Influence Of Efflux Pump Inhibitors On The Multidrug Resistance Of Helicobacter Pylori

Background and ObjectivesHelicobacter pylori (H.pylori) is an important human pathogen that infects 50% of the world's population. H.pylori infection in human is associated with the development of numerous gastric pathologies, such as peptic ulcer, chronic gastritis and gastric cancer. Once H.pylori is established in the gastric submucosa, infected individuals usually carry it for all life unless treated. In recent years, the rate of resistance of H. pylori to standard therapies has increased as a result of the widespread use of antibiotics. H.pylori resistance to metronidazole and clarithromycin has increased worldwide and multidrug resistant (MDR) strains that are simultaneously resistant to 3 or more kinds of antibiotics, such as amoxicillin, metronidazole and clarithromycin, have been reported. Boyanova et al. reported that 26.4% of the strains were resistant to metronidazole and clarithromycin among antibiotic-treated patients, Wueppenhorst et al. found that 15% of the H.pylori isolated from patients showed resistance to 3 or more types of antibiotic.Efflux pump systems in bacteria which can actively eject the drugs arid toxic compounds, including antibiotics, have a critical role in the development of multidrug resistance. We have observed previously that the efflux pump gene hefA of H.pylori has valuable applications in multidrug resistance. Until now, efflux pump inhibitors (EPIs) as promising therapeutic agents have been widely investigated in other bacteria, where they could decrease the intrinsic bacterial resistance to antibiotics, and reverse the acquired resistance associated with efflux pump overexpression. Different classes of EPIs have been exploited and studied, including analogues of antibiotic substrates and new molecules. However, whether the EPIs can also reverse the multidrug resistance of H.pylori has not been fully researched. The carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and reserpine were employed in the present study as different types of EPI, and a chloramphenicol-induced multidrug resistance model was developed. The aim of this investigation was to elucidate whether EPIs can influence the antibiotic susceptibilities of H.pylori MDR strains.Materials and MethodsH.pylori strains were isolated and cultured on Brucella agar plates with 10% sheep's blood. The multidrug resistant (MDR) strains of H.pylori were obtained with the inducer chloramphenicol by doubling up to the concentration in which no colony were seen, then the susceptibilities of the MDR strains and their parents to 9 antibiotics were assessed with agar dilution tests. The present study included periods before and after the advent of the EPIs:CCCP, reserpine and pantoprazole, and the minimum inhibitory concentrations (MIC) were determined accordingly. In the same way, the effects of 5 proton pump inhibitors (PPIs), used in treatment of H.pylori infection, on MIC of antibiotics were evaluated.Results1. From 65 H.pylori isolates,9 strains with no antibiotic resistance phenotype were selected randomLy, and the NCTC11637 strain was taken as the control. Following a doubling increase in the concentration of chloramphenicol, MICs of chloramphenicol-induced strains to 9 antibiotics were determined. The MICs in 4 MDR strains (including 3 clinical isolates and H.pylori NCTC11637) were significantly increased (=4-fold) compared with their parent strains.2. The expression of hefA in MDR strains and their parent isolates was assessed by real-time RT-PCR. Each relative expression value was the mean of 3 replications. The relative expression of hefA versus gyrB in 3 clinical isolates and H.pylori 11637 was significantly higher in MDR strains (5.83±0.91) than in parent strains (1.90±0.45). The difference in hefA expression was statistically significant (P<0.001).3. When treated with CCCP (100μmol/L), MICs of the 9 antibiotics were tested. MICs of 5 antibiotics decreased at least 4-fold in MDR strains:19-fold with chloramphenicol,10-fold with tetracycline,7-fold with erythromycin,4-fold with cefotaxime and ceftriaxone (geometric mean), while there was little difference in the MIC of antibiotics in parent strains (sensitive strains) after challenge with CCCP. After treatment with pantoprazole (10μg/mL), MICs of the 9 antibiotics were tested. MICs of 5 antibiotics decreased at least 4-fold in MDR strains:6-fold with erythromycin,13-fold with tetracycline,9-fold with cefotaxime and ceftriaxone, 4-fold with clindamycin (geometric mean), while MICs with erythromycin and ceftriaxone were also decreased 4-fold in their parent strains. MICs of antibiotics in MDR strains and parent strains had no obvious difference after reserpine (20μg/mL) was added to the agar.4. To further clarify the effect of PPIs on MICs of antibiotics used in eradicating H.pylori in the clinic, and to determine which PPI was the most efficient in H.pylori treatment when combined with 2 antibiotics, MICs of metronidazole, amoxicillin, furazolidone and clarithromycin in MDR H.pylori strains were measured after treatment with each of the 5 PPIs (10μg/mL). Our data indicated that MICs of metronidazole and amoxicillin were decreased 4-fold and 3-fold by treatment with rabeprazole, while there was a half decrease with pantoprazole. There was little effect of omeprazole, lansoprazole and esomeprazole.on the MICs of antibiotics.ConclusionsWith the extended use of antibiotics, the rate of resistance of antibiotics for H.pylori has increased worldwide, and appearance of MDR strain has been reported. The results of this study show that:1. With induced by chloramphenicol, the sensitivity of H.pylori strains to some antibiotics has significantly changed, resulting in a multidrug resistance phenotype. We has not only established the model of multidrug resistant H.pylori, but also confirmed that abuse of antibiotics would lead more harm for human.2. HefABC, as a RND efflux pump system in MDR-H.pylori, plays a crucial role in the MDR appearance.3. A part of the efflux pump inhibitors can selectively lower the MIC value of antibiotics, by the mechanism of inhibiting the efflux pump system of MDR-H.pylori to restore sensitivity.4. Because of interference with the electrochemical proton gradient between inside and outside of bacteria, proton pump inhibitors can alter the sensitivity of MDR-H.pylori to some antibiotics, and rabeprazole is the most influential one in vitro.