To characterize two uncommon, patient-derived *Orientia tsutsugamushi* strains from South Korea and define their relationships with established reference strains to expand genomic resources for molecular surveillance and diagnostic evaluation.
Approach:
Bacterial reactivation and identification: Two archived isolates, Chuncheon17-2 and Chuncheon17-13, obtained from patients with scrub typhus in 2017, were reactivated in Vero cells and preliminarily identified using 56-kDa type-specific antigen (TSA) gene PCR and Sanger sequencing.
Whole-genome sequencing: Hybrid sequencing using PacBio HiFi and Illumina platforms was followed by genome assembly, annotation, 56-kDa TSA sequence analysis, phylogenetic reconstruction, average nucleotide identity analysis, repeated coding-sequence and insertion-sequence–related profiling, and whole-genome dot-plot comparisons.
Key Findings:
Both strains were assembled as single circular chromosomal contigs and exhibited highly repetitive genomes.
The 56-kDa TSA analysis linked Chuncheon17-2 with Je-Cheon and Chuncheon17-13 with Young-Worl, although neither strain was identical to its corresponding reference sequence.
Core-genome analysis placed Chuncheon17-13 near the Ikeda and Kato strains, whereas Chuncheon17-2 formed a distinct genome-wide lineage, indicating that 56-kDa TSA relationships did not fully reflect genome-wide phylogeny.
Neither strain had an average nucleotide identity above 99.5% with any available genome, indicating that neither was nearly identical to currently available *O. tsutsugamushi* genomes.
Repetitive coding-sequence and insertion-sequence–related profiles differed between strains, suggesting variation in mobile element–associated genome architecture.
Interpretation:
These genomes expand resources for uncommon South Korean *O. tsutsugamushi* lineages and demonstrate that whole-genome sequencing can complement 56-kDa TSA genotyping by resolving broader phylogenetic relationships, genome structure, and repeat-associated features. The biological significance of the observed mobile element–associated variation remains undetermined.
Limitations:
Whole-genome sequences for the closest 56-kDa TSA-related strains, Je-Cheon and Young-Worl, were unavailable, restricting genome-wide comparisons to other available reference genomes.
Repeat and mobile element analyses relied primarily on annotation-derived coding-sequence features and could not fully resolve repeat architecture or exact insertion-sequence and RAGE boundaries.
Only two uncommon strains were newly sequenced, making associations between mobile element variation, genome plasticity, and strain diversification hypothesis-generating.
Detailed patient data and direct assessments of virulence, growth kinetics, host-cell interactions, and clinical severity were unavailable, preventing clinical correlations.
Conclusion:
Whole-genome characterization identified Chuncheon17-2 and Chuncheon17-13 as genomically distinct South Korean strains. Their sequences provide reference data for future genomic surveillance, diagnostic evaluation, and studies integrating strain diversity with clinical characteristics.
