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 t hat are used against Helicobacter pylori, the major cause of peptic ul cer disease. Many H. pylori strains are Mtz resistant (Mtz(R)), howeve r, and here we show that Mtz(R) results from loss of oxygen-insensitiv e NADPH nitroreductase activity, The underlying gene (called 'rdxA') w as identified in several steps: transformation of Mtz-susceptible (Mtz (S)) H. pylori with cosmids from a Mtz(R) strain, subcloning, polymera se 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 r dxA gene; (ii) introduction of rdxA on a shuttle vector plasmid into f ormerly 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 resu lted 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 u niform in overall genotype, were sequenced. In each case, the paired r dxA genes differed from one another by one to three base substitutions . Typical rdxA genes from unrelated isolates differ by approximate to 5% in DNA sequence. Therefore, the near identity of rdxA genes from pa ired Mtz(R) and Mtz(S) isolates implicates de novo mutation, rather th an horizontal gene transfer in the development of Mtz(R), Horizontal g ene transfer could readily be demonstrated under laboratory conditions with mutant rdxA alleles. RdxA is a homologue of the classical nitror eductases (CNRs) of the enteric bacteria, but differs in cysteine cont ent (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 e arly risk factor for gastric cancer; the possibility that its carcinog enic effects are exacerbated by Mtz use, which is frequent in many soc ieties, or the reduction of nitroaromatic compounds to toxic, mutageni c and carcinogenic products, may be of significant concern in public h ealth.