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Penetration of Candida Biofilms by Antifungal Agents. Mohammed A. Al-Fattani, 2004.A filter disk assay was used to investigate the penetration of antifungal agents through biofilms containing single and mixed-species biofilms containing Candida . Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine . The rates of diffusion of either drug through biofilms of three strains of Candida albicans were similar . However, the rates of drug diffusion through biofilms of C . glabrata or C . krusei were faster than those through biofilms of C . parapsilosis or C . tropicalis . In all cases, after 3 to 6 h the drug concentration at the distal edge of the biofilm was very high (many times the MIC) . Nevertheless, drug penetration failed to produce complete killing of biofilm cells . These results indicate that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms . The abilities of flucytosine, fluconazole, amphotericin B, and voriconazole to penetrate mixed-species biofilms containing C . albicans and Staphylococcus epidermidis (a slime-producing wild-type strain, RP62A, and a slime-negative mutant, M7) were also investigated . All four antifungal agents diffused very slowly through these mixed-species biofilms . In most cases, diffusion was slower with biofilms containing S . epidermidis RP62A, but amphotericin B penetrated biofilms containing the M7 mutant more slowly . However, the drug concentrations reaching the distal edges of the biofilms always substantially exceeded the MIC . Thus, although the presence of bacteria and bacterial matrix material undoubtedly retarded the diffusion of the antifungal agents, poor penetration does not account for the drug resistance of Candida biofilm cells, even in these mixed-species biofilms . Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays. Adelaide E. Warsen, 2004.We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides . Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer . PCR products (ca . 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55°C) to the microarray . Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546 . This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens . With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 106 genomes; 5 ng) or by the addition of up to 500 ng of fish DNA . Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens . The Phosphotransferase System of Streptomyces coelicolor Is Biased for N-Acetylglucosamine Metabolism. Harald Nothaft, 2003.Mutation of the crr-ptsI gene locus revealed that Streptomyces coelicolor uses the phosphotransferase system (PTS) for N-acetylglucosamine uptake . crr, ptsI, and ptsH, which encode the three general PTS phosphotransferases, are induced by N-acetylglucosamine but not by other PTS substrates . Thus, the S . coelicolor PTS is biased for N-acetylglucosamine utilization, a novel feature that distinguishes this PTS from others . Membrane Fluidity Adjustments in Ethanol-Stressed Oenococcus oeni Cells. M. Graça Da Silveira, 2003.The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated . Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells . To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy . Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface . Fluidity increased with increasing concentrations of ethanol . Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity . Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol . Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol . Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage . This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved . We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested . We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface .
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