Fluoroquinolone and multidrug resistance phenotypes associated with the overexpression of AcrAB and an orthologue of MarA in Yersinia enterocolitica

Abstract

Quinolone resistance among clinical isolates is of increasing importance. This phenotype particularly affects the nalidixic acid resistance levels and is also increasing among Yersinia enterocolitica strains. This study investigated the quinolone resistance mechanisms acquired in vitro by a Y. enterocolitica clinical isolate exposed to increasing concentrations of ciprofloxacin in a multi-step selection process. The fluoroquinolone-susceptible clinical isolate, Y.83-wt, the fluoroquinolone-resistant mutant, Y.83-64, and intermediate mutants were analysed by QRDR sequencing and MIC determination. Four different QRDRs (quinolone resistance-determining regions) mutations were characterised in Y.83-64: one in gyrA, one in gyrB, and two in parC. A significant increase in the MICs of ciprofloxacin, norfloxacin, nalidixic acid, and other unrelated antibiotics was detected in Y.83-64. Furthermore, the bacterial growth rate was assessed for strains Y.83-wt and Y.83-64. The analysis reported no significant differences between both strains. Cell envelope protein approach revealed an overexpression of both AcrA and AcrB proteins in Y.83-64. RT-PCR analyses were also carried out as was sequencing of known AcrAB regulators in Yersinia. The RT-PCR analysis showed an increased transcriptional level of a marA orthologue, marA(Ye), in Y.83-64. The sequencing results reported no change in the acrR gene or in the promoter sequence of the acrAB operon when comparing Y.83-wt with Y.83-64. One differential mutation was detected within the marA(Ye) promoter in Y.83-64. Thus, the fluoroquinolone resistance phenotype acquired by Y.83-64 relies on the acquisition of 4 QRDR mutations in addition to the overexpression of AcrAB, which is likely triggered by increased expression levels of marA(Ye).