The role of active efflux in antibiotic - resistance of clinical isolates of Helicobacter pylori.

PURPOSE

In gram-negative bacteria, active efflux pumps that excrete drugs can confer resistance to antibiotics however, in Helicobacter pylori this role is not well established. The purpose of this study is to evaluate the role of active efflux in resistance of H. pylori isolates to antibiotics.

MATERIALS AND METHODS

Twelve multiple antibiotic resistant (MAR) isolates resistant to at least four antibiotics, including beta-lactams, metronidazole, tetracycline, erythromycin, and ciprofloxacin; three resistant to only beta-lactams, and two hyper-susceptible isolates, were obtained from screening of 96 clinical isolates of H. pylori . Their minimal inhibitory concentrations (MICs) for antibiotics and ethidium-bromide (EtBr) were compared in the presence- and absence of a proton-conductor, carbonyl cyanide-m chlorophenyl-hydrazone (CCCP) using agar-dilution and disc diffusion. Drug accumulation studies for EtBr and antibiotics were assessed in the presence and absence of CCCP using spectrofluorometry.

RESULTS

MIC of EtBr for eight MAR-isolates was decreased two- to four-folds in the presence of CCCP, of which five showed reduced MICs for beta-lactam, metronidazole, tetracycline, and ciprofloxacin with CCCP. Accumulation of EtBr by the MAR-isolates was rapid and not dependant on the pattern of multiple resistance. Antibiotic accumulation assay confirmed the presence of energy-dependant efflux of beta-lactam, metronidazole, tetracycline, and ciprofloxacin, but no erythromycin in five MAR isolates. Energy-dependant efflux of EtBr or antibiotics was not observed for four MAR-isolates, and three isolates were resistant only to beta-lactams.

CONCLUSION

Energy-dependant efflux plays a role in the resistance of H. pylori clinical isolates to structurally unrelated antibiotics in a broadly specific multidrug efflux manner. Difference in the efflux potential of MAR isolates may be related to the presence or absence of functional efflux-pumps in diverse H. pylori isolates.