Reclassification of Borrelia Spp. Isolated in South Korea Using

Jpn. J. Infect. Dis., 71, 350–353, 2018

Original Article Reclassification of Borrelia spp. Isolated in South Korea Using Multilocus Sequence Typing Kyung-Hee Park1, Yeon-Joo Choi1,2, Jeoungyeon Kim1,2, Hye-Jin Park1,2, Dayoung Song1,2, and Won-Jong Jang1,2* 1Department of Microbiology and 2Institute of Glocal Disease Control, College of Medicine, Konkuk University, Seoul, Korea

SUMMARY: Here, we used multilocus sequence typing (MLST) to evaluate 3 intergenic genes (16S rRNA, ospA, and 5S-23S IGS) in Borrelia isolated from South Korea to analyze the relationships between host, vector, and molecular background. We identified B. afzelii, B. yangtzensis, B. garinii, and B. bavariensis. This study is the first report for the identification of B. yangtzensis using MLST in South Korea.

related to B. tanukii and B. valaisiana (23). We postu- INTRODUCTION lated that such reclassifications can be explained by both Lyme disease is a common arthropod-borne epidemic the discovery and addition of new reference species as inflammatory disorder prevalent in Europe and North well as the development of experimental techniques. America (1,2). It is a multi-systemic zoonotic disorder Multilocus sequence typing (MLST) was originally caused by motile, helically-shaped Borrelia spp. bacteria proposed as a tool for the epidemiological study of infec- (3,4). Borrelia spp. is comprised of 2 major clades that tious diseases (24,25). MLST has been shown to be a form a monophyletic group in the Order Spirochetes (5). powerful and pragmatic molecular method for typing The Lyme borreliosis B. burgdorferi (sensu lato), trans- large numbers of microbial strains for population genetic mited by various species of Ixodid ticks, forms one studies, delineation of species, and assignment of strains monophyly. Relapsing fever Borrelia, mostly transmitted to defined bacterial species (26–31). Single-locus ap- by Argasid ticks, a few species of Ixodid ticks, and lice, proaches may result in distorted evolutionary patterns forms the other monophyly (6–11). There are several due to different evolutionary processes on different re- species in the genus Borrelia, all of which are parasites, gions of the genome. In contrast, MLST may more accu- either as specialists or generalists, on one or more verte- rately reflect evolutionary relationships (29,32), and has brate species, and are transmitted between these hosts by been advocated as an alternative method to determine a hematophagous arthropod (12). Borrelia spp. (26) because of its simplicity compared to Cases of Lyme disease have been widely reported in traditional DNA-DNA hybridization. A previous study North America, Europe, and East Asian countries includ- used MLST to define B. bavariensis as a new species ing South Korea (13–19). In South Korea, 4 isolates of that was sufficiently genetically distinct from B. garinii. Borrelia spp. were initially identified as both B. afzelii Another study reported that Japanese isolates of B. and B. garinii using polymerase chain reaction (PCR) valaisiana-like strains were closely related to Chinese analysis (20). Subsequently, 15 more Borrelia spp. isolates of B. yangtzensis, forming a monophyletic group strains were isolated in Korea, which included HN6, (33). HN7, HN8, HN9, HNM14, and HNM19 (21). All of However, MLST has not yet been used to study these strains were identified as B. garinii, except for Korean Borrelia spp. although it has been applied to HN9, which was identified as B. afzelii using PCR Campylobacter spp. and Staphylococcus aureus (34,35). analysis. However, when these isolates were classified In this study, we used an MLST scheme based on bac- by rRNA gene restriction fragment length polymor- terial housekeeping genes as described previously (26) phisms (RFLPs), HN6, HN8, HNM14, and HNM19 in addition to previously studied 16S rRNA, 5S-23S exhibited some degree of variation from the previously intergenic spacer (IGS), and the gene encoding the outer known reference strain, indicating that they may be a surface protein A (ospA) in the first examination of the new strain (22). A UPGMA phylogeny constructed based evolutionary position of Korean Borrelia strain isolates. on 16S rRNA further supported the view that HN6 and The data suggest the reclassification and possible mis- HN8 were clearly separated from B. garinii but closely identification of the previously studied strains.

