The emergence of multidrug-resistant strains ofMycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions ofgyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons ofgyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was >2 ,ug/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 107 to 108 at ciprofloxacin concentrations of 2 ,ug/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.The resurgence of tuberculosis and its incidence in human immunodeficiency virus-positive populations in both developing countries and the industrialized world have been accompanied by the alarming emergence of virulent multidrugresistant tuberculosis (MDR-TB) strains in North American cities (7). Many of these strains have acquired resistance to almost all first-and second-line antituberculosis agents. For this reason, there is an increasing interest in the antimycobacterial actions of the fluoroquinolones (FQs). Against Mycobacterium tuberculosis, the FQs show moderate in vitro activity (4), with sparfloxacin (MIC, 0.25 to 0.5 ,ug/ml) perhaps being the most effective compound (17). The principal target of the quinolones is the DNA gyrase, a type II DNA topoisomerase that is composed of two A and two B subunits (30) encoded by gyrA and gyrB, respectively. Mutations in the putative FQbinding region of the A subunit have been found to confer high-level FQ resistance in several bacterial species (8,19,22,31,33). Other mutations that confer resistance to quinolones have been found in gyrB, in genes that lower the intracellular concentration of the drug (although these tend to confer lower-level resistance than do the gyrA mutations [32,34]), or

The insertion sequence IS1245 is a novel mycobacterial repetitive element identified in Mycobacterium avium. It encodes a transposase which exhibits a 64% amino acid similarity with IS1081, an insertion element present in the M. tuberculosis complex. The host range of IS1245 appears limited to M. avium as this element was not identified in M. intracellulare or in any other of 18 mycobacteria species tested. When IS1245 was used for restriction fragment length polymorphism (RFLP) analysis, human isolates characteristically presented a high number of copies (median, 16; range, 3 to 27) and a diversity of RFLP patterns comparable to that found by pulsed-field gel electrophoresis. Isolates from nonhuman sources differed both in number of copies and in RFLP pattern diversity: while swine isolates shared the characteristics of human strains, those from several avian sources exhibited a very low copy number of IS1245 and appeared clonal on the basis of RFLP.

A strategy aimed at increasing repositioning frequency (2 versus 4 h) in patients under MV and on an APAM did not reduce the incidence of pressure ulcers. However, it did increase device-related adverse events and daily nursing workload.

The guanine nucleotide releasing protein C3G was initially identi®ed as a Crk SH3-binding protein and recently shown to exhibit exchange activity on Rap1 proteins. Overexpression in NIH3T3 cells of a full-length C3G cDNA isolated from human placenta markedly reduced the focus forming activity of cotransfected, malignantly activated, ras oncogenes (5 ± 7-fold). C3G also had a reverting eect on sis-mediated transformation, decreasing the number of c-sis-induced foci by a factor of 5 ± 10-fold. The observed inhibitory eect of C3G on focus-forming activity of Ras and Sis was always higher than that observed with Rap1A, a known target of C3G. The inhibition of focus formation observed in the presence of C3G was not due to toxic eects on cell viability, since transfected C3G cells exhibited the same survival and growth rates as untransfected NIH3T3 cells or cells transfected with plasmid vector alone. Surprisingly, as opposed to Rap1A, which has no eect on Raf-1 oncogene-mediated transformation, C3G also reduced dramatically (6 ± 8-fold) the number of v-raf-induced foci in transfected NIH3T3 cells. The inhibitory eect on Raf-induced transformation suggests that C3G has other functional targets in addition to Rap1. A C3G mutant (C3G DCat) lacking the catalytic domain (CDC25-H) but retaining the rest of the N-terminal sequences, including the Crkbinding domain, exhibited similar ability than full length C3G to inhibit focus formation. In contrast, a C3G mutant (C3G Cat), containing the catalytic domain only but lacking the rest of the N-terminal sequences, did not have any inhibitory eect on transformation mediated by the oncogenes tested. The C3G-derived gene products overexpressed in our transfected cell lines localized to the cytoplasm and did not change the basal MAPK or JNK activity of those cell lines nor their ability to activate the kinases in response to agonists. Our results suggest that the N-terminal region of C3G, and not its catalytic domain, may be responsible for the inhibitory eects observed.