Sequential inactivation of rdxA (HP0954) and frxA (HP0642) nitroreductase genes causes moderate and high-level metronidazole resistance in Helicobacter pylori

Abstract

Helicobacter pylori is a human-pathogenic bacterial species that is subdivided geographically, with different genotypes predominating in different parts of the world. Here we test and extend an earlier conclusion that metronidazole (Mtz) resistance is due to mutation in rdxA (HP0954), which encodes a nitroreductase that converts Mtz from prodrug to bactericidal agent. We found that (i) rdxA genes PCR amplified from 50 representative Mtz(r) strains from previously unstudied populations in Asia, South Africa, Europe, and the Americas could, in each case, transform Mtz(s) H. pylori to Mtz(r); (ii) Mtz(r) mutant derivatives of a cultured Mtz(s) strain resulted from mutation in rdxA; and (iii) transformation of Mtz(s) strains with rdxA-null alleles usually resulted in moderate level Mtz resistance (16 microg/ml). However, resistance to higher Mtz levels was common among clinical isolates, a result that implicates at least one additional gene. Expression in Escherichia coli of frxA (HP0642; flavin oxidoreductase), an rdxA paralog, made this normally resistant species Mtz(s), and frxA inactivation enhanced Mtz resistance in rdxA-deficient cells but had little effect on the Mtz susceptibility of rdxA(+) cells. Strains carrying frxA-null and rdxA-null alleles could mutate to even higher resistance, a result implicating one or more additional genes in residual Mtz susceptibility and hyperresistance. We conclude that most Mtz resistance in H. pylori depends on rdxA inactivation, that mutations in frxA can enhance resistance, and that genes that confer Mtz resistance without rdxA inactivation are rare or nonexistent in H. pylori populations.

FIG. 1

Profiles of intrinsic susceptibility and resistance to Mtz of reference strains 26695 and J99. Young exponentially growing cultures were diluted, aliquots were spotted on BHI agar with indicated concentrations of Mtz, and surviving colonies were counted from appropriate dilutions, as detailed in Materials and Methods. Presented are the average and range of results with two single colony isolates (cultures) of each strain, with assays repeated three times with each culture.

FIG. 2

DNA transformation strategy for testing involvement of rdxA gene mutation in Mtz^r clinical isolates.

FIG. 3

Mtz resistance profiles of rdxAΔ111 or rdxAΔ601 deletion mutant transformants of strain 26695 and of rdxAΔ601-containing transformants of three 26695 derivatives that had exhibited an unusual 3R 8S phenotype that was not attributable to mutation in rdxA (201, 116, and 619 [Table 4]). Transformants containing the rdxAΔ111 and rdxAΔ601 alleles were selected using BHI agar with 8 μg of Mtz/ml. Presented are the average and range of results with three single colony isolates (cultures) of each strain, with each culture assayed two times.

FIG. 4

Mtz resistance profiles of the unusual highly resistant mutant of strain 26695 (designated 26695 mutant 161 [Table 4]) and also a 26695 derivative that contains only the rdxA gene from this mutant (designated rdxA.161). The rdxA.161 transformant was selected on BHI agar with 8 μg of Mtz/ml. The presence of the expected GAG-to-TAG change in rdxA (Table 4) was verified by DNA sequencing. Presented are the average and range of results with five single colony isolates (cultures) from each strain, with each culture assayed once.

FIG. 5

Effect of frxA inactivation on Mtz susceptibility and resistance depends on whether rdxA is functional or not. (A) Profile of Mtz susceptibility of an frxA-null mutant of an rdxA::cam strain and of its frxA⁺ parent; (B) profile of Mtz susceptibility of an frxA-null mutant derivative of 26695 (rdxA⁺) and of its wild-type parent. Presented are the average and range of results with three single colony isolate cultures of each strain, with each culture assayed two times.