Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region

BACKGROUND & OBJECTIVES: There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. METHODS: Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥0.25 µg/ml), SFM2 (≥4 µg/ml) and SFM3 (.32 µg/ml) were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥0.25 µg/ml) and SDM2 (≥4 µg/ml) were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. RESULTS: Mutations were observed in gyrA Ser(83) →Leu, Asp(87) →Asn/Gly, Val(196) →Ala and in parC Phe(93) →Val, Ser(80) →Ile, Asp(101) →Glu and Asp(110) →Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance (P<0.05); while tolC and acrR expression levels did not. INTERPRETATION & CONCLUSIONS: Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val(196) →Ala in gyrA in clinical isolates and Phe(93) →Val, Asp(101) →Glu, Asp(110) →Glu and in parC in majority of laboratory-grown mutants.