Whole Genome Characterization of Leptospira kirschneri Serogroup Pomona in Croatia: Insights into Its Diversity and Evolutionary Emergence

Leptospirosis is a worldwide zoonosis caused by pathogenic <i>Leptospira</i> spp. with small rodents serving as the main reservoir. In Croatia, the serogroup Pomona has been detected most frequently, but its genomic diversity remains insufficiently characterized. This study presents the first whole genome sequencing analysis of 48 Croatian <i>Leptospira</i> spp. isolates collected from small rodents over a 14-year period. Serological typing confirmed that all the isolates belonged to the serogroup Pomona. Genomic analysis assigned them to <i>L. kirschneri</i> based on high genomic similarity using average nucleotide identity (ANI). The isolates were assigned to ST-98 using traditional multilocus sequence typing (MLST), while cgMLST identified seven genotype clusters, many of which showed geographic structuring. Phylogenetic analyses based on single nucleotide polymorphisms (SNPs) supported this structure and revealed a monophyletic clade of Croatian isolates distinct from other global <i>L. kirschneri</i> strains. Serological typing, MLST, and phylogenetic clustering support classification of the isolates as <i>L. kirschneri</i>, serogroup Pomona, most likely serovar Mozdok, although serovar Tsaratsovo cannot be excluded. These results indicate the existence of a geographically restricted and potentially host-adapted lineage of <i>L. kirschneri</i> in Croatia. The integration of ecological, serological, and genomic data in this study emphasizes the value of whole genome sequencing for understanding the population biology of <i>Leptospira</i> spp. serogroup Pomona. Moreover, it supports targeted, country-specific surveillance and control strategies for leptospirosis through the identification of circulating serovars and species in reservoir hosts, in line with a One Health approach.