DETECTION OF GYRA AND GYRB MUTATIONS IN QUINOLONE-RESISTANT CLINICAL ISOLATES OF ESCHERICHIA-COLI BY SINGLE-STRAND CONFORMATIONAL POLYMORPHISM ANALYSIS AND DETERMINATION OF LEVELS OF RESISTANCE CONFERRED BY 2 DIFFERENT SINGLE GYRA MUTATIONS

Twelve quinolone-resistant clinical isolates of Escherichia coli (nalidixic acid MICs, 64 to 512 mu g/ml; norfloxacin MICs; 0.25 to 8 mu g/ml) were transformed with plasmid pJSW101 carrying the gyrA(+) gene and with plasmid pJB11 carrying the gyrB(+) gene to examine the proportion of gyrA and gyrB mutations. Transformation with pJSW101 resulted in complementation (nalidixic acid MICs, 4 to 32 mu g/ml; norfloxacin MICs, 0.06 to 0.25 mu g/ml). In contrast, no change in MICs were observed after transformation with pJB11, A 418-bp fragment of gyrA from the 12 strains was amplified by PCR. Direct DNA sequencing of that fragment identified the causes of quinolone resistance in eight strains as a single point mutation leading to a substitution of the serine at position 83 (Ser-83) to Leu and in four strains as a single point mutation leading to a substitution of Asp-87 to Gly. Exchange of the fragment from one of these strains with that of gyrA(+) and transformation of resistance with the hybrid gyrA plasmid indicated the contribution of Gly-87 to resistance and the stabilities of mutants containing GyrA (Gly-87), Thus, gyrA gene mutations are probably encountered more often than gyrB gene mutations in clinical isolates off. coli, In addition, the Substitution of Asp-87 to Gly can be encountered in such strains, On the basis of the level of resistance found in the fragment exchange experiment, the quinolone resistance attributable to Gly-87 appears to be comparable to that attributable to Leu-83, The levels of resistance found in the clinical isolates shown to have a Gly-87 mutation (nalidixic acid MICs, 64 to 512 mu g/ml; norfloxacin MICs, 0.5 to 4 mu g/ml) suggest that the Gly-87 mutation causes resistance at the lei el of the nalidixic acid MIC (64 mu g/ml) or the norfloxacin MIC (0.5 mu g/ml or less) and that the additional increments in resistance seen in the other strains with higher levels of resistance may be attributable to additional mutations, The single-strand conformational polymorphism analysis with PCR products readily detected the Leu 83 and Gly-87 mutations.