Next Article in Journal
Description of a Virulent Systemic Feline Calicivirus Infection in a Kitten with Footpads Oedema and Fatal Pneumonia
Previous Article in Journal
Bovine Clinical E. coli Mastitis in Italian Dairy Herds Is Not Associated with a Specific Pathotype
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pathogens
Volume 14
Issue 11
10.3390/pathogens14111182
pathogens-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparative Genomics of Two Newly Sequenced Rodent-Derived and One Previously Reported Tick-Derived Borrelia garinii Strains from South Korea Reveals Plasmid Variation and Virulence Gene Diversity

by
Hyungsuk Kang
1,2,3,†,
Yeon-Joo Choi
1,2,3,†,
Ji-Young Park
4,†,
Kwangjun Lee
4 and
Won-Jong Jang
1,2,3,*
1
Department of Microbiology, Konkuk University School of Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea
2
Research Institute of Medical Science, Konkuk University School of Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea
3
Institute of Biomedical Sciences & Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea
4
Division of Zoonotic and Vector Borne Disease Research, National Institute of Health, Korea Disease Control and Prevention Agency, Osong, Cheongju 28160, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Pathogens 2025, 14(11), 1182; https://doi.org/10.3390/pathogens14111182
Submission received: 30 October 2025 / Revised: 14 November 2025 / Accepted: 16 November 2025 / Published: 18 November 2025
Browse Figures
Versions Notes

Abstract

Borrelia garinii is a spirochete associated with Lyme borreliosis and is widely distributed across Eurasia. Although its genomic features have been well characterized in Europe, genomic data from East Asian isolates remain limited. Two B. garinii strains, HN13 and HN18, were isolated from a wild rodent (Apodemus agrarius) in South Korea and subjected to whole-genome sequencing and comparative genomic analysis. Their genomic features were compared with those of a tick-derived Korean strain 935 and additional global reference genomes. Phylogenetic analyses revealed that B. garinii strain HN18 clustered closely with French strains CIP103362 and 20047, whereas B. garinii strain HN13 showed high chromosomal similarity to the Korean strain 935. Both rodent-derived strains harbored plasmids carrying virulence-associated genes, including vlsE and vls silent cassettes, which were absent in B. garinii strain 935. This study provides new genomic insights into B. garinii circulating in East Asia and reveals host-associated plasmid variation linked to virulent potential. This study also suggests possible trans-Eurasian gene flow and underscores the need for continued genomic surveillance to better understand the evolution and epidemiology of Borrelia species.

1. Introduction

Borrelia garinii is a Gram-negative, microaerophilic, slowly growing, extracellular spirochete that belongs to the family Spirochaetaceae [1,2,3,4]. It is one of the main causative agents of Lyme borreliosis [5] and is transmitted primarily by Ixodes ticks that are known vectors of the pathogen [5,6]. The first isolation of B. garinii was reported in 1992 and named after the French physician ‘Charles Garin’ [7]. B. garinii is now known to be widely distributed across the Northern Hemisphere [8], which includes countries in Europe (e.g., Germany [9], France [10], England, the Czech Republic, Switzerland, Slovakia, Serbia, Finland [11], Italy [12], Austria [13], Russia [14], Denmark, Slovenia [15], Sweden [7], the Netherlands [16], and Estonia [17]); North America (USA [18] and Canada [19]); and Asia (China [20], Japan [21], Mongolia [22], and Taiwan [23]), including South Korea [24,25,26].
In South Korea, B. garinii was first reported in 1993 [27]. Since then, it has been detected in ticks [28,29], clinical samples [24], and wild rodents such as Apodemus agrarius [26]. However, national surveillance studies suggested that Borrelia afzelii is the dominant Lyme borreliosis species in South Korea, accounting for up to 62.5% of cases, while B. garinii comprises up to 3.7% [28,30,31].
Borrelia garinii has been associated with severe clinical manifestations, including skin lesions, arthritis and neuroborreliosis (e.g., meningitis, peripheral facial palsy) [2,32,33]. Its pathogenicity is partly attributed to its ability to evade the host immune system through mechanisms such as antigenic variation via the vls locus and modulation of outer surface proteins like OspC [2,34,35,36,37]. Among the genes involved in immune evasion and tissue adhesion, genes like ospC [38,39], vlsE [36,40], dbpA [41], and PFam54/60 [42] are commonly used as molecular markers for strain-specific virulence and antigenic variation.
The Borrelia genome comprises a highly conserved linear chromosome and a variable set of linear and circular plasmids that encode host-adaptive and virulence-associated genes [2,43,44]. Despite global efforts to characterize the genetic diversity of B. garinii, plasmid-borne virulence genes remain highly variable [2,45]. As of 2025, 172 whole-genome sequences (WGS) of B. garinii strains have been deposited in the NCBI, including one Korean strain (935) isolated from a tick (accession number: GCF_000714705.1) [25]. Despite the identification of B. garinii in ticks, rodents, and clinical samples in South Korea, high-quality whole-genome sequences from this region remain limited. Moreover, the extent of genomic variation, particularly at the plasmid level, and its relationship to host or vector origin is not well understood.
In this study, we conducted whole-genome sequencing of two B. garinii strains isolated from wild rodents (A. agrarius) in South Korea and performed comparative genomic analyses with a tick-derived Korean strain (935) and other globally reported B. garinii genomes. Our analysis focused on genome-wide phylogenetic placement, plasmid structural variation, and the distribution of virulence-associated genes to investigate host-associated genomic diversity among B. garinii strains.

