Analysis of rdxA and Involvement of Additional Genes Encoding NAD(P)H Flavin Oxidoreductase (FrxA) and Ferredoxin-Like Protein (FdxB) in Metronidazole Resistance of Helicobacter pylori

Abstract

Metronidazole (Mtz) is a critical ingredient of modern multidrug therapies for Helicobacter pylori infection.Mtz resistance reduces the effectiveness of these combinations. Although null mutations in a rdxA gene that encodes oxygen-insensitive NAD(P)H nitroreductase was reported in Mtz-resistant H. pylori, an intact rdxA gene has also been reported in Mtz-resistant H. pylori, suggesting that additional Mtz resistance mechanisms exist in H. pylori. We explored the nature of Mtz resistance among 544 clinical H. pylori isolates to clarify the role of rdxA inactivation in Mtz resistance and to identify another gene(s) responsible for Mtz resistance in H. pylori. Mtz resistance was present in 33% (181 of 544) of the clinical isolates. There was marked heterogeneity of resistance, with Mtz MICs ranging from 8 to >256 g/ml. rdxA inactivation resulted in Mtz MICs of up to 32 g/ml for 6 Mtz-sensitive H. pylori strains and 128 g/ml for one Mtz-sensitive strain. Single or dual (with rdxA) inactivation of genes that encode ferredoxin-like protein (designated fdxB) and NAD(P)H flavin oxidoreductase (frxA) also increased the MICs of Mtz for sensitive and resistant strains with low to moderate levels of Mtz resistance. fdxB inactivation resulted in a lower level of resistance than that from rdxA inactivation, whereas frxA inactivation resulted in MICs similar to those seen with rdxA inactivation. Further evidence for involvement of the frxA gene in Mtz resistance included the finding of a naturally inactivated frxA but an intact rdxA in an Mtz-resistant strain, complementation of Mtz sensitivity from an Mtz-sensitive strain to an Mtz-resistant strain or vice versa by use of naturally inactivated or functional frxA genes, respectively, and transformation of an Mtz-resistant Escherichia coli strain to an Mtz sensitive strain by a naturally functional frxA gene but not an inactivated frxA gene. These results are consistent with the hypothesis that null mutations in fdxB, frxA, or rdxA may be involved in Mtz resistance.

Genetic analysis of Helicobacter pylori clinical isolates suggests resistance to metronidazole can occur without the loss of functional rdxA

“…This result is in agreement with Marais et al 33 who suggested that an MTZ resistance phenotype may arise in H. pylori without mutation in rdxA or frxA. Interestingly, several groups 22,[34][35][36][37] and genome sequence annotation [38][39][40] suggest other putative redox systems that may play a role in MTZ resistance. These include fdxB (encoding a ferredoxin-like protein), fdxA (ferredoxin), fldA (flavodoxin), oorD (the g-subunit of 2-oxoglutarate oxidoreductase and porD (the g-subunit of pyruvate ferredoxin oxidoreductase).…”

“…Kwon et al 22 reported that frxA inactivation resulted in MICs (32 or 128 mg ml À1 ) similar to those seen with rdxA inactivation, whereas a study by Jeong et al 21 showed that frxA inactivation enhanced MTZ resistance in rdxA-deficient H. pylori but had little effect on the MTZ susceptibility of strains carrying a functional rdxA allele. In addition, Yang et al 23 recently identified the truncation of FrxA in an MTZ-sensitive H. pylori isolate.…”

“…However, with the exception of fdxB, research to identify other MTZ important redox systems has been hampered because the deletion of the genes seems to be lethal for H. pylori. 22 Recently, van Amsterdam et al 41 reported that double mutation of HP0605 and HP0971 (TolC-like proteins) results in decreased MTZ resistance (from wild-type MIC, 4256 mg ml À1 to 8 mg ml À1 ), indicating that the TolC efflux pump may confer resistance to MTZ. Therefore, H. pylori possessing TolC efflux pumps may be resistant to MTZ independent of rdxA or frxA mutations.…”

“…However, the absolute association of H. pylori MTZ resistance with mutations of rdxA or frxA is still a debated topic. [21][22][23] To conclusively prove this association, the contribution of specific rdxA or frxA mutations to MTZ resistance needs to be confirmed using classical genetic techniques. However, genetic tools and strategies for manipulation of H. pylori still lag behind those available for other model organisms.…”

Structural Basis of 5-Nitroimidazole Antibiotic Resistance

“…As a consequence, species with altered, absent, or elevated redox potential pathways are resistant to 5-Ni drugs (see Ref. 12 and references therein). For H. pylori, the most convincing data regarding MTR resistance relate to inactivation of the RdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase (13).…”