Evolution of Amoxicillin Resistance ofHelicobacter pyloriIn Vitro: Characterization of Resistance Mechanisms

Abstract

Helicobacter pylori is the major cause of peptic ulcers and gastric cancer in humans. Treatment involves a two or three drug cocktail, typically including amoxicillin. Increasing levels of resistance to amoxicillin contribute to treatment failures, and higher levels of resistance are believed to be due to multiple genetic mutations. In this study, we examined the progression of spontaneous genetic mutations that contribute to amoxicillin resistance in H. pylori when exposed to increasing concentrations of amoxicillin in vitro. During the selection process, we isolated five strains each of which had progressively higher levels of resistance. Using a whole genome sequencing approach, we identified mutations in a number of genes, notably pbp1, pbp2, hefC, hopC, and hofH, and by sequencing these genes in each isolate we were able to map the order and gradual accumulation of mutations in these isolates. These five isolates, each expressing multiple mutated genes and four transformed strains expressing individually mutated pbp1, hefC, or hofH, were characterized using minimum inhibitory concentrations, amoxicillin uptake, and efflux studies. Our results indicate that mutations in pbp1, hefC, hopC, hofH, and possibly pbp2 contribute to H. pylori high-level amoxicillin resistance. The data also provide evidence for the complexity of the evolution of amoxicillin resistance in H. pylori and indicate that certain families of genes might be more susceptible to amoxicillin resistance mutations than others.

Helicobacter pylori and Antibiotic Resistance, A Continuing and Intractable Problem

“…In combination with the amino acid substitutions of pbp1, hopB and hopC, mutations confer further AMX resistance [60]. A later study, however, did identify hefC as important here and was interpreted to mean that mutations in pbp1, hefC, hopC, hofH, and possibly pbp2 contribute to high-level resistance to AMX [65]. As omp25 acts as a porin, this change may be involved in increased AMX efflux as well [62,63].…”

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

“…In an interesting study, H. pylori cultured in the presence of cholesterol became resistant to bile salts and their analogs, and this phenotype required the HefC pump (688). 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).…”

Review article: the global emergence ofHelicobacter pyloriantibiotic resistance

“…Expression of active efflux pumps that excrete drugs and point mutations in the pbp1A gene may contribute to the mechanisms of resistance to beta‐lactams. Other mutations such as pbp2, hefC, hopC and hofH have been identified among H. Pylori ‐resistant strains …”