New Genotypes and Diversity of Orientia tsutsugamushi Isolated from Scrub Typhus Patients in South Korea as Determined by Multilocus Sequence Typing

Orientia tsutsugamushi, an obligate intracellular organism, is the causative agent of scrub typhus, which is endemic in the Asia-Pacific region. No comparative studies on the genotypic properties of O. tsutsugamushi have been performed using multilocus sequence typing (MLST) in South Korea. Here, we characterized 51 clinical isolates from Jeonju, in southwestern Korea, and we compared them to isolates from Thailand, Laos, and Japan. We also identified 10 new alleles and six novel sequence types. Overall, our results suggest that the relative genetic stability and the clonal populations of O. tsutsugamushi strains in South Korea have remained mostly conserved. Author summary Scrub typhus is a life-threatening disease, caused by infection with O. tsutsugamushi, a Gram-negative intracellular bacterium. Approximately one million people are infected globally every year, especially in the Asia-Pacific region. Strains of O. tsutsugamushi are typically distinguished serologically on the basis of sequences of the highly polymorphic 56-kDa outer membrane protein. Multilocus sequence typing (MLST) is a generic typing method that provides a unified bacterial isolate characterization approach that can be used for evolutionary and population studies of bacteria. In this study, we describe the development and application of a MLST scheme that was applied to 51 O. tsutsugamushi isolates. We found 10 new alleles and six new STs, which yielded a total of seven O. tsutsugamushi STs in South Korea. Among seven different STs (ST 48, 93-98), ST 48 account for the largest proportion (49.0%) of O. tsutsugamushi STs in South Korea. With the exception of the appearance of six novel STs, the clonal populations have remained conserved but further study of population structure and evolutionary trends is warranted.


Introduction
Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi, which is transmitted by a chigger bite [1]. The clinical course of scrub typhus can range from a self-limiting to a fatal illness with an estimated mortality of 6.0% (median, range 0%-70.0%) [2]. Disease severity appears to be strain dependent, although the basis for this is unknown and is multifactorial [3,4]. tsutsugamushi isolated in Thailand was first proposed in 2010 [5]; in the past decade, only two studies have been conducted that used an MLST approach for O. tsutsugamushi typing; they evaluated isolates from Cambodia and Laos [4,6].
Here, using an MLST approach, we present the results of a prospective study to determine the geographic structure of O. tsutsugamushi isolated from scrub typhus patients in South Korea and we conducted a comparative analysis with previous data collected from Thailand, Laos, and Japan.

Patients and data collection
A prospective study was conducted in two tertiary hospitals in Jeonju, in southwestern Korea, between January 2016 and December 2017. Patients ≥18 years old who were clinically suspected of having scrub typhus were eligible. Laboratory diagnosis of scrub typhus was made according to one of the following criteria: (1) increase in indirect immunofluorescence assay

DNA extraction from scrub typhus patients
Whole blood samples from scrub typhus patients were collected and peripheral blood mononuclear cells (PBMC) were isolated using Lymphoprep ™ density gradient medium and SepMate ™ tubes (Stemcell Technologies, Vancouver, Canada). The isolated PBMC were aliquoted and stored at -80°C.
After isolation, DNA was purified using the QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions [7]. Purified DNA was aliquoted and GAT GCA CTA TTA GGC-3') were used in the second PCR amplification to generate a 483bp fragment. Nested PCR was performed as described by Lee et al. [8]. The amplified PCR products were confirmed by agarose gel electrophoresis and purified from the agarose gel using the QIAquick gel extraction kit (QIAGEN) and the Expin TM Gel SV kit (GeneAll, Seoul, Korea). Each PCR product was analyzed and confirmed by sequencing.

Multilocus sequence typing (MLST) and data analyses
Genomic DNA of O. tsutsugamushi was characterized using MLST as previously described [5]. Housekeeping genes (gpsA, mdh, nrdB, nuoF, ppdK, sucB, sucD) were amplified by PCR and sequenced in forward and reverse directions with their associated primers (Supplementary Table S1). The amplified PCR products were confirmed by agarose gel electrophoresis, purified, and sequenced.
Sequence data and chromatograms were edited and analyzed using Bioedit 7.0.4. Allele numbers were assigned by querying the PubMLST database, and new alleles and profiles were submitted to the PubMLST database to obtain new allele and ST numbers (http://pubmlst.org/otsutsugamushi/). The relationships between sequence types (STs) were visualized using goeBURST [9]. The ratio of non-synonymous to synonymous nucleotide substitutions (dN/dS) of the partial sequences of seven housekeeping genes were analyzed and calculated using START2 (http://pubmlst.org/software/analysis/start2/) [10]. The phylogenetic tree of the STs was constructed based on 2,700 base pairs (bp) from concatenated sequences of all genes analyzed in specific order-gpsA-mdh-nrdB-nuoF-ppdK-sucB-sucD-using the neighbor-joining tree-building algorithm implemented in MEGA X [11]. We calculated the Fstatistic (F ST ) value, which indicates the degree of genetic differentiation and gene flow among populations from different geographical locations [12]. The index of diversity (D), as defined by Simpson [13], was estimated using a previously published formula [14].   Table   3). The MLST dendrogram generated by concatenating the seven housekeeping gene sequences (2,700 bp) showed 288 polymorphic sites. Regarding our findings, moderate nucleotide diversity of all sequenced genes was observed (π = 0.01 for mdh, nrdB, sucB, and sucD; 0.02 for gpsA; 0.03 for nuoF and ppdK) ( Table 3).
Tajima's D value is a widely used test of neutrality in population genetics. In this study, Tajima's D value for the dataset containing all O. tsutsugamushi isolates yielded negative values, which indicate a high level of low frequency polymorphisms compared with the expected level in a neutral model.
The ratio of dN/dS was much lower than for the seven housekeeping genes, and it varied from 0.0011 for nrdB to 0.2790 for sucD (Table 3). In the South Korea isolates, the dN/dS ratio corresponded to a range of 0.0000-0.3593 (Table 4). These results suggest that all the housekeeping genes are predominantly evolving by purifying selection. These also showed that synonymous substitutions predominated over nonsynonymous substitutions for all O.
tsutsugamushi genes we tested. We noticed that the dN/dS ratios for nrdB and sucD were lower than for the other genes. These results indicate that housekeeping genes are under strong selective constraint in the O. tsutsugamushi genome.

Discussion
In In conclusion, this study demonstrated the relative genetic stability of O. tsutsugamushi strains in South Korea over the study period. With the exception of the appearance of six novel STs, the clonal populations have remained conserved but further study of population structure and evolutionary trends is warranted.