Within-host diversity and phased variant analysis reveal structures and recombination of Helicobacter pylori subpopulations in stomach

Abstract Helicobacter pylori (H. pylori) has a highly plastic genome and can generate substantial within-host diversity during chronic gastric colonization. However, the delineation of its within-host subpopulations, particularly regarding the emergence and spread of antibiotic resistance-conferring mutations, remains poorly understood. In this study, we enrolled 25 chronic gastritis patients from southern China, collecting multiple isolates from distinct gastric regions. Among them, 14 patients exhibited heterogeneity in antibiotic susceptibility across isolates (heteroresistant), while the remaining 11 showed consistent profiles (homoresistant). Using ultra-deep short- and long-read sequencing, we showed that co-existing H. pylori subpopulations were prevalent in these patients, particularly within the same anatomical niche. Two patients presented mixed infections involving different strains as subpopulations, while others exhibited microevolution from a common ancestor. We reconstructed the subpopulation structures and found that isolates from heteroresistant patients had greater within-host diversity compared to these from homoresistant patients. Notably, subpopulations in the antrum demonstrated higher diversity than those in the gastric corpus and incisura angularis. Through a custom-developed phasing bioinformatics workflow, we resolved subpopulation-level genomic regions and directly observed extensive homologous recombination among them. Importantly, we traced the distribution of levofloxacin- and clarithromycin-associated resistance mutations across subpopulations, which was mainly mediated by recombination. To our knowledge, this study provides the first detailed depiction of H. pylori subpopulation distribution within the human stomach, illustrating how recombination drives within-host diversification and contributed to the spread of antibiotic resistance mutations.