Received March 27, 2018. Accepted May 11, 2018. MATERIALS AND METHODS J-STAGE Advance Publication May 31, 2018. DOI: 10.7883/yoken.JJID.2018.139 Strains and DNA extraction: Three samples (HN7, *Corresponding author: Mailing address: Department of HN9, and KW3) were isolated from Ixodes granulatus, Microbiology, College of Medicine, Konkuk University, and the remaining 4 samples (HN12, HN13, HNM17, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Korea. and KM4) were separated from Apodemus agrarius and Tel: +82-2-2030-7816, Fax: +82-2-2030-7845, E-mail: named according to the name of the locality where they [email protected] were found. Five Hae Nam strains (HN7, HN9, HN12,

350 Reclassification of Borrelia in South Korea

HN13, and HNM17), one Kangwon strain (KW3), and at the Borrelia MLST online under ID 2032–2036. for characterization. Genomic DNA was extracted and Sequence alignment and phylogenetic trees: Multi- purified as described previously (20,36). ple sequence alignment was generated with the ClustalW MLST and sequencing: The Borrelia MLST scheme algorithm and MegAlign software by manual inspection. suggested previously (26) is comprised of 8 housekeep- The alignment was made on the translated amino acid ing genes (clpA, clpX, pepX, pyrG, nifS, recG, rplB, and sequences and then back-translated to nucleotide se- uvrA). The PCR conditions for the housekeeping genes, quences to ensure in frame alignment. except for recG, were as follows: 95°C for 15 min, 94°C To construct the phylogenetic tree, the maximum like- for 30 s, annealing temperature from 55°C to 48°C for lihood method based on the Tamura-Nei model of nucle- 60 s, and an extension step of 72°C for 60 s. An addi- otide substitution was determined. An initial tree was tional 30 cycles were performed at 94°C for 30 s, anneal- obtained by applying the neighbor-joining and BioNJ ing temperature of 48°C for 60 s, and extension at 72°C algorithms to a matrix of pairwise distances estimated for 60 s. After a final extension step for 5 min at 72°C, using the maximum composite likelihood approach, and the samples were kept at 15°C until further analysis. For then selecting the topology with superior log likelihood recG, the PCR conditions for the first set of cycles were value. Evolutionary analyses were conducted in 95°C for 15 min; followed by 30 cycles at 94°C for 30 s, MEGA6, and bootstrap with 1,000 replications was 55°C for 30 s, 72°C for 30 s; and a final extension at performed. 72°C for 5 min. The PCR reactions were performed with a VertiTM 96 well Thermal Cycler (Applied Biosystems, RESULTS Foster City, CA, USA). PCR products of all housekeeping genes were sequenced in the forward and reverse Identification of Borrelia isolates: The partial se- directions (Sequencing was performed by Bioneer Co. quence of the 16S rRNA gene (1,482 bp) of the isolates Ltd., Daejeon, South Korea). The obtained sequences was identical to those of B. burgdorferi (HN13), B. val- were compared and aligned with reference Borrelia spp. aisiana (HN7, HN9, HN12, HNM17, and KM4) and B. gene sequences deposited in the GenBank database to garinii (KW3). Different and partial 5S-23S IGS gene identify published sequences with a high degree of simi- (350 bp) sequences were identical to those of B. valaisi- larity using a BLAST search. MLST sequences were ana (HN12), B. garinii (HN13 and KW3), and B. afzelii compared with the described alleles in the B. burgdorferi (HN7, HN9, HNM17, and KM4). In addition, the ospA MLST database , and new al- gene (402 bp) sequences were identical to those of B. leles were assigned arbitrary number designations. Al- valaisiana (HN12), B. garinii (HN13 and KW3), and B. lelic profiles already present in the B. burgdorferi MLST afzelii (HN7, HN9, HNM17, and KM4) (Table 1). database were assigned the corresponding sequence type MLST results: In this study, MLST analysis identi- (ST) number. Novel combinations of allele were as- fied 7 samples, 2 distinct STs, and 5 new ST profiles signed ST numbers, and the sequence data are available (Table 2). Briefly, HN7, HN9, HNM17, and KM4 were

Table 1. Summary of Borrelia genospecies by MLST and 3 genes typing Strain 16S rRNA1) Genospecies2) ospA1) Genospecies2) IGS1) Genospecies2) MLST type HN7 1 B. afzelii 3 B. afzelii 1 B. afzelii B. afzelii HN9 1 B. afzelii 3 B. afzelii 1 B. afzelii B. afzelii HN12 11 B. burgdorferi 23 B. valaisiana 31 B. valaisiana B. yangtzensis HN13 15 B. garinii 41 B. garinii 13 B. garinii B. garinii HNM17 11 B. burgdorferi 3 B. afzelii 1 B. afzelii B. afzelii KW3 5 B. garinii 11 B. garinii 15 B. garinii B. bavariensis KM4 11 B. burgdorferi 3 B. afzelii 1 B. afzelii B. afzelii 1): Alleles of 16S rRNA, ospA, and 5S–23S IGS were assigned numbers based on Borrelia database. (http://www.pubMLST.org/bburgdorferi/). 2): Result of genospecies based on BLAST based on uploaded references.

