Functional determination of site-mutations in involved in metronidazole resistance of .

BACKGROUND

Metronidazole (MTZ) is among the first-line drugs against the human gastric pathogen (). MTZ is used as a prodrug that is activated by an oxygen-insensitive enzyme NADPH nitroreductase (RdxA). Loss-of-function mutations in make MTZ resistant; however, experimental proof is lacking.

METHODS

We collected 139 gastric biopsy samples from patients suspected of infection in Shanghai, and amplified -specific gene from 134 samples. All these genes were sequenced and phylogenetically compared. The effect of mutations on RdxA function was measured by expressing them in DH5α by using the MTZ sensitivity test.

RESULTS

In total, 134 gastric biopsy samples were identified as positive. Of the 134 samples, 74 and 6 had point mutations at the various sites or promoter region of , generating truncated and extended fused proteins, respectively. The remaining 54 were full-length with single nucleotide variation (SNV) compared with the wild-type RdxA from , with 49 clustering with hpEastAsia, 3 with hpEurope, and 2 with hpNEAfrica. All 134 were expressed in DH5α; 22 and 112 resultant strains showed MTZ-sensitive and MTZ-resistant phenotypes, respectively. Comparative analysis of single nucleotide polymorphisms (SNPs) in the functional and inactivated RdxA revealed 14 novel mutations in RdxA, 5 of which conferred MTZ resistance: S18F, D59S, L62I, S79N, and A187V.

CONCLUSION

The occurrence of MTZ resistance induced by site-mutation of RdxA in patients with infection was 83.6% (112/134) in the Shanghai region. The major form of loss-of-function mutation was truncation of RdxA translation at a rate of 58/112 (51.8%). Molecular detection reliably determined the resistance of to MTZ. Thus, the functional mutants involved in MTZ resistance facilitate clinical diagnosis and medication based on sequence analysis.