2. Materials and Methods

2.1. Bacterial Sample Preparation

Two B. garinii strains were isolated from a rodent species (A. agrarius) captured in the Haenam region (latitude: 39.1543, longitude: 127.4457) of South Korea. The spirochetes were cultured using heart tissue from an individual rodent in BSK II medium at 32 °C under microaerophilic conditions [46]. Species identification was performed using multilocus sequence typing (MLST) targeting 8 housekeeping genes: clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA [26]. The isolates were designated B. garinii strain HN13 and B. garinii strain HN18.
Genomic DNA was extracted and purified by Macrogen Inc. (Humanizing Genomics Macrogen, Seoul, Republic of Korea) using a commercially available DNA extraction kit (Wizard® HMW DNA Extraction Kit, Promega, Fitchburg, WI, USA). The concentration of the extracted genomic DNA from B. garinii strain HN13 was 71.66 ng/µL in an elution volume of 164 µL with an average fragment size of 17,507 bp, whereas that from B. garinii strain HN18 was 41.11 ng/µL in 100 µL with an average fragment size of 39,545 bp. The A260/A280 ratios for both samples ranged between 1.8 and 2.0.

2.2. Whole-Genome Sequencing, Assembly, and Annotation

A hybrid sequencing approach was used, combining long-read and short-read platforms. Long-read sequencing was performed using the Revio system (Pacific Biosciences, Menlo Park, CA, USA), and short-read sequencing was performed with the NovaSeq X series (Illumina, San Diego, CA, USA). Long-read libraries were prepared using the commercially available kits, SMRTbell prep kit 3.0 (Pacific Biosciences, Menlo Park, CA, USA), with DNA concentration of 10 ng/µL and fragment sizes between 5000 bp and 20,000 bp. Short-read libraries were prepared using the TruSeq DNA Nano Kit (Illumina, San Diego, CA, USA), with average fragment sizes of 654 bp (HN18) and 625 bp (HN13).
PacBio HiFi reads were assembled using the Hifiasm 0.19.9 tool [47] with the default settings. Contigs shorter than 1000 bp were discarded. Error correction was performed using short Illumina reads with Inspector v1.0.1 [48]. Illumina reads were filtered at a Phred score of 30 or higher and trimmed using the Trimmomatic 0.38 tool [49]. Three rounds of polishing were conducted using the Pilon 1.22 tool [50].
Genome completeness was computed via the BUSCO 5.1.3 tool [51] with the bacterial ortholog database version 10 (Bacteria_odb10). Gene prediction and functional annotation were performed using the Prokka 1.14.6 tool [52]. Protein domain annotation was completed using InterProScan 5.34–73.0 [53] and PSI-BLAST 2.14.0 [54] with the EggNOG v4.5 database [55].

