Phylogeographic diversity of <i>Orientia tsutsugamushi</i> strains from clinical isolates in South Korea

<i>Orientia tsutsugamushi</i>, the causative agent of scrub typhus, is endemic to the Asia-Pacific region. In South Korea, the Boryong strain is considered dominant; however, nationwide phylogeographic distribution and genetic diversity based on clinical isolates remain incompletely characterized. In this study, 121 <i>O. tsutsugamushi</i> clinical isolates were collected from scrub typhus patients at 11 hospitals across South Korea between 2015 and 2024. Isolates were genotyped using <i>56-kDa</i> gene sequencing and multilocus sequence typing (MLST) of seven housekeeping genes. Sequence analysis and phylogenetic reconstruction were performed using BLAST, PubMLST, BURST, MEGA11, DnaSP6, and R-based tools. Five <i>56-kDa</i> genotypes were identified: Boryong (93.4%), Ikeda, Je-cheon, Young-worl, and Yeo-joo. MLST revealed 11 sequence types (STs), including five novel STs. While the Boryong strain and related STs were distributed nationwide, minor strains showed restricted distribution in northern regions. Several isolates sharing the same <i>56-kDa</i> genotype exhibited different MLST STs, indicating possible recombination or local microevolution. This study provides the first nationwide MLST-based characterization of <i>O. tsutsugamushi</i> in South Korea and demonstrates the dominance of the Boryong strain alongside localized diversity. Our findings underscore the utility of MLST for higher-resolution typing and support the need for continued molecular surveillance to inform regional epidemiology and disease management.