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Activities of Different Fluoroquinolones against Bacillus anthracis Mutants Selected In Vitro and Harboring Topoisomerase Mutations. Patrick Grohs, 2004.Three sets of mutants of Bacillus anthracis resistant to fluoroquinolones were selected on ciprofloxacin and moxifloxacin in a stepwise manner from a nalidixic acid-resistant but fluoroquinolone-susceptible plasmidless strain harboring a Ser85Leu GyrA mutation . A high level of resistance to fluoroquinolones could be obtained in four or five selection steps . In each case, ParC was the secondary target . However, in addition to the GyrA mutation, expression of high-level resistance required (i) in the first set of mutants, active drug efflux associated with a mutation in the QRDR of ParC; (ii) in the second set, two mutations in the QRDR of ParC associated with a mutation in GyrB; and (iii) in the third set, two QRDR mutations, one in ParC and one in GyrA . Interestingly, several selection steps occurred without obvious mutations in the QRDR of any topoisomerase, thereby implying the existence of other resistance mechanisms . Among the fluoroquinolones tested, garenoxacin showed the best activity . Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from Pyrococcus furiosus. Corné H. Verhees, 2002.The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized . The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a Km of 0.32 mM and a Vmax of 12.2 U/mg . The P . furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li+ (50% inhibitory concentration, 1 mM) . Based on the presence of conserved sequence motifs and the substrate specificity of the P . furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase . The Pseudomonas aeruginosa rhlAB Operon Is Not Expressed during the Logarithmic Phase of Growth Even in the Presence of Its Activator RhlR and the Autoinducer N-Butyryl-Homoserine Lactone. Gerardo Medina, 2003.The Pseudomonas aeruginosa rhlAB operon encodes the enzyme rhamnosyltransferase 1, which produces the biosurfactant mono-rhamnolipid; rhlAB induction is dependent on the quorum-sensing transcription activator RhlR complexed with the autoinducer N-butyryl-homoserine lactone (C4-HSL) . In this work we studied rhlAB induction in a P . aeruginosa and Escherichia coli background . We found that, in both bacteria, its expression is not induced during the logarithmic phase of growth even in the presence of RhlR and C4-HSL . Additionally, we found that rhlAB expression is partially  s dependent .
Growth of Escherichia coli in Model Distribution System Biofilms Exposed to Hypochlorous Acid or Monochloramine. Margaret M. Williams, 2003.Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers . Six flow chambers were also inoculated with an Escherichia coli isolate obtained from potable water . The effect of disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of hypochlorous acid (ClOH) for 67 min or 4 ppm of monochloramine (NH2Cl) for 155 min . To test the ability of bacterial populations to initiate biofilm formation in the presence of disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of ClOH or 4 ppm of NH2Cl . Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of ClOH after 5 days of growth in the absence of disinfectant . Whole-cell in situ hybridizations using fluorescently tagged, 16S rRNA-targeted oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species . The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of disinfectants . Specifically, Legionella pneumophila, E . coli, and ß and  proteobacteria were identified within biofilms both before and after treatment . Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing E . coli within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems . The persistence of diverse bacterial species within disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting waterborne disease .
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