2.3. Comparative Genomic Analysis of Korean B. garinii Strains and Other B. garinii Strains

The assembled genomes of B. garinii strains HN13 and HN18 were compared with the previously registered Korean strain B. garinii strain 935 (accession number: GCF_000714705.1), which was isolated from Ixodes persulcatus [25]. In addition, 14 publicly available B. garinii WGS were retrieved from the NCBI database, filtered to include only assemblies at the ‘chromosome’ or ‘complete genome’ level. Genomes flagged as suppressed by RefSeq due to annotation failure were excluded.
Plasmid types of B. garinii strains HN13, HN18 and 935 were identified by BLAST (BLAST+ version 2.17.0; BLASTDB v5 database format) [54] searches against full-length contigs and gene annotations, combined with results from MOB-recon v3.1.9 implemented in the MOB-suite [56].
Phylogenetic relationships among the 17 B. garinii genomes were inferred based on core-genome single-nucleotide polymorphisms (SNPs) using chromosome sequences on the Galaxy web-based platform (https://usegalaxy.org/) [57]. For core-genome alignment, Prokka-generated ‘.gff’ files were used as input for Roary [58] with a minimum BLASTP identity threshold of 95% and a core gene definition of presence in 99% of genomes, resulting in 775 core genes. Recombination was identified and masked with Gubbins [59] using 5 iterations, and convergence was assessed using the weighted Robinson–Foulds metric. Analyses were run without an outgroup (unrooted), and the recombination-filtered alignment generated by Gubbins was used for subsequent tree inference. The maximum-likelihood (ML) phylogeny was reconstructed in IQ-TREE v2.4.0 [60] under the GTR + F+ASC + R2 model, with 1000 nonparametric bootstrap replicates performed to assess branch support. A consensus tree was generated from all bootstrap replicates, which showed an identical topology to the ML tree (Robinson–Foulds distance = 0). The resulting tree was visualized in MEGA12 [61].
As ospC and multilocus sequence typing (MLST) sequence types (STs) are commonly employed for Borrelia strain typing [62], ospC gene sequences from the 17 genomes were obtained from EggNOG annotation results and the NCBI WGS database [63]. MLST allelic profiles were determined using eight housekeeping genes, nifS, clpA, rplB, pyrG, recG, clpX, pepX, and uvrA, through the PubMLST Borrelia spp. database [64]. The ospC and concatenated MLST nucleotide sequences were aligned using the MUSCLE algorithm implemented in MEGA12, and ML phylogenetic trees were constructed with 1000 bootstrap replicates to assess branch support.
Whole-genome average nucleotide identity (ANI) values among the 17 genomes were calculated using FastANI v1.3 [65] in the Galaxy platform, and the resulting heatmap was visualized in Rstudio v2025.09.2 [66] using ggplot2 v3.5.2 [67] and reshape2 v1.4.4 [68].
The ospC-containing plasmids (cp26) and vls locus-containing plasmids (lp28 and lp36 types) were aligned using MAFFT v7.526 [69]. Because cp26 is circular, sequence ends were manually inspected and, when necessary, reverse-complemented to ensure proper orientation and alignment. Once terminal regions were corrected, the sequences were re-aligned with MAFFT, and a phylogenetic tree was constructed in IQ-TREE with 1000 bootstrap replicates and visualized in MEGA12.
The vls locus (vlsE and/or vls cassette) was identified using Prokka-generated ‘.gff’, ‘.ffn’, and ‘.faa’ files in conjunction with EggNOG and InterProScan results. The vls loci of B. garinii strains HN13 and HN18 were visualized using Prokka-generated ‘.genbank’ files, and their synteny plots were produced with Easyfig v. 2.2.5 [70].

3. Results

3.1. General Genomic Features of the Three Korean B. garinii Strains HN13, HN18, and 935

The general genome characteristics of the two newly isolated B. garinii strains HN13 and HN18 and the previously reported B. garinii strain 935 are summarized in Table 1. The total genome sizes were 1191,250 bp for B. garinii strain HN13, 1140,642 bp for HN18, and 1176,739 bp for 935, with a consistent chromosomal GC content of 28.4%. Although B. garinii strain HN13 had the largest total genome size, the chromosomal sequence of B. garinii strain 935 (918,136 bp) was slightly longer than those of HN13 (906,429 bp) and HN18 (906,038 bp), suggesting subtle differences.
All three strains harbored both linear and circular plasmids. B. garinii strains HN13 and HN18 shared 8 plasmids, including lp54, lp32-10, and lp28-4, whereas B. garinii strain 935 contained a fused plasmid, lp54_lp32-10, and lacked lp28-4. The number of predicted coding sequences (CDSs) was highest in B. garinii strain HN18 (1360), followed by B. garinii strain 935 (1332) and B. garinii strain HN13 (1312). All strains carried 3 rRNA and 32 tRNA genes.

