Helicobacter pylori, a dominant member of the gastric microbiota, shares co-evolutionary history with humans. This has led to the development of genetically distinct H. pylori subpopulations associated with the geographic origin of the host and with differential gastric disease risk. Here, we provide insights into H. pylori population structure as a part of the Helicobacter pylori Genome Project (HpGP), a multi-disciplinary initiative aimed at elucidating H. pylori pathogenesis and identifying new therapeutic targets. We collected 1011 well-characterized clinical strains from 50 countries and generated high-quality genome sequences. We analysed core genome diversity and population structure of the HpGP dataset and 255 worldwide reference genomes to outline the ancestral contribution to Eurasian, African, and American populations. We found evidence of substantial contribution of population hpNorthAsia and subpopulation hspUral in Northern European H. pylori. The genomes of H. pylori isolated from northern and southern Indigenous Americans differed in that bacteria isolated in northern Indigenous communities were more similar to North Asian H. pylori while the southern had higher relatedness to hpEastAsia. Notably, we also found a highly clonal yet geographically dispersed North American subpopulation, which is negative for the cag pathogenicity island, and present in 7% of sequenced US genomes. We expect the HpGP dataset and the corresponding strains to become a major asset for H. pylori genomics.

H. pylori (HP) genetics may determine its clinical outcomes. Despite high prevalence of HP infection in Latin America (LA), there have been no phylogenetic studies in the region. We aimed to understand the structure of HP populations in LA mestizo individuals, where gastric cancer incidence remains high. The genome of 110 HP strains from Mexico, Nicaragua and Colombia were analyzed with 59 worldwide available genomes. To study bacterial relationship on whole genome level we propose a virtual hybridization technique using thousands of high-entropy 13 bp DNA probes to generate fingerprints. Phylogenetic virtual genome fingerprint (VGF) was compared with Multi Locus Sequence Analysis (MLST) and with phylogenetic analyses of cagPAI virulence island sequences. With MLST some Nicaraguan and Mexican strains clustered close to Africa isolates, whereas European isolates were spread without clustering and intermingled with LA isolates. VGF analysis resulted in increased resolution of populations, separating European from LA strains. Furthermore clusters with exclusively Colombian, Mexican, or Nicaraguan strains were observed, where the Colombian cluster separated from Europe, Asia and Africa, while Nicaraguan and Mexican clades grouped close to Africa. In addition, a mixed large LA cluster including Mexican, Colombian, Nicaraguan, Peruvian and Salvadorian strains was observed; all LA clusters separated from the Amerind clade. With cagPAI sequence analyses LA clades clearly separated from Europe, Asia and Amerind and Colombian strains formed a single cluster. A NeighborNet analyses suggested frequent and recent recombination events particularly among LA strains. Results suggests that in the new world, H. pylori has evolved to fit mestizo LA populations, already 500 years after the Spanish colonization. This co-adaption may account for regional variability in gastric cancer risk.

Helicobacter pylori is a common component of the human stomach microbiota, possibly dating back to the speciation of Homo sapiens. A history of pathogen evolution in allopatry has led to the development of genetically distinct H. pylori subpopulations, associated with different human populations, and more recent admixture among H. pylori subpopulations can provide information about human migrations. However, little is known about the degree to which some H. pylori genes are conserved in the face of admixture, potentially indicating host adaptation, or how virulence genes spread among different populations. We analyzed H. pylori genomes from 14 countries in the Americas, strains from the Iberian Peninsula, and public genomes from Europe, Africa, and Asia, to investigate how admixture varies across different regions and gene families. Whole-genome analyses of 723 H. pylori strains from around the world showed evidence of frequent admixture in the American strains with a complex mosaic of contributions from H. pylori populations originating in the Americas as well as other continents. Despite the complex admixture, distinctive genomic fingerprints were identified for each region, revealing novel American H. pylori subpopulations. A pan-genome Fst analysis showed that variation in virulence genes had the strongest fixation in America, compared with non-American populations, and that much of the variation constituted non-synonymous substitutions in functional domains. Network analyses suggest that these virulence genes have followed unique evolutionary paths in the American populations, spreading into different genetic backgrounds, potentially contributing to the high risk of gastric cancer in the region.

