Amplified fragment length polymorphism genotyping of metronidazole-resistant Helicobacter pylori infecting dyspeptics in England

Abstract

Intra-specific diversity of Helicobacter pylori infecting stomachs of different individuals was investigated by numerical analysis of amplified fragment length polymorphisms (AFLP), to determine the existence of clones within the strain population and the effect that antibiotic treatment, particularly with metronidazole (Mtz), had on the balance of types/subtypes present before and after treatment. The 92 cultures studied comprised 89 single or multiple (pre- and post-treatment) isolates from gastric biopsies from 35 dyspeptic patients at two geographical locations in England, and three reference strains. HindIII restriction fragments tagged with specific adaptors were used as template DNA for AFLP. Patterns were coded in binary format according to deduced sizes of amplified fragments, and numerical analysis was performed. H. pylori isolated from different individuals were highly diverse (43 AFLP types) with a continuum of similarities that included three putative strain clusters at the 55% similarity level. Twelve sets each comprised identical isolates but subclonal variants with similarities of 82-99% coexisted in isolate sets from 19 patients. Seven sets contained strains with different AFLP types which for several corresponded with vacA/cagA genotypic differences. Mtz resistance was a feature of clonal as well as unrelated isolates. AFLP profiling was a robust, reproducible and highly discriminatory means of indexing H. pylori strain diversity, and the numerical analysis enabled clonal/subclonal variants infecting an individual to be defined and contrasted with the general species diversity. The majority (65%) of patients had co-infections with different strain types/subtypes but antibiotic treatment apparently did not markedly modify H. pylori population diversity in individual stomachs. Mtz sensitivity was generally associated with greater strain diversity as several subtypes often coexisted in sensitive pretreatment strain sets. In contrast, Mtz-resistant strain populations were less diverse, which was attributed to selection by previous exposure to nitroimidazoles in the same or a different host.