3.2. Phylogeny and Comparative Analysis

The phylogenetic relationships among B. garinii strains are shown in Figure 1. Based on ospC-based phylogenetic analysis (Figure 1a), B. garinii strain HN13 was identical (100% sequence similarity) to B. garinii strain 935, while B. garinii strain HN18 formed a distinct branch. Most Norwegian strains clustered together, except for the B. garinii strain 17-63N1, while the South Korean and Russian strains were broadly grouped in the same cluster. In contrast, the German and French strains formed separate clusters. Notably, B. garinii strains SZ and NMJW1 lacked plasmids, and the ospC gene was also absent in these strains.
In the MLST-based phylogenetic analysis (Figure 1b), the three Korean strains clustered closely together, with B. garinii strains HN13 and 935 again showing 100% sequence identity, whereas B. garinii strain HN18 formed an independent branch. In this tree, Chinese and Russian strains were closely grouped, while the European strains formed a broad cluster that also included the three Korean strains.
The core-genome SNP (chromosomal) phylogeny (Figure 1c) showed a similar overall pattern; B. garinii strains HN13 and 935 were again 100% core-genome sequence identity, while B. garinii strain HN18 remained distinct from the other two Korean strains. The Chinese and Russian strains were again grouped together, consistent with the MLST-based tree, whereas the European strains clustered primarily according to their geographic origin.
These phylogenetic patterns suggest that the rodent-derived Korean strain HN13 likely shares a recent common ancestor with 935 or has undergone limited diversification, consistent with local clonal expansion within South Korea. In contrast, B. garinii strain HN18, although also isolated from a rodent in the same region, formed an independent lineage, implying either a distinct evolutionary origin or possible gene flow with non-Korean populations. The clustering of Chinese and Russian strains together, as well as the partial overlap of European isolates, reflects the complex geographic distribution of B. garinii and may indicate multiple introduction or migration events across Eurasia.
The ANI analysis showed consistent results (Figure 2). Borrelia garinii strains HN13 and 935 exhibited nearly 100% chromosomal sequence identity, suggesting clonal or recently diverged origin within South Korea. In contrast, the B. garinii strain HN18 showed higher genomic similarity to the French strains CIP103362 and 20047, indicating a potential evolutionary connection between East Asian and European lineages. These patterns suggest that multiple B. garinii lineages, possibly representing both locally maintained and introduced strains, may coexist in the Korean Peninsula. The Chinese and Russian strains again formed a distinct cluster, and the Norwegian strains were also grouped together but separated into two subclusters.

3.3. Plasmid Content, Virulence Gene Distribution, and Comparative Analysis

The plasmid compositions of the 17 B. garinii strains are summarized in Table 2. Except for B. garinii strains NMJW1 and SZ, which lacked any plasmid sequences, all strains contained both lp54 and cp26. In addition, five strains possessed multiple lp28-type plasmids.
It is common for most B. garinii strains to possess the plasmids lp54, cp26, and multiple lp28s, which harbor several virulence-related genes. Specifically, ospA, ospB, dbpA, and dbpB were consistently located on the lp54 plasmid, whereas ospC was encoded on the cp26 plasmid. The vlsE gene and multiple vls silent cassettes were mainly detected on lp28-type plasmids (Table 3).
Among the three Korean strains, only B. garinii strains HN13 and HN18 carried the vlsE gene and multiple vls silent cassettes, both located on plasmid lp28-3 (Supplementary Table S1). Borrelia garinii strain HN13 additionally possessed one vls cassette on lp28-4.
Across all 17 B. garinii strains analyzed in this study, only 6 strains—two Korean strains, HN13 and HN18; two French strains, 20047 and CIP103362; a German strain, Pbes; and a Norwegian strain, 17-63N1—harbored vlsE and at least one vls silent cassette, primarily on lp28-type plasmids, although some French strains carried them on plasmid lp36.
The vlsE-carrying lp28-3 plasmids in B. garinii strains HN13 and HN18 also contained multiple vls silent cassettes (Figure 3). The vlsE gene was located near the 5’ end of the plasmid and was oriented in the opposite transcriptional direction to the downstream vls silent cassettes, highlighting the structural polarity of the vls locus.
Two phylogenetic trees based on cp26 plasmid (ospC-containing plasmid) and lp28 and lp36 plasmid types (vls locus-containing plasmid) are shown in Figure 4. The phylogenetic tree constructed from the cp26 plasmid (Figure 4a), which encodes ospC, exhibited a circular topology and showed a high degree of similarity to both the ospC-based phylogenetic tree (Figure 1a) and the core-genome SNP-based tree (Figure 1c). The two Korean strains HN13 and 935 clustered together, whereas B. garinii strain HN18 formed a distinct branch. The French strains CIP103362 and 20047 showed 100% sequence identity in cp26, while the Russian strain BgVir again formed a long, separate branch, consistent with the ospC and core-genome SNP-based phylogenies (Figure 1a,c). This phylogenetic concordance indicates that the evolutionary trajectories of the cp26 plasmid and its encoded ospC gene are highly linked, potentially reflecting co-inheritance or selective pressures that maintain plasmid-gene linkage. Such consistency across plasmid- and chromosome-based trees may indicate that the cp26 replicon contributes to the evolutionary diversification and ecological adaptation of B. garinii lineages.
A similar pattern was observed for the vls locus-containing plasmids (lp28 and lp36 types) (Figure 4b). In contrast to the cp26 tree, the two Korean strains HN13 and HN18 clustered closely with the French strains 20047 and CIP103362, as well as the Norwegian strain 17-63N1 lp28-9, which also carries the vlsE gene. However, the Korean strains formed their own distinct plasmid branches, whereas the French strains again shared 100% identical plasmid sequences in cp26, lp28-4 and lp36. Similarly, two Norwegian strains (17-54Z3 and FNG-2Z14) showed 100% identity in cp26 and lp28-3. These findings suggest that although the Korean strains share general plasmid similarity with European lineages, their plasmids have diverged into separate, lineage-specific variants rather than remaining 99–100% identical, implying independent circulation and local evolutionary differentiation.

