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Complex Multiple Antibiotic and Mercury Resistance Region Derived from the r-det of NR1 (R100). Sally R. Partridge, 2004.The sequence of the 45.2-kb multidrug and mercury resistance region of pRMH760, a large plasmid from a clinical isolate of Klebsiella pneumoniae collected in 1997 in Australia, was completed . Most of the modules found in the resistance determinant (r-det), or Tn2670, region of NR1 (also known as R100), isolated from a Shigella flexneri strain in Japan in the late 1950s, were present in pRMH760 but in a different configuration . The location was also different, with the Tn2670-derived region flanked by the transposition module of Tn1696 and a mercury resistance module almost identical to one found in the plasmid pDU1358 . This arrangement is consistent with a three-step process . First, the r-det was circularized via homologous recombination between the IS1 elements and reincorporated at a new location, possibly in a different plasmid, via homologous recombination between the 5'-conserved (5'-CS) or 3'-CS of the In34 integron in the r-det and the same region of a second class 1 integron in a Tn1696 relative . Subsequently, resolvase-mediated recombination between the res sites in the r-det and a second mercury resistance transposon removed one end of the Tn1696-like transposon and part of the second transposon . Other events occurring within the r-det-derived portion have also contributed to the formation of the pRMH760 resistance region . Tn2 or a close relative that includes the blaTEM-1b gene had moved into the Tn21 mercury resistance module with subsequent deletion of the adjacent sequence, and all four 38-bp inverted repeats corresponding to Tn21 family transposon termini have been interrupted by an IS4321-like element . Invasive Streptococcus pneumoniae in France: Antimicrobial Resistance, Serotype, and Molecular Epidemiology Findings from a Monthly National Study in 2000 to 2002. Jean-Winoc Decousser, 2004.A study of 257 French invasive pneumococci isolated between 2000 and 2002 showed high rates of nonsusceptibility to penicillin and macrolides (50%), contrasting with a low frequency of resistance to amoxicillin or levofloxacin (<1%) and tolerance to vancomycin (0%) . The genetic homogeneity of some serogroups, including serogroup 1, enhanced the risk of epidemiological changes . Essential Nature of the mreC Determinant of Bacillus subtilis. Joong-Chul Lee, 2003.The mre genes of Escherichia coli and Bacillus subtilis are cell shape determination genes . Mutants affected in mre function are spheres instead of the normal rods . Although the mre determinants are not required for viability in E . coli, the mreB determinant is an essential gene in B . subtilis . Conflicting results have been reported as to whether the two membrane-associated proteins MreC and MreD are essential proteins . Furthermore, although the MreB protein has been studied in some detail, the roles of the MreC and MreD proteins in cell shape determination are unknown . We constructed a strain of B . subtilis in which expression of the mreC determinant is dependent upon the addition of isopropyl-ß-D-thiogalactopyranoside to the culture medium . Utilizing this conditional strain, it was shown that mreC is an essential gene in B . subtilis . Furthermore, it was shown that cells lacking sufficient quantities of MreC undergo morphological changes, namely, swelling and twisting of the cells, which is followed by cell lysis . Electron microscopy was utilized to demonstrate that a polymeric material accumulated at one side of the division septum of the cells and that the presence of this material correlated with the bending of the cell . The best explanation for the results is that the MreC protein is involved in the control of septal versus long-axis peptidoglycan synthesis, that cells lacking MreC perform aberrant septal peptidoglycan synthesis, and that lysis results from a deficiency in long-axis peptidoglycan synthesis . A Field Investigation of Bacillus anthracis Contamination of U.S . Department of Agriculture and Other Washington, D.C., Buildings during the Anthrax Attack of October 2001. James A. Higgins, 2003.In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis . The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples . Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents . Surface swabs and air samples were also cultured for B . anthracis at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa . From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings . None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002 . DNA extracted from these suspected B . anthracis colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for capA and vrr genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of B . anthracis, suggesting they arose from cross-contamination during the attack through the mail . The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates . An Alternative Efficient Procedure for Purification of the Obligate Intracellular Fish Bacterial Pathogen Piscirickettsia salmonis. Vitalia Henríquez, 2003.Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome . Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile . Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P . salmonis DNA free from contaminating host cell DNA . In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis . The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P . salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell . This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter .
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