The role of intra- and extragenic compensatory mutations in the suppression of fluoroquinolone resistance in a Salmonella Typhimurium gyrA mutant (D87G).

OBJECTIVES: The live vaccine strain TAD Salmonella vacT (vacT) carrying gyrA mutations W59R, G75A, D87G and A866S shows resistance to nalidixic acid and rifampicin, but increased susceptibility to macrolides, fluoroquinolones and phenylalanyl-arginyl-beta-naphthylamide. This phenotype contrasts with the presence of the gyrA mutation D87G usually associated with reduced susceptibility to fluoroquinolones. Thus, a possible compensatory effect on the suppression of gyrA-mediated resistance by gyrA mutations within the quinolone resistance-determining region alone or in combination (intragenic) or by a mutation affecting AcrAB-TolC (extragenic), the major multidrug resistance efflux pump in Salmonella, was investigated. METHODS: Site-specifically introducing combinations of novel mutations into the chromosomal gyrA gene of the vacT parent strain M415 yielded defined gyrA mutants. These were characterized by determining fluoroquinolone susceptibilities and the degree of DNA supercoiling. DNA sequences of acrR, acrA and acrB of vacT were determined. A role of acrB mutations in increased fluoroquinolone susceptibility of vacT was investigated by a complementation test. RESULTS: All in vitro generated mutants carrying the gyrA D87G mutation showed increased fluoroquinolone MICs and reduced degrees of DNA supercoiling, irrespective of the presence of an additional gyrA mutation, W59R or G75A. DNA sequence analysis revealed two deletions in acrB of vacT. Complementation of vacT with an acrB wild-type gene decreased the susceptibilities to fluoroquinolones and macrolides. CONCLUSIONS: The increased fluoroquinolone susceptibility of the gyrA mutant vacT is most plausibly explained by a reduced efflux pump activity caused by acrB deletions in vacT.

Role of novel gyrA mutations in the suppression of the fluoroquinolone resistance genotype of vaccine strain Salmonella Typhimurium vacT (gyrA D87G).

OBJECTIVES: This study was aimed at characterizing the gyrA locus and determining its impact on fluoroquinolone susceptibility, DNA supercoiling degree and growth rate of Salmonella Typhimurium live vaccine strain vacT in comparison with its parent M415. Furthermore, the role of multiple drug resistance efflux in the susceptibility of vacT to fluoroquinolones and macrolides was investigated. METHODS: DNA sequences were determined for genes gyrA, gyrB, parC and parE of M415 and three consecutive mutants Nal2ori, Nal2passage and vacT. The impact of gyrA mutations on the fluoroquinolone susceptibilities and relative DNA supercoiling degrees was investigated by a complementation assay using wild-type gyrA (gyrA+) and a reporter gene system, respectively. Doubling times of the strains and MICs of different antibiotics in the absence and presence of an efflux pump inhibitor (EPI) were determined. RESULTS: Besides the gyrA mutation D87G, two novel mutations (G75A and A866S) were identified in the three mutants and a third novel mutation W59R in vacT. Fluoroquinolone susceptibilities and DNA supercoiling degrees of all three mutants were reduced compared with those of M415. Introduction of the gyrA+ allele restored fluoroquinolone susceptibilities of the two intermediate strains to the wild-type level; however, for vacT, MICs of fluoroquinolones were reduced below those of M415. VacT had a higher susceptibility to macrolides and the EPI compared with M415. CONCLUSIONS: The data point to a combination of at least one non-gyrA mutation and novel gyrA mutation(s) as the basis for the unusual fluoroquinolone susceptibility of vacT.