4. Discussion

In this study, two B. garinii strains, HN13 and HN18, were isolated from wild rodents (A. agrarius) in South Korea and compared with the previously sequenced B. garinii strain 935, which was isolated from the tick I. persulcatus [25] in South Korea. Whole-genome analysis revealed that B. garinii strain HN13 shared high chromosomal similarity with 935, as supported by ospC, MLST, and core-genome SNP analyses. However, the two strains differed substantially in plasmid composition and virulent gene profiles.
Borrelia garinii strain HN13 possessed plasmids lp28-3 and lp28-4, both of which harbored vlsE and multiple vls silent cassettes. In contrast, B. garinii strain 935 lacked the vlsE system entirely, despite possessing plasmid lp28-3. In addition, B. garinii strain 935 carried a unique hybrid plasmid (lp54_lp32-10) and lacked lp28-4. These findings aligned with previous reports that demonstrate high conservation in Borrelia chromosomes but extensive plasmid variability in Borrelia, particularly in plasmids carrying virulence determinants [43,44,71]. Loss of virulence-associated plasmids, including the absence of vlsE, has been associated with reduced infectivity and adaptation to non-vertebrate hosts such as ticks [40,72]. Although B. garinii is widespread across East Asia [73,74], most surveillance studies have been performed at the species level rather than at the strain level. Thus, this work represents a comparative genomic analysis of East Asian B. garinii strains and establishes a foundation for future evolutionary and epidemiological investigations.
Phylogenetic analysis of the vlsE-containing plasmids indicated that B. garinii strains HN13 and HN18 clustered closely with several European strains, including B. garinii strains 20047 and CIP103362 (France) and strain 17-63N1 (Norway). In contrast, the cp26 plasmid encoding ospC was conserved across all three Korean strains. The phylogenetic concordance between the cp26 plasmid and ospC-based trees further supports the notion that the evolution of cp26 is tightly linked to that of its encoded ospC, likely reflecting co-inheritance or selective pressures that maintain gene–plasmid compatibility.
Notably, B. garinii strain HN18 clustered with French B. garinii strains 20047 and CIP103362 across multiple analyses, including core-genome SNPs, ANI heatmap, and plasmid phylogeny, suggesting a potential historical connection between South Korean and French B. garinii populations. Combined with the near-identical chromosomal similarity between HN13 and 935, these findings indicate the coexistence of at least two distinct B. garinii lineages in South Korea: one locally maintained (HN13/935) and another genetically affiliated with French strains (HN18). A plausible mechanism for this connectivity is migratory birds, which serve as vertebrate hosts for Ixodes ticks across overlapping habitats in Europe and Asia [19,75,76,77,78,79]. For example, B. garinii has been detected in Ixodes ricinus on Fringilla coelebs, a bird species migrating between Europe, Central Asia, and western China (https://www.datazone.birdlife.org, accessed on 15 September 2025) [80], and in Ixodes persulcatus feeding on Turdus naumanni, which occurs across East Asia and the Korean Peninsula [81,82]. Rodents, including A. agrarius, are also known reservoirs for Borrelia species and likely maintain local transmission cycles [77,82].
The virulence gene profiles further support differential pathogenic potential among the strains. Borrelia garinii strain HN18 possessed a full complement of key virulence genes, including ospA, ospB, dbpA, dbpB, vlsE, and PFam54/60 family protein, suggesting a high virulence potential. Borrelia garinii strain HN13 also carried vlsE but lacked dbpA, whereas B. garinii strain 935 lacked both vlsE and dbpA. Because vlsE is associated with persistent infection and immune evasion [83,84], and vlsE and dbpA with tissue adhesion and Lyme arthritis [36,41], these genetic differences may have clinical relevance. Interestingly, all three Korean B. garinii strains encoded ospC, typically linked to the early stage of Lyme borreliosis [84], whereas only the rodent-derived strains (HN13 and HN18) carried vlsE, which is essential for long-term persistence in vertebrate hosts [83,84]. These findings support the hypothesis that 935 may have adapted to a non-vertebrate (tick) host and lost certain virulence traits.
In human Lyme borreliosis, vlsE expression promotes antigenic variation and immune evasion during chronic infection, contributing to disease persistence and treatment resistance [37,72]. Although clinical data for Korean B. garinii infections are not yet available, the presence of vlsE-harboring plasmids in rodent-derived strains suggests that such lineages may possess an enhanced capacity for persistent infection in vertebrate hosts. Comparative analyses with human-derived B. garinii isolates will be important to evaluate whether these virulence determinants correlate with clinical severity or treatment outcomes.
Despite the insights gained, the number of Korean B. garinii genomes available in public databases remains limited, restricting broader regional comparisons. To ensure analytical robustness, this study included only complete or chromosome-level assemblies with well-annotated plasmid structures, enabling accurate assessment of gene content and plasmid diversity. This approach revealed both conserved and lineage-specific genomic features among B. garinii strains from Europe, Russia, China and South Korea. Further sequencing of additional isolates from diverse hosts and geographic regions will be necessary to more fully resolve the evolutionary dynamics and pathogenic heterogeneity of B. garinii in East Asia.
The genomic differences observed among the Korean B. garinii strains also have potential epidemiological and public health relevance. The coexistence of rodent-derived vlsE-positive strains (HN13 and HN18) and a tick-adapted vlsE-negative strain (935) suggests the presence of multiple transmission cycles involving both vertebrate and arthropod hosts in the region. Such genomic heterogeneity may influence transmission dynamics, host range, and the risk of human exposure. From a public health perspective, the detection of vlsE-harboring plasmids in local rodent isolates underscores the need for continued surveillance to monitor the emergence of potentially more virulent B. garinii lineages and to strengthen early warning systems for Lyme borreliosis in East Asia.

