TITLE:
Stability of Helicobacter pylori Resistance to Clarithromycin
AUTHORS:
Esther Nina Ontsira Ngoyi, Elvire Berthenet, Lucie Benejat, Claire Guilloteau, Jamunarani Vadivelu, Khean Lee Goh, Philippe Lehours, Ange Antoine Abena, Matthew D. Hitchings, Ben Pascoe, Sam Sheppard, Francis Mégraud
KEYWORDS:
H. pylori, Clarithromycin, Competition, Compensatory Mutation
JOURNAL NAME:
Open Journal of Medical Microbiology,
Vol.15 No.4,
December
11,
2025
ABSTRACT: The aim of this work was to study the competition between Helicobacter pylori isolates susceptible to clarithromycin and their resistant mutants, in terms of growth, by co-culture over an extended time period and to look for compensatory mutations eventually occurring in resistant mutants by whole genome sequencing. A mixture of susceptible and resistant isolates to clarithromycin was detected in gastric biopsies from two patients by real-time FRET-PCR and the H. pylori isolates from these two patients were obtained by culture on blood agar with and without macrolide supplement and were named 3657S/3657R and 3695S/3695R. Random amplified polymorphism DNA confirmed that the susceptible and resistant isolates belonged to the same strain and sequencing showed that the 23S rDNA mutations were A2143G (isolate 3657) and A2142G (isolate 3695). The susceptible isolate and its resistant mutant were cultured in the short term in brucella broth where the resistant mutants grew faster, and co-cultured over the long term (30 - 40 days) with AGS gastric eucaryotic cells. Although after 15 days, the growth of the resistant mutant was better than the growth of the susceptible isolate in both pairs, after 30 or 40 days a difference was observed: the growth of the resistant mutant was still better for strain 3657 (R/S ratio: 1.7) but not for strain 3695 (R/S: 0.1). Whole genome sequencing indicated that resistant and susceptible isolates of the same strain clustered closely on a phylogenetic tree. However, concerning strain 3657, changes were detected in two genes between the resistant mutant studied at the start and the same mutant after 40 days of co-culture: HP-0316, a hypothetical protein and HP-1342, a putative outer membrane protein. These changes may correspond to putative compensatory mutations. In conclusion, different molecular adaptations may occur following the selection of an H. pylori mutant resistant to macrolides, confirming the in vivo observation.