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In Vitro Evolution of Itraconazole Resistance in Aspergillus fumigatus Involves Multiple Mechanisms of Resistance. Márcia Eliana da Silva Ferreira, 2004.We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions . For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole . After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations . These mutant strains had different growth rates and different levels of itraconazole resistance . Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole . The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes . Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains . Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole . Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug . Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity. Eric Cascales, 2002.The Tol-Pal system of gram-negative bacteria is composed of five proteins . TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane . In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli . lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles . In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain . Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes . The density of Pal was measured and found to be lower than that of Lpp . However, Pal was present in larger amounts compared to TolA and TolR proteins . The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated . Comparison of UV Inactivation of Spores of Three Encephalitozoon Species with That of Spores of Two DNA Repair-Deficient Bacillus subtilis Biodosimetry Strains. Marilyn M. Marshall, 2003.When exposed to 254-nm UV, spores of Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m2, respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates . The results indicate that spores of Encephalitozoon spp . are readily inactivated at low UV fluences and that spores of UV-sensitive B . subtilis strains can be useful surrogates in evaluating UV reactor performance . Incidence and Diversity of Microorganisms within the Walls of an Active Deep-Sea Sulfide Chimney. Matthew O. Schrenk, 2003.A large, intact sulfide chimney, designated Finn, was recovered from the Mothra Vent Field on the Juan de Fuca Ridge in 1998 . Finn was venting 302°C fluids on the seafloor and contained complex mineralogical zones surrounding a large open central conduit . Examination of microorganisms within these zones, followed by community analysis with oligonucleotide probes, showed that there were variations in the abundance and diversity of eubacteria and archaea from the exterior to the interior of the chimney . The microbial abundance based upon epifluorescence microscopy and quantitative fatty acid analyses varied from >108 cells/g of sulfide 2 to 10 cm within the chimney wall to <105 cells/g in interior zones . Direct microscopic observation indicated that microorganisms were attached to mineral surfaces throughout the structure . Whole-cell hybridization results revealed that there was a transition from a mixed community of eubacteria and archaea near the cool exterior of the chimney to primarily archaea near the warm interior . Archaeal diversity was examined in three zones of Finn by cloning and sequencing of the 16S rRNA gene . The majority of sequences from the exterior of the chimney were related to marine group I of the Crenarchaeota and uncultured Euryarchaeota from benthic marine environments . In contrast, clone libraries from interior regions of the chimney contained sequences closely related to methanogens, Thermococcales, and Archaeoglobales, in addition to uncultured crenarchaeal phylotypes obtained from deep subsurface sites . These observations of microbial communities within an active hydrothermal chimney provide insight into the microbial ecology within such structures and may facilitate follow-up exploration into expanding the known upper temperature limits of life . Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities. R. Temmerman, 2003.The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes . None of the currently available identification techniques are able to visualize a  complete community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential . This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem . The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene . A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains . The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples . Except for the species Bifidobacterium coryneforme and B . indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner . Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community .
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