Genetic Diversity and Spatial Distribution of Yersinia pestis by Core Genome-Based Multilocus Sequence Typing Analysis

Yersinia pestis is the etiological agent of plague, a severe and often fatal disease in humans when left untreated. Because of the high genetic clonality of Y. pestis, high-resolution genotyping assays are necessary to differentiate between individual strains. Here, we report on the development and validation of a robust and reproducible core-genome multilocus sequence typing (cgMLST) assay for Y. pestis comprising 3139 gene targets, enabling high-resolution typing at the strain level. The assay was validated using 222 publicly available Y. pestis genomes, including 45 recently sequenced outbreak isolates from Madagascar and 21 isolates from Mongolia. The cgMLST analysis revealed primary clustering aligned with known biovar-associated branches and sub-branches. Additional geographically structured sub-clusters illustrate its application for regional diversification analysis. Yersinia pestis strains from different geographic regions were clearly distinguished, consistent with spatial clustering. Within the analyzed dataset, closely related or epidemiologically linked strains differed by zero to three alleles, suggesting this range as an operational reference for identifying highly similar isolates. The cgMLST showed clustering patterns concordant with previously described single-nucleotide polymorphism (SNP) assays. It therefore provides a standardized high-resolution typing approach, with demonstrated applicability for outbreak investigations, source tracking, and comparative genomic surveillance of Y. pestis.