Table 2. Borrelia MLST sequence types (ST) and 8 allelic profiles Strain Source ST1) clpA clpX nifS pepX pyrG recG rplB uvrA HN7 I. granulatus 7362) 241 202 185 214 220 231 195 215 HN9 I. granulatus 7362) 241 202 185 214 220 231 195 215 HN12 A. agrarius 152 83 61 59 74 69 63 62 65 HN13 A. agrarius 7372) 242 203 29 215 221 232 196 216 HNM17 A. agrarius 7382) 243 204 47 216 222 233 197 215 KW3 I. persulcatus 122 60 45 64 54 50 46 41 45 KM4 A. agrarius 7392) 243 205 47 57 220 234 198 28 1): STs are based on eight housekeeping genes according to the MLST website, www.mlst.net/borrelia.mlst. 2): Novel combination with ST number assigned.

351 Fig. 1. Phylogenetic analyses of concatenated sequences of 8 housekeeping gene fragments. Concatenated sequences of 8 housekeeping genes were aligned and analyzed in MEGA6. Phylogenetic tree was constructed using the Maximum Likelihood method. The percentage of trees in which the associated taxa clustered together is shown next to branches. Bootstrap test was for 1,000 repetitions (†, Novel ST). identified as B. afzelii, HN12 was identified as B. yangt- as B. garinii. The 16S rRNA gene typing identified 3 zensis, HN13 was identified as B. garinii, and KW3 was isolates as B. burgdorferi, 2 as B. afzelii, and 2 as B. identified as B. bavariensis. Interestingly, HN12 classi- garinii. This classification was likely due to mislabeling fied as B. valaisiana in the ospA and 5S-23S IGS analy- of sequences in GenBank. Indeed, the MLST analysis ses were re-classified as B. yangtzensis in the MLST on the 16S rRNA, ospA, and 5S-23S IGS sequences analysis (Tables 1 and 2). revealed that these isolates belonged to 4 species: B. Phylogenetic tree of Borrelia strains: The 7 Borrelia afzelii, B. garinii, B. yangtzensis and B. bavariensis, as strains were classified into 4 genospecies: B. afzelii shown by the phylogenetic analysis of 8 housekeeping (HN7, HN9, HNM17, and KM4,), B. yangtzensis loci. Using single locus analyses, KW3 was identified (HN12), B. bavariensis (KW3), and B. garinii (HN13) as B. garinii. However, this isolate was identified as B. (Fig. 1). In addition to the experimental samples, other bavariensis—a species most closely related to B. STs were inserted into groups (ST330, ST364, ST411, garinii—in the MLST analysis. ST18, ST400, ST133, ST585, ST106, and ST424) and Furthermore, a previous study has reported a high ge- the tree was rooted with sequences of the B. parkeri netic heterogeneity of Borrelia within each genospecies, (ST670). including B. burgdorferi, B. garinii, and B. afzelii (37). B. afzelii reportedly has the highest haplotype diversity, which greatly contributes to increased genetic diversity. DISCUSSION Presently, B. afzelii remains a potent genospecies (57.1%, The 7 strains (5 HN, one KW, and one KM) that we n = 4), which displayed genetic heterogeneity with mul- re-analyzed using MLST for the 16S rRNA, ospA, and tiple branches (Fig. 1). Of these 4 strains, HN7 and HN9 5S-23S IGS sequences that have been previously classi- showed the same ST profile (241-202-185-214-220-231- fied through various analyses. For example, one study 195-215), and the rest were analyzed to have different identified HN7 and HN12 as B. lusitaniae and B. valaisi- ST profiles (Table 2). This genetic information may also ana, HN9 and KM4 strains as B. afzelii, and HN7, appear for other types of ST phenotypes according to HNM17, and KM4 as B. afzelii based on a groEL RFLP reservoir. For example, I. granulatus (HN7, HN9) analysis (21). showed the same phenotype as ST706, but A. agrarius To corroborate the affiliation of Borrelia isolates from (HNM17, and KM4) showed different ST phenotypes. South Korea, we performed MLST/16S rRNA, ospA, Originally found in Europe, B. valaisiana is mainly and 5S-23S IGS using conserved housekeeping loci transmitted by I. ricinus and birds (33). However, (intra-species level) and inter-species levels, respec- slightly different B. valaisiana-related species were iden- tively. Typing using 5S-23S IGS and ospA genes identi- tified as B. yangtzensis in different reservoir hosts (38). fied 4 isolates as B. afzelii, one as B. valaisiana, and one As found presently, HN12 has been identified as B.

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