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 pat terns. Type 1 shelved a low level of resistance to ofloxacin (MIC of 8 mu g/ml), whereas a high level of resistance to this drug (MICs of 32 to 64 mu g/ml) characterized type 2. By using two oligonucleotide pri mers homologous to DNA sequences flanking the quinolone resistance-det ermining region (QRDR) in the gyrA gene of Escherichia coli and Staphy lococcus aureus, a 150-bp DNA fragment was obtained by PCR amplificati on from total DNA of two wild type and five mutant strains of M. smegm atis. The nucleotide sequences of the amplified fragments were determi ned. The deduced amino acid sequence from the wild-type strains showed ca. 79% similarity with the QRDR in the gyrase A subunit from other g ram-positive and gram-negative bacteria. The DNA sequences obtained fr om the fluoroquinolone-resistant mutants of M. smegmatis exhibited nuc leotide 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 mu tants had a Val-83 mutation or both Val-83 and Gly-87 mutations detect ed in the type 1 mutants. These results suggest that point mutations i n the QRDR of the mycobacterial gyrA gene are responsible for acquired quinolone resistance in M. smegmatis.