Genome-scale insights into the genetic diversity and pan-genome architecture of Orientia tsutsugamushi

Scrub typhus is a neglected vector-borne zoonosis caused by Orientia tsutsugamushi (OT), an obligate intracellular bacterium with a heterogeneous, repeat-rich genome. Molecular epidemiology of OT has long relied on single marker loci, but whether marker-based classifications reflect genome-wide evolutionary relationships remains unclear. We collected publicly available OT sequences of the three standard marker loci ( tsa56 , htrA , and groEL ) along with 19 quality-controlled genomes, and evaluated marker-based inferences against genome-scale references. We first compared the two most widely used markers in 35 isolates carrying both sequences: the tsa56 and htrA phylogenies were markedly incongruent (Mantel ρ = 0.103, P = 0.433; normalized Robinson–Foulds (RF) distance = 0.938). We then evaluated whether any of the three marker loci recovered the core-genome topology across the same genomes. None did (normalized RF distance = 0.750–0.800). The conserved loci groEL and htrA retained significant genome-wide distance signals (Mantel ρ = 0.845 and 0.613, both P < 0.001), whereas tsa56 did not (Mantel ρ = 0.146, P = 0.313). Core-genome SNP and core-protein phylogenies showed a significant positive correlation (Mantel ρ = 0.625, P < 0.001) but different topologies (normalized RF distance = 0.500). Pan-genome analysis identified 4,350 gene clusters (13.8% core) with an open accumulation curve; most accessory (60.1%) and strain-specific (81.3%) genes were uncharacterized. Accessory-gene repertoires were highly heterogeneous, yet showed no statistically detectable structuring by geographic origin or core-genome lineage. Marker-based classifications of OT are strongly locus-dependent and show only weak genome-wide topological concordance. Genotype labels and strain relationships should therefore be interpreted within a locus-specific framework and should not be considered directly interchangeable across loci. In these small matched genome sets, groEL and htrA better reflected genome-wide pairwise distances than tsa56 . Accessory-gene composition showed no statistically detectable association with core-genome relatedness or geographic origin in the present dataset. Together with the open, accessory-dominated pan-genome, these findings provide a curated overview of OT diversity in public datasets and a reproducible framework for future surveillance.