Characterization of mutations in Mycobacterium smegmatis involved in resistance to fluoroquinolones.
Fluoroquinolone-resistant mutants of Mycobacterium smegmatis have been obtained in vitro by using ofloxacin as a selecting agent. Two types of mutants were identified according to their quinolone resistance patterns. Type 1 showed a low level of resistance to ofloxacin (MIC of 8 micrograms/ml), whereas a high level of resistance to this drug (MICs of 32 to 64 micrograms/ml) characterized type 2. By using two oligonucleotide primers homologous to DNA sequences flanking the quinolone resistance-determining region (QRDR) in the gyrA gene of Escherichia coli and Staphylococcus aureus, a 150-bp DNA fragment was obtained by PCR amplification from total DNA of two wild-type and five mutant strains of M. smegmatis. The nucleotide sequences of the amplified fragments were determined. The deduced amino acid sequence from the wild-type strains showed ca. 79% similarity with the QRDR in the gyrase A subunit from other gram-positive and gram-negative bacteria. The DNA sequences obtained from the fluoroquinolone-resistant mutants of M. smegmatis exhibited nucleotide modifications compared with the wild-type QRDR. The QRDR from type 1 mutants had a C-T or an A-G transition leading to a change from Ala-83 to Val or Asp-87 to Gly, respectively. The QRDR from type 2 mutants had a Val-83 mutation or both Val-83 and Gly-87 mutations detected in the type 1 mutants. These results suggest that point mutations in the QRDR of the mycobacterial gyrA gene are responsible for acquired quinolone resistance in M. smegmatis.
Revel
V
V
Laboratoire de Bactériologie-Virologie, Faculté de Médecine Pitié-Salpêtrière, Université Paris VI, France.
Cambau
E
E
Jarlier
V
V
Sougakoff
W
W
D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001483:Base Sequence; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005784:Gene Amplification; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000187:drug effects* / Q000235:genetics*; D015242:Ofloxacin / Q000494:pharmacology; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid
D000890:Anti-Infective Agents; D004269:DNA, Bacterial; D015242:Ofloxacin; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II