5. Conclusions

This study provides the first comparative genomic analysis of rodent- and tick-derived B. garinii strains circulating in South Korea. Although B. garinii strains HN13 and 935 shared highly similar chromosomal sequences, they differed markedly in plasmid composition, and virulence gene profiles suggest host-associated adaptation within local transmission cycles. In contrast, the B. garinii strain HN18 represents a genetically distinct lineage that clustered closely with European B. garinii strains and carried a broader repertoire of virulence determinants. These findings highlight the coexistence of multiple B. garinii lineages in South Korea, including both locally maintained and potentially trans-Eurasian lineages, whose circulation may be shaped by migratory birds, rodents, and shared Ixodes vectors. Given the limited genomic data available from East Asia, continued genomic surveillance of Borrelia species across Eurasia is essential to better understand their evolutionary dynamics and assess their potential public health impact.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/pathogens14111182/s1, Table S1: Borrelia garinii plasmids carrying vlsE gene and/or vls silent cassettes identified through genome annotation. References cited in Supplementary Materials [52,53].

Author Contributions

Conceptualization, W.-J.J.; methodology, W.-J.J., Y.-J.C., J.-Y.P.; formal analysis, H.K., Y.-J.C., J.-Y.P.; data curation, H.K.; writing—original draft preparation, H.K.; writing—review and editing, J.-Y.P., Y.-J.C., K.L., W.-J.J.; visualization, H.K.; supervision, W.-J.J.; funding acquisition, W.-J.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by two grants from the Korea National Institute of Health, grant numbers 2024-ER2103-00 and 2025-NI-025-00.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The whole-genome sequencing datasets supporting this study have been deposited in the NCBI database under BioProject accessions PRJNA1250628 & PRJNA1250701 (BioSamples: SAMN47947845 & SAMN47947906). These data are currently under embargo and will be released to the public upon publication of this article or one year after submission. Access may be granted earlier upon reasonable request to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
WGSWhole-genome sequencing
NCBINational Center for Biotechnology Information
MLSTMultilocus sequence typing
PacBioPacific Biosciences
STSequence type
ANIAverage nucleotide identity
bpBase pair
CDSCoding sequence
SNPSingle-nucleotide polymorphism

