Search for Novel Candidate Mutations for Metronidazole Resistance in Helicobacter pylori Using Next-Generation Sequencing

Abstract

ABSTRACTMetronidazole resistance is a key factor associated withHelicobacter pyloritreatment failure. Although this resistance is mainly associated with mutations in therdxAandfrxAgenes, the question of whether metronidazole resistance is caused by the inactivation offrxAalone is still debated. Furthermore, it is unclear whether there are other mutations involved in addition to the two genes that are associated with resistance. A metronidazole-resistant strain was cultured from the metronidazole-susceptibleH. pyloristrain 26695-1 by exposure to low concentrations of metronidazole. The genome sequences of both susceptible and resistantH. pyloristrains were determined by Illumina next-generation sequencing, from which putative candidate resistance mutations were identified. Natural transformation was used to introduce PCR products containing candidate mutations into the susceptible parent strain 26695-1, and the metronidazole MIC was determined for each strain. Mutations infrxA(hp0642),rdxA(hp0954), andrpsU(hp0562) were confirmed by the Sanger method. The mutated sequence inrdxAwas successfully transformed into strain 26695-1, and the transformants showed resistance to metronidazole. The transformants containing a single mutation inrdxAshowed a low MIC (16 mg/liter), while those containing mutations in bothrdxAandfrxAshowed a higher MIC (48 mg/liter). No transformants containing a single mutation infrxAorrpsUwere obtained. Next-generation sequencing was used to identify mutations related to drug resistance. We confirmed that the mutations inrdxAare mainly associated with metronidazole resistance, and mutations infrxAare able to enhanceH. pyloriresistance only in the presence ofrdxAmutations. Moreover, mutations inrpsUmay play a role in metronidazole resistance.