Differentiation of metronidazole-sensitive and -resistant clinical isolates of Helicobacter pylori by immunoblotting with antisera to the RdxA protein

Antimicrobial resistance in Helicobacter pylori is a serious and increasing problem, and the development of rapid, reliable methods for detecting resi stance would greatly improve the selection of antibiotics used to treat gas tric infection with this organism. We assessed whether detection of the Rdx A protein could provide the basis for determining the susceptibility of IT. pylori to metronidazole. In order to raise polyclonal antisera to RdxA, we cloned the rdxA gene from H. pylori strain 26695 into the commercial expre ssion vector pMAL-c2, purified the resultant fusion protein by affinity chr omatography, and used this recombinant RdxA preparation to immunize rabbits . We then used this specific anti-RdxA antibody to perform immunoblotting o n whole bacterial cell lysates of 17 metronidazole-sensitive and 27 metroni dazole-resistant clinical isolates of H. pylori. While a 24-kDa immunoreact ive band corresponding to the RdxA protein was observed in all metronidazol e-sensitive strains, this band was absent in 25 of 27 resistant isolates. O ur results indicate that testing for the absence of the RdxA protein would identify the majority of clinical isolates that will respond poorly to metr onidazole-containing eradication regimens and have implications for the dev elopment of assays capable of detecting metronidazole resistance in H. pylo ri.