References

  1. Parija, S.C. Treponema, Borrelia and Leptospira. In Textbook of Microbiology and Immunology; Parija, S.C., Ed.; Springer Nature Singapore: Singapore, 2023; pp. 617–639. [Google Scholar]
  2. Strnad, M.; Rudenko, N.; Rego, R.O.M. Pathogenicity and virulence of Borrelia burgdorferi. Virulence 2023, 14, 2265015. [Google Scholar] [CrossRef]
  3. Petnicki-Ocwieja, T.; Kern, A. Mechanisms of Borrelia burgdorferi internalization and intracellular innate immune signaling. Front. Cell. Infect. Microbiol. 2014, 4, 175. [Google Scholar] [CrossRef] [PubMed]
  4. Barbour, A.G.; Qiu, W. Borreliella. In Bergey’s Manual of Systematics of Archaea and Bacteria; Barbour, A.G., Qiu, W., Eds.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2019. [Google Scholar]
  5. Bush, L.M.; Vazquez-Pertejo, M.T. Tick borne illness—Lyme disease. Disease-A-Month 2018, 64, 195–212. [Google Scholar] [CrossRef] [PubMed]
  6. Steere, A.C.; Strle, F.; Wormser, G.P.; Hu, L.T.; Branda, J.A.; Hovius, J.W.R.; Li, X.; Mead, P.S. Lyme borreliosis. Nat. Rev. Dis. Primers 2016, 2, 16090, Erratum in 2017, 3, 17062. [Google Scholar] [CrossRef]
  7. Baranton, G.; Postic, D.; Saint Girons, I.; Boerlin, P.; Piffaretti, J.-C.; Assous, M.; Grimont, P.A.D. Delineation of Borrelia burgdorferi Sensu Stricto, Borrelia garinii sp. nov., and Group VS461 Associated with Lyme Borreliosis. Int. J. Syst. Evol. Microbiol. 1992, 42, 378–383. [Google Scholar] [CrossRef]
  8. Lochhead, R.B.; Strle, K.; Arvikar, S.L.; Weis, J.J.; Steere, A.C. Lyme arthritis: Linking infection, inflammation and autoimmunity. Nat. Rev. Rheumatol. 2021, 17, 449–461. [Google Scholar] [CrossRef]
  9. Fingerle, V.; Schulte-Spechtel, U.C.; Ruzic-Sabljic, E.; Leonhard, S.; Hofmann, H.; Weber, K.; Pfister, K.; Strle, F.; Wilske, B. Epidemiological aspects and molecular characterization of Borrelia burgdorferi s.l. from southern Germany with special respect to the new species Borrelia spielmanii sp. nov. Int. J. Med. Microbiol. 2008, 298, 279–290. [Google Scholar] [CrossRef]
  10. Duffau, P.; Korbi, S.; Guillotin, V.; Talagrand-Reboul, E.; Ménard, A.; Peuchant, O. An unexpected case of Borrelia garinii liver infection. Ann. Clin. Microbiol. Antimicrob. 2022, 21, 15. [Google Scholar] [CrossRef]
  11. Hansford, K.M.; Wheeler, B.W.; Tschirren, B.; Medlock, J.M. Questing Ixodes ricinus ticks and Borrelia spp. in urban green space across Europe: A review. Zoonoses Public Health 2022, 69, 153–166. [Google Scholar] [CrossRef]
  12. Cinco, M.; Murgia, R.; Costantini, C. Prevalence of IgG reactivity in Lyme borreliosis patients versus Borrelia garinii and Borrelia afzelii in a restricted area of Northern Italy. FEMS Immunol. Med. Microbiol. 1995, 12, 217–222. [Google Scholar] [CrossRef]
  13. Ruivo, M.; Kovács, N.Z.; Schötta, A.-M.; Stelzer, T.; Hermann, L.; Mündler, V.; Bergthaler, A.; Reiter, M.; Wijnveld, M. Optimising Transformation Efficiency in Borrelia: Unravelling the Role of the Restriction-Modification System of Borrelia afzelii and Borrelia garinii. Int. J. Mol. Sci. 2024, 25, 11343. [Google Scholar] [CrossRef] [PubMed]
  14. Brenner, E.V.; Kurilshikov, A.M.; Stronin, O.V.; Fomenko, N.V. Whole-Genome Sequencing of Borrelia garinii BgVir, isolated from Taiga ticks (Ixodes persulcatus). J. Bacteriol. 2012, 194, 5713. [Google Scholar] [CrossRef] [PubMed]
  15. Wilske, B.; Preac-Mursic, V.; Gobel, U.B.; Graf, B.; Jauris, S.; Soutschek, E.; Schwab, E.; Zumstein, G. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J. Clin. Microbiol. 1993, 31, 340–350. [Google Scholar] [CrossRef] [PubMed]
