Whole-genome sequencing of clarithromycin resistant Helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes

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.

Genetic variants of Helicobacter pylori type IV secretion system components CagL and CagI and their association with clinical outcomes

“…Consistent with our earlier report, the WGS data in this study had high sequencing coverage, and were of sufficiently high quality to allow detection of SNVs in the H. pylori genome [20]. …”

“…Using WGS, we and others detected potential mutations throughout the H. pylori genome and identified variants when sequence changes were present [20, 24, 25]. Here, we used WGS technology to detect novel variants in uncharacterized cag PAI genes associated with H. pylori pathogenicity.…”

In Vitro Activity of Sertraline, an Antidepressant, Against Antibiotic-Susceptible and Antibiotic-Resistant Helicobacter pylori Strains

“…For other antibiotics, an additivity with SER may be related to an interference with efflux pumps and thus a sensitization of H. pylori to these substances. The participation of efflux pumps in antibiotic resistance of H. pylori has been demonstrated in many studies [65][66][67][68][69][70][71][72][73], while the use of efflux pump inhibitors (carbonyl cyanide m-chlorophenylhydrazone and Phe-Arg-beta-naphthylamide) correlated with an increased sensitivity to antibiotics [66,69,70,73].…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…Intriguingly, hefC missense mutations were among the several gene mutations in in vitro-selected isolates with high-level resistance to amoxicillin, and the introduction of a mutated allele to the wild type increased resistance (689). Through examining the whole-genome sequences of clarithromycin-resistant isolates, a new study revealed the gene clusters of TolC homologs involved in clarithromycin susceptibility profiles in individual isolates (690). Given the extremely acidic environments where H. pylori colonizes, the effect of this environment on secondary transporters in in vivo antimicrobial resistance remains to be determined.…”