Helicobacter pylori uptake and efflux: Basis for intrinsic susceptibility to antibiotics in vitro

We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun, 63: 1567-1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the oute r membrane but forms large channels. Ii pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibio tic polymyxin B. We demonstrate here that H. pylori is able to take up high er basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamin e (NPN) than Pseudomonas aeruginosa or Escherichia coli, consistent with it s enhanced susceptibility to hydrophobic agents. Addition of polymyxin B le d to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin;in B to yield a 50% increase in NPN uptake in H. pylori (6 to 8 mu g/ml) than in P, aerugi nosa or E, coli (0.3 to 0.5 mu g/ml), suggesting that II. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involve s the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic p-lactamase (which H, pylori lacks ) or efflux, we examined the possible role of efflux in antibiotic suscepti bility, We had previously identified in H, pylori 11637 the presence of por tions of three genes with homology to potential restriction-nodulation-divi sion (RND) efflux systems. It was confirmed that H. pylori contained only t hese three putative RND efflux systems, named here hefABC, hefDEF, and hefG HI, and that the hefGHI system was expressed only in vivo while the two oth er RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three put ative RND efflux operons in the chromosome of H. pylori had no effect on th e in vitro susceptibility of H. pylori to 19 antibiotics. These results, in contrast to what is observed in E. coli, P. aeruginosa, and other clinical ly important gram-negative bacteria, suggest that active efflux does not pl ay a role in the intrinsic resistance of H. pylori to antibiotics.