Efflux pump gene hefA of Helicobacter pylori plays an important role in multidrug resistance

Abstract

To determine whether efflux systems contribute to multidrug resistance of H pylori. A chloramphenicol-induced multidrug resistance model of six susceptible H pylori strains (5 isolates and H pylori NCTC11637) was developed. Multidrug-resistant (MDR) strains were selected and the minimal inhibitory concentration (MIC) of erythromycin, metronidazole, penicillin G, tetracycline, and ciprofloxacin in multidrug resistant strains and their parent strains was determined by agar dilution tests. The level of mRNA expression of hefA was assessed by fluorescence real-time quantitative PCR. A H pylori LZ1026 knockout mutant (delta H pylori LZ1026) for (putative) efflux protein was constructed by inserting the kanamycin resistance cassette from pEGFP-N2 into hefA, and its susceptibility profiles to 10 antibiotics were evaluated. The MIC of six multidrug-resistant strains (including 5 clinical isolates and H pylori NCTC11637) increased significantly (>or=4-fold) compared with their parent strains. The expression level of hefA gene was significantly higher in the MDR strains than in their parent strains (P=0.033). A H pylori LZ1026 mutant was successfully constructed and the delta H pylori LZ1026 was more susceptible to four of the 10 antibiotics. All the 20 strains displayed transcripts for hefA that confirmed the in vitro expression of these genes. The efflux pump gene hefA plays an important role in multidrug resistance of H pylori.