Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase

Metronidazole (Mtz) is a critical component of combination therapies that are used against Helicobacter pylori, the major cause of peptic ulcer disease. Many H. pylori strains are Mtz resistant (Mtz(R)), however, and here we show that Mtz(R) results from loss of oxygen-insensitive NADPH nitroreductase activity. The underlying gene (called 'rdxA') was identified in several steps: transformation of Mtz-susceptible (Mtz(S)) H. pylori with cosmids from a Mtz(R) strain, subcloning, polymerase chain reaction (PCR) and DNA sequencing. We also found that (i) E. coli (normally Mtz(R)) was rendered Mtz(S) by a functional H. pylori rdxA gene; (ii) introduction of rdxA on a shuttle vector plasmid into formerly Mtz(R) H. pylori rendered it Mtz(S); and (iii) replacement of rdxA in Mtz(S) H. pylori with an rdxA::camR null insertion allele resulted in a Mtz(R) phenotype. The 630 bp rdxA genes of five pairs of H. pylori isolates from infections that were mixed (Mtz(R)/Mtz(S)), but uniform in overall genotype, were sequenced. In each case, the paired rdxA genes differed from one another by one to three base substitutions. Typical rdxA genes from unrelated isolates differ by ~5% in DNA sequence. Therefore, the near identity of rdxA genes from paired Mtz(R) and Mtz(S) isolates implicates de novo mutation, rather than horizontal gene transfer in the development of Mtz(R). Horizontal gene transfer could readily be demonstrated under laboratory conditions with mutant rdxA alleles. RdxA is a homologue of the classical nitroreductases (CNRs) of the enteric bacteria, but differs in cysteine content (6 vs. 1 or 2 in CNRs) and isoelectric point (pI = 7.99 vs. 5.4-5.6), which might account for its reduction of low redox drugs such as Mtz. We suggest that many rdxA (Mtz(R)) mutations may have been selected by prior use of Mtz against other infections. H. pylori itself is an early risk factor for gastric cancer; the possibility that its carcinogenic effects are exacerbated by Mtz use, which is frequent in many societies, or the reduction of nitroaromatic compounds to toxic, mutagenic and carcinogenic products, may be of significant concern in public health.