Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment. Substitutions Ser-833Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found. The levels of resistance to quinolones for each mutant were determined.Fluoroquinolones are potent antibacterial agents, derived from nalidixic acid, that inhibit DNA gyrase (11,12). Active DNA gyrase is a tetramer composed of two A and two B subunits, with molecular masses of 97 and 90 kDa for each monomer, respectively (17, 26), encoded by the gyrA and gyrB genes (10,12 DNA gyrase activity can be selectively inhibited by quinolones (8,25). Apparently, quinolones bind to the gyrase-DNA complex at the site of cleaved DNA, where they interact with the single-stranded DNA region (20,25). It has been proposed that magnesium ions are needed for quinolone-DNA binding and possibly for formation of the quinolone-gyrase-cleavable DNA complex (30). Single point mutations in gyrA that confer resistance to quinolone drugs have been described for a variety of microorganisms (14, 21-23, 31, 32, 36, 38). These mutations occur in a conserved region of the N-terminal domain of the A subunit close to the catalytic Tyr-122 site, termed the quinolone resistance-determining region (QRDR).In E. coli, replacement of Ser-83 by Leu or Trp confers high-level resistance to quinolones. Mutations in other residues also result in resistance phenotypes (29,39).Fluoroquinolone resistance is rarely found among Salmonella species. A clinical isolate of Salmonella typhimurium in which resistance to ciprofloxacin was associated with alterations in both gyrase subunits has recently been described (15). The exact locations and genetic changes associated with these mutations have not been defined.In this study, we analyzed the DNA sequences of gyrA fragments from spontaneous resistant mutants of S. typhimurium Su694 and their corresponding fluoroquinolone susceptibilities. Strain Su694 is a derivative of S. typhimurium LT2 (described elsewhere [2]). Spontaneous mutants were obtained by plating an overnight culture of Su694 on nalidixic acid-or pefloxacin-containing agar. The quinolone concentrations used were 10 and 20 times the MIC for Su694 (9). Several individual colonies were collected from these plates to purify DNA and amplify an 800-bp gyrA fragment containing the QRDR. Primers for PCR amplification were designed on the basis of the known E. coli gyrA sequence (35) and correspond to nucleotides 93 to 110 and 874 to 890 for forward and reverse reactions, respectively.PCR amplifications were performed according to the manufacturer's instructions, with 100 ng of DNA template and final concentrations of 2 mM MgCl 2 , 200 M (each) deoxynucleotide triphosphate, 10 pmol of each primer, and 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer Cet...

The production of extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae is the most prevalent resistance mechanism to third-generation cephalosporins. The aim of this study was to identify the ESBLs produced in Escherichia coli and Klebsiella pneumoniae clinical isolates from two hospitals of the Colombian Caribbean Region. A total of 30 clinical isolates of K. pneumoniae (21) and E. coli (9) ESBL-producers were collected in two hospitals from January, 2001 to June, 2003. Isoelectric point values were indicative of SHV-, and CTX-M-type beta-lactamases. PCR amplification and sequencing of SHV genes revealed that SHV-12 was the most prevalent ESBL followed by SHV-5, SHV-2a, the novel SHV-86 and CTX-M-12. There was a geographic distribution of two particular PFGE subtypes in these two distant hospitals. Clonal and horizontal dissemination of resistance was observed.

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