  16. Nohlmans, L.M.K.E.; De Boer, R.; Van Den Bogaard, A.E.J.M.; Van Boven, C.P.A. Genotypic and phenotypic analysis of Borrelia burgdorferi isolates from The Netherlands. J. Clin. Microbiol. 1995, 33, 119–125. [Google Scholar] [CrossRef]
  17. MÄKinen, J.; Vuorinen, I.; Oksi, J.; Peltomaa, M.; He, Q.; MarjamÄKi, M.; Viljanen, M.K. Prevalence of granulocytic Ehrlichia and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from Southwestern Finland and from Vormsi Island in Estonia. J. Pathol. Microbiol. Immunol. 2003, 111, 355–362. [Google Scholar] [CrossRef]
  18. Rudenko, N.; Golovchenko, M.; Horak, A.; Grubhoffer, L.; Mongodin, E.F.; Fraser, C.M.; Qiu, W.; Luft, B.J.; Morgan, R.G.; Casjens, S.R.; et al. Genomic Confirmation of Borrelia garinii, United States. Emerg. Infect. Dis. 2023, 29, 64–69. [Google Scholar] [CrossRef]
  19. Smith, J.R.P.; Muzaffar, S.B.; Lavers, J.; Lacombe, E.H.; Cahill, B.K.; Lubelczyk, C.B.; Kinsler, A.; Mathers, A.J.; Rand, P.W. Borrelia garinii in seabird ticks (Ixodes uriae), Atlantic Coast, North America. Emerg. Infect. Dis. 2006, 12, 1909–1912. [Google Scholar] [CrossRef]
  20. Jiang, B.; Yao, H.; Tong, Y.; Yang, X.; Huang, Y.; Jiang, J.; Cao, W. Genome Sequence of Borrelia garinii Strain NMJW1, Isolated from China. J. Bacteriol. 2012, 194, 6660–6661. [Google Scholar] [CrossRef]
  21. Uesaka, K.; Maezawa, M.; Inokuma, H. Serological survey of Borrelia infection of dogs in Sapporo, Japan, where Borrelia garinii infection was previously detected. J. Vet. Med. Sci. 2016, 78, 463–465. [Google Scholar] [CrossRef]
  22. Fomenko, N.V.; Stronin, O.V.; Khasnatinov, M.N.; Danchinova, G.A.; Bataa, J.; Gol’tsova, N.A. Heterogeneity of the ospA gene structure from isolates of Borrelia garinii and Borrelia afzelii from Western Siberia and Mongolia. Mol. Genet. Microbiol. Virol. 2009, 24, 183–188. [Google Scholar] [CrossRef]
  23. Chao, L.-L.; Lu, C.-F.; Shih, C.-M. Molecular detection and genetic identification of Borrelia garinii and Borrelia afzelii from patients presenting with a rare skin manifestation of prurigo pigmentosa in Taiwan. Int. J. Infect. Dis. 2013, 17, e1141–e1147. [Google Scholar] [CrossRef]
  24. Oh, W.; Kim, J.; Choi, Y.-J.; Kang, T.; Park, H.-J.; Lee, K.; Jang, W.-J. Human Co-infection with Rickettsia spp., Borrelia garinii, and Orientia tsutsugamushi in South Korea. Syst. Appl. Acarol. 2024, 29, 1201–1206. [Google Scholar] [CrossRef]
  25. Noh, Y.; Kim, S.Y.; Lee, Y.S.; Kim, D.-W.; Kwon, T.; Hwang, K.-J. Whole-Genome Sequence of Borrelia garinii Strain 935T Isolated from Ixodes persulcatus in South Korea. Genome Announc. 2014, 2, e01298-14. [Google Scholar] [CrossRef] [PubMed]
  26. Park, K.-H.; Choi, Y.-J.; Kim, J.; Park, H.-J.; Song, D.; Jang, W.-J. Reclassification of Borrelia spp. Isolated in South Korea Using Multilocus Sequence Typing. Jpn. J. Infect. Dis. 2018, 71, 350–353. [Google Scholar] [CrossRef] [PubMed]
  27. Park, K.H.; Chang, W.H.; Schwan, T.G. Identification and characterization of lyme disease spirochetes, Borrelia burgdorferi sensu lato, isolated in Korea. J. Clin. Microbiol. 1993, 31, 1831–1837. [Google Scholar] [CrossRef]
  28. Kim, K.G.; Hwang, D.J.; Park, J.W.; Ryu, M.G.; Kim, Y.; Yang, S.-J.; Lee, J.-E.; Lee, G.S.; Lee, J.H.; Park, J.S.; et al. Distribution and pathogen prevalence of field-collected ticks from south-western Korea: A study from 2019 to 2022. Sci. Rep. 2024, 14, 12336. [Google Scholar] [CrossