Genomic Epidemiology and Microevolution of the Zoonotic Pathogen Corynebacterium ulcerans

Corynebacterium ulcerans is an emerging zoonotic pathogen that belongs to the Corynebacterium diphtheriae (Cd) species complex (CdSC), and that causes diphtheria-like infections in humans. Our understanding of the transmission, phylogeography and evolution of C. ulcerans remains limited, in part due to the lack of a standardized genomic epidemiology toolkit. The aim of this work was to develop a core genome multi-locus sequence typing (cgMLST) scheme for high-resolution genotyping and classification of C. ulcerans strains, and to explore transmission, spatial spread and genomic evolution among 582 C. ulcerans isolates from sporadic clinical cases and reported case clusters. The cgMLST scheme combines 1,628 loci with highly reproducible allele calls and shows high strain subtyping resolution. We demonstrate its utility for capturing population structure by defining sublineages (SL, maximum 940 allele differences) and clonal groups (CG, 194 allele differences, AD) and for epidemiological surveillance by defining genetic clusters, i.e., previously undetected chains of transmission (25 AD). Genetic clusters correspond to cryptic and case clusters that were associated with specific geographical regions within France. Major C. ulcerans sublineages (SL325, SL331, SL339) and clonal groups (CG325, CG331, CG583) showed strong associations with diphtheria toxin variants and tox-carrying prophages or other genetic elements. The evolutionary dynamics of tox gene presence or absence varied sharply among clonal groups. The cgMLST scheme is publicly available (https://bigsdb.pasteur.fr/diphtheria) and provides a common framework for investigating the ecology, evolution and variations in virulence among C. ulcerans strains. The implementation of a standardized high-resolution genotyping method will also facilitate the tracing of C. ulcerans transmission and spread across hosts and from local to global spatial scales.