info)
Investigation of two evolutionarily unrelated halocarboxylic acid dehalogenase gene families.
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Dehalogenases are key enzymes in the metabolism of halo-organic compounds. This paper describes a systematic approach to the isolation and molecular analysis of two families of bacterial alpha-halocarboxylic acid (alphaHA) dehalogenase genes, called group I and group II deh genes. The two families are evolutionarily unrelated and together represent almost all of the alphaHA deh genes described to date. We report the design and evaluation of degenerate PCR primer pairs for the separate amplification and isolation of group I and II deh genes. Amino acid sequences derived from 10 of 11 group I deh partial gene products of new and previously reported bacterial isolates showed conservation of five residues previously identified as essential for activity. The exception, DehD from a Rhizobium sp., had only two of these five residues. Group II deh gene sequences were amplified from 54 newly isolated strains, and seven of these sequences were cloned and fully characterized. Group II dehalogenases were stereoselective, dechlorinating L- but not D-2-chloropropionic acid, and derived amino acid sequences for all of the genes except dehII degrees P11 showed conservation of previously identified essential residues. Molecular analysis of the two deh families highlighted four subdivisions in each, which were supported by high bootstrap values in phylogenetic trees and by enzyme structure-function considerations. Group I deh genes included two putative cryptic or silent genes, dehI degrees PP3 and dehI degrees 17a, produced by different organisms. Group II deh genes included two cryptic genes and an active gene, dehIIPP3, that can be switched off and on. All alphaHA-degrading bacteria so far described were Proteobacteria, a result that may be explained by limitations either in the host range for deh genes or in isolation methods. (+info)
Characterization of two novel type I ribosome-inactivating proteins from the storage roots of the andean crop Mirabilis expansa.
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Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop. The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C4 reverse-phase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others. (+info)
Comparison of peritoneal fluid culture results from adults and children undergoing CAPD.
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BACKGROUND: Peritonitis is a common complication in patients with end-stage renal disease treated by continuous ambulatory peritoneal dialysis (CAPD). Empirical treatment is based on the organisms that are most frequently isolated and their susceptibilities. OBJECTIVE: To analyze and then compare peritoneal fluid culture results from adult and pediatric patients on CAPD, with respect to micro-organisms and antimicrobial susceptibilities. DESIGN: Three-year retrospective review of peritoneal fluid cultures from adults and children on CAPD. RESULTS: We isolated 481 organisms from 378 peritoneal fluid specimens, collected from 135 patients (45 children, 90 adults). There were 191 episodes of peritonitis in children (mean 4.2+/-3.5, range 1 - 15) compared to 187 in adults (2.1+/-1.9, range 1 - 10) (p< 0.001). Two or more episodes occurred in 30 of 45 children (67%) compared to 33 of 90 adults (37%) (p < 0.001).The number of different organisms/patient as well as the total number of isolates/patient were significantly greater in children (respectively, 2.8+/-2.3, range 1 - 12; and 5.3+/-5.2, range 1 - 27) than in adults (2.0+/-1.3, range 1 - 6; and 2.7+/-2.4, range 1 - 10) (p< 0.005). After Staphylococcus epidermidis, S. aureus was the most frequently isolated organism, occurring in 18% of episodes in adults and 12% of episodes in children (p< 0.01). Twenty-two of 33 fungal isolates (67%) in children were Candida parapsilosis compared to 3 of 24 (12%) in adults (p < 0.001). Subanalysis of multiple episodes revealed that Pseudomonas and Candida occurred significantly more often in children (p< 0.01), whereas S. aureus occurred more often in adults (p< 0.001). In polymicrobial episodes S. epidermidis occurred more often in adults (p < 0.05). Significant differences in susceptibilities to ampicillin, ceftriaxone, chloramphenicol, and gentamicin were found between children and adults (p< 0.05 - 0.001). CONCLUSIONS: CAPD-associated peritonitis occurs significantly more often in children than adults. Significant differences in microbial etiology and susceptibilities were found between pediatric and adult patients. Each dialysis unit should periodically analyze peritoneal fluid culture results from its CAPD patients. These data can then be used for optimization of empirical antimicrobial therapy of peritonitis. (+info)
Clinical and financial benefits of rapid bacterial identification and antimicrobial susceptibility testing.
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To assess the expected clinical and financial benefits of rapid reporting of microbiology results, we compared patients whose cultured samples were processed in the normal manner to patients whose samples were processed more rapidly due to a minor change in work flow. For the samples tested in the rapid-reporting time period, the vast majority of bacterial identification and antimicrobial susceptibility testing (AST) results were verified with the Vitek system on the same day that they were available. This time period was called rapid AST (RAST). For RAST, a technologist on the evening shift verified the data that became available during that shift. For the control time period, cultures were processed in the normal manner (normal AST [NAST]), which did not include evening-shift verification. For NAST, the results for approximately half of the cultures were verified on the first day that the result was available. The average turnaround time for the reporting of AST results was 39.2 h for RAST and 44.4 h for NAST (5.2 h faster for RAST [P = 0.001]). Subsequently, physicians were able to initiate appropriate antimicrobial therapy sooner for patients whose samples were tested as part of RAST (P = 0.006). The mortality rates were 7. 9 and 9.6% for patients whose samples were tested as part of RAST and NAST, respectively (P = 0.45). The average length of stay was 10. 7 days per patient for RAST and 12.6 days for NAST, a difference of 2.0 days less for RAST (P = 0.006). The average variable cost was $4, 927 per patient for RAST and $6,677 for NAST, a difference of $1,750 less per patient for RAST (P = 0.001). This results in over $4 million in savings in variable costs per year in our hospital. (+info)
Detection of unculturable bacteria in periodontal health and disease by PCR.
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Recently developed molecular methods have made it possible to characterize mixed microflora in their entirety, including the substantial numbers of bacteria which do not grow on artificial culture media. In a previous study, molecular analysis of the microflora associated with acute oral infections resulted in the identification of three phylotypes, PUS3.42, PUS9.170, and PUS9.180, representing as-yet-uncultured organisms. The aim of this study was to design and validate specific PCR primers for these phylotypes and to determine their incidences in samples collected from healthy and diseased periodontal tissues. Two specific reverse primers were devised for each phylotype, and these were used in duplex PCRs with universal forward and reverse primers. All three phylotypes were detected in periodontal sites; PUS9.170, related to oral asaccharolytic Eubacterium spp., was significantly associated with disease. This study demonstrates the possibility of using unculturable, and therefore uncharacterized, organisms as markers of disease. (+info)