Abstract
BackgroundClarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori. The known mechanism of CLR resistance has been established in A2146G and A2147G mutations in the 23S rRNA gene, but evidence of the involvement of other genetic mechanisms is lacking. Using the MiSeq platform, whole-genome sequencing of the 19 clinical strains and the reference strain ATCC26695 was performed to identify single nucleotide variants (SNVs) of multi-drug resistant efflux pump genes in the CLR-resistant phenotype.ResultsBased on sequencing data of ATCC26695, over one million sequencing reads with over 50-fold coverage were sufficient to detect SNVs, but not indels in the bacterial genome. Sequencing reads of the clinical isolates ranged from 1.82 to 10.8 million, and average coverage ranged from 90.9- to 686.3-fold, which were acceptable criteria for detecting SNVs. Utilizing the conventional approach of allele-specific PCR, point mutations in the 23S rRNA gene were detected in 12 clinical resistant isolates, but not in 7 clinical susceptible isolates. All sequencing reads of CLR-resistant strains had a G mutation in an identical position of the 23S rRNA gene. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of TolC homologues, which have been implicated in multi-drug resistance, were examined. Specific SNVs were dominantly found in resistant strains.ConclusionsGene clusters of TolC homologues are involved in CLR susceptibility profiles in individual H. pylori strains. Whole-genome sequencing has yielded novel understanding of genotype-phenotype relationships.
Highlights
Clarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori
Allele-specific PCR (AS-PCR) results were in agreement with the outcome of eradication therapy based on clinical information
When all data were mapped to the theoretical nucleotide length of ATCC26695 (1,667,867 bp) using Genomics Workbench 6.0.1, the total consensus coverage (%) ranged from 87.38 to 99.99%, and average coverage was from 4.2- to 81.7-fold
Summary
Clarithromycin (CLR) is the key drug in eradication therapy of Helicobacter pylori (H. pylori) infection, and widespread use of CLR has led to an increase in primary CLR-resistant H. pylori. The known mechanism of CLR resistance has been established in A2146G and A2147G mutations in the 23S rRNA gene, but evidence of the involvement of other genetic mechanisms is lacking. It is accepted that triple therapy should not be used to manage H. pylori infection when the prevalence of CLR resistance is higher than 15–20% [3]; in such cases, other therapeutic alternatives should be considered. Acquired resistance of CLR in H. pylori has been associated with point mutations in the peptidyl transferase region of domain V of the 23S rRNA. CLR resistance seems to be related with other factors, such as rRNA methylase production [14], the actions of macrolide-inactivating enzymes, and active efflux pumps, which have been described in several bacterial species [15]. There is no genetic evidence for other bacterial factors in H. pylori
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