Association of AcrAB–tolC efflux pump and marA–soxS–rob regulon with ciprofloxacin–resistance in Shigella flexneri isolates

Abstract

Objective To investigate the association of AcrAB–tolC efflux pump and marA–soxS–rob regulon with ciprofloxacin–resistance in Shigella flexneri isolates. Methods Forty five strains of Shigella flexneri were isolated in stool samples collected from outpatient diarrhea clinics in Tianjin from 2009 to 2013. The antimicrobial susceptibility of bacteria was analyzed by Kirby–Bauer disk diffusion method. Ten ciprofloxacin–resistant Shigella flexneri isolates and 10 ciprofloxacin–sensitive isolates were randomly selected. The gyrA and parC genes, plasmid mediated quinolone resistant (PMQR) determinants, efflux pump genes (acrA, acrB) and regulation genes (marA, soxS, rob) were screened and sequenced. Minimum inhibition concentrations (MICs) of the strains were determined before and after efflux pump inhibitor carbonyl cyanide m–chlorophenylhydrazone (CCCP) was added. The expression of acrA, acrB, marA, soxS and rob genes in ciprofloxacin–resistant and ciprofloxacin–sensitive strains was detected by real–time fluorescence quantitative RCR (RT–PCR), and the differences of expression were evaluated using t test. Results Both gyrA and parC mutations were detected in all ciprofloxacin–resistant strains; qnrS1 was positive in ciprofloxacin–resistant strain CR2 and aac(6′)–Ib–cr was positive in ciprofloxacin–resistant strain CR5. Efflux pump genes and regulation genes were not detected in ciprofloxacin–sensitive strains, while soxRS mutation was detected in all ciprofloxacin–resistant strains except CR10. MICs of quinolones in ciprofloxacin–resistant strains decreased to one–fourth or one–eighth when CCCP added, while not changed or only decreased to one–half in ciprofloxacin–sensitive strains. Expressions of acrA, acrB, marA and soxS were significantly higher in ciprofloxacin–resistant strains than those in ciprofloxacin–sensitive strains (P<0.05 or P<0.01). Conclusion Efflux pump may involve in the high–level ciprofloxacin resistance in Shigella flexneri through activating transcription of efflux pump genes by marA–soxS–rob regulon, in which soxRS mutation may play an important role. Key words: Shigella; Drug resistance, multiple; Ciprofloxacin; Efflux pump; marA–soxS–rob regulon