Characterization of rdxA, frxA, fdxA and hefA genes in metronidazole-resistant Helicobacter pylori

Abstract

Metronidazole is a bactericidal antibiotic that one component of the triple drug therapy that commonly used for treating Helicobacter pylori infection. However, metronidazole resistance rate has been incleasingly report. The mechanism of metronidazole resistance in H. pylori is still unclear. The acquisition of metronidazole resistance is reported to be associated with mutational inactivation of the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase. Recent evidence has suggested that mutations in other nitroreductase-encoding genes including frxA and fdxA may contribute to the resistance phenotype. The aims of this study are to determine mutations and expression of rdxA, frxA and fdxA in metronidazole-resistant H. pylori and to determine expression of hefA gene in metronidazole-resistant H. pylori. There were 30 metronidazole-resistant and 5 metronidazole-susceptible isolates included in this study. The results showed that 30 amino acid substitutions were found in RdxA. There were 15 amino acid substitution found in both susceptible and resistant isolates. Frameshift mutation leading to premutured stop codon was found in 10 metronidazole-resistant isolates. Few alterations in amino acid sequences of FdxA were observed. One amino acid substitution was found in metronidazole-resistant isolate,828, and amino acid deletion by nucleotide TGA deletion was found in 6 metronidazole-resistant isolates. No mutation in FdxA amino acid sequences was found in metronidazole-susceptible isolates. In FrxA, 15 metronidzole-resistant isolates had the frameshift mutation, leading to stop codon, and amino acid substitutions were present in 4 metronidazole-susceptible isolates. The results showed that mutations in RdxA together with FdxA and FrxA were observed in 7(23.33%) and 30(100%) of 30 metronidazole-resistant isolates, respectively, and combination of 3 gene mutations was found in 7(23.33%) metronidazole-resistant isolates, and all of metronidazole-resistant isolates were found at least two mutation in these nitroreductase genes (100%). For study of the efflux pump mechanism (HefA), no detection the decreasing of MIC level of metronidazole with CCCP which the efflux pump inhibitor. RT-PCR could not detect mRNA expression of nitroreductase genes and HefA in both susceptible and resistant isolates. A sensitive method, Real-time RT-PCR, is considered to be used for further investigation. Our results demonstrated that mutations in nitro reductase genes including rdxA, frxA and fdxA were associated with metronidazole resistance in H. pylori