Helicobacter pylori disturbs the stomach lining during long-term colonization of its human host, with sequelae including ulcers and gastric cancer^1,2. Numerous H. pylori virulence factors have been identified, showing extensive geographic variation¹. Here we identify a ‘Hardy’ ecospecies of H. pylori that shares the ancestry of ‘Ubiquitous’ H. pylori from the same region in most of the genome but has nearly fixed single-nucleotide polymorphism differences in 100 genes, many of which encode outer membrane proteins and host interaction factors. Most Hardy strains have a second urease, which uses iron as a cofactor rather than nickel³, and two additional copies of the vacuolating cytotoxin VacA. Hardy strains currently have a limited distribution, including in Indigenous populations in Siberia and the Americas and in lineages that have jumped from humans to other mammals. Analysis of polymorphism data implies that Hardy and Ubiquitous coexisted in the stomachs of modern humans since before we left Africa and that both were dispersed around the world by our migrations. Our results also show that highly distinct adaptive strategies can arise and be maintained stably within bacterial populations, even in the presence of continuous genetic exchange between strains.

We conducted a global-scale study to identify H. pylori antimicrobial-resistant genes (ARG), address their global distribution, and understand their effect on the antimicrobial resistance (AMR) phenotypes of the clinical isolates. We identified ARG using several well-known tools against extensive bacterial ARG databases, then analyzed their correlation with clinical antibiogram data from dozens of patients across countries. This revealed that combining multiple tools and databases, followed by manual selection of ARG from the annotation results, produces more conclusive results than using a single tool or database alone. After curation, the results showed that H. pylori has 42 ARG against 11 different antibiotic classes (16 genes related to single antibiotic class resistance and 26 genes related to multidrug resistance). Further analysis revealed that H. pylori naturally harbors ARG in the core genome, called the ‘Set of ARG commonly found in the Core Genome of H. pylori (ARG-CORE)’, while ARG-ACC—the ARG in the accessory genome—are exclusive to particular strains. In addition, we detected 29 genes of potential efflux pump-related AMR that were mostly categorized as ARG-CORE. The ARG distribution appears to be almost similar either by geographical or H. pylori populations perspective; however, some ARG had a unique distribution since they tend to be found only in a particular region or population. Finally, we demonstrated that the presence of ARG may not directly correlate with the sensitive/resistance phenotype of clinical patient isolates but may influence the minimum inhibitory concentration phenotype.

The increasing antibiotic resistance of Helicobacter pylori has had a serious impact on gastric cancer prevention. Our study aimed to profile the genomic characteristics and explore variants associated with resistance in H. pylori strains from a high-risk area of gastric cancer in China. We isolated 153 strains from a community-based cohort and assessed their susceptibility to six antibiotics by MIC Test Strip and genomic characteristics by whole-genome sequencing. Phylogenetic analysis identified the strains as an independent cluster within H. pylori East Asian population (hpEastAsia). HefA , an efflux pump gene, showed the highest differentiation in the Linqu strains compared with the other Chinese strains. Bacterial genome-wide association study (GWAS) identified 86 resistance variants covering 44 genes. Novel resistance variants were found in lon and babA for metronidazole, HP1168 for clarithromycin, hcpC for levofloxacin, and sabA for rifamycin. Two newly identified hefA mutations (R229K and A283V) showed significant associations with metronidazole ( P = 0.012) and tetracycline ( P = 0.044) resistance, respectively. HefA mutations and GWAS variants were integrated with the significant literature-reported mutations to optimize the prediction models for metronidazole, levofloxacin, clarithromycin, and tetracycline resistance with area under the receiver operating characteristic curves of 0.82–0.93. Double-antibiotic resistance models were established for clinical applicability. Furthermore, hefA expression may play a potential mediating role in the associations between mutations and resistance. This study identified genetic independence in the representative H. pylori strains from a high-risk area of gastric cancer. Optimized resistance prediction panels, including novel hefA mutations and GWAS variants, may provide preliminary guidance for localized precise treatment and helpful experiences for the similar high-risk populations. IMPORTANCE: Helicobacter pylori is a remarkable pathogen due to its virulence in gastric cancer and high genetic plasticity. Linqu County in China, a high-risk area of gastric cancer, faces serious antibiotic resistance issues and necessitates genomic profiling of local H. pylori strains. Phylogenetic analysis revealed the Linqu strains as a relatively independent cluster within the hpEastAsia population. Novel antibiotic resistance-associated hefA mutations and variants from our bacterial genome-wide association study in the Linqu strains were optimized to improve the prediction performances for single antibiotic and double-drug combination resistance compared with traditional literature-reported mutations. This study identified relative genetic independence and high differentiation in the representative H. pylori strains from a population with high risk of gastric cancer and high prevalence of antibiotic resistance. The optimized panels with novel variants improve antibiotic resistance prediction models compared with literature-reported mutations, providing guidance for localized precise treatment and suggesting prevention strategies for similar high-risk populations.