A core genome multilocus sequence typing scheme for Bacteroides fragilis : insights into population structure and genomic epidemiology

ABSTRACT Bacteroides fragilis strains are important members of the human gut microbiota and represent major opportunistic pathogens responsible for a wide range of anaerobic infections. Their clinical relevance is reinforced by diverse antimicrobial resistance mechanisms and a complex population structure comprising two major phylogenetic lineages, Division I and Division II. Although whole-genome sequencing is increasingly used for bacterial surveillance, no standardized core genome multilocus sequence typing (cgMLST) scheme is available for B. fragilis , limiting high-resolution and comparable genomic analyses. In this study, we developed a cgMLST scheme comprising 2,342 highly conserved loci applicable across both Divisions. The scheme was developed from 433 publicly available genomes with diverse geographic origins, sample types, and isolation periods. It was subsequently evaluated using 118 newly sequenced infection-associated isolates from France not included in scheme development. Clustering analysis identified an accurate single-linkage threshold of 400 allelic mismatches, providing balanced resolution for population structure analyses and high concordance with core genome phylogeny and classical MLST sequence types. Well-defined clusters were identified within both Divisions, with genetic profiles indicative of long-standing evolutionary divergence. These clusters showed no clear association with geographic origin, isolation source, or sampling period. Notably, some clusters were enriched in nim -positive and multidrug-resistant (MDR) isolates, including one in particular that emerged as a genetically divergent MDR-associated lineage within Division II. This cgMLST scheme provides a standardized and high-resolution framework for B. fragilis genotyping. Its implementation in the publicly accessible BIGSdb-Pasteur platform ( https://bigsdb.pasteur.fr/ ) will support global genomic surveillance and future epidemiological studies. IMPORTANCE Bacteroides fragilis strains hold a position of relevance within the human gut microbiota, as they represent a major cause of severe anaerobic infections. Defining genotypes is essential for tracking infections and monitoring the emergence and spread of antimicrobial resistance. In this study, we developed a core genome multilocus sequence typing scheme for B. fragilis and applied it to a large collection of isolates from diverse origins. Our results reveal a structured population with well-defined and genetically conserved clusters across both Divisions, consistent with a long-standing population structure. Notably, several clusters were enriched in antimicrobial resistance determinants and multidrug-resistant isolates. This study also provides an easy-to-use resource for global surveillance and future epidemiological investigations of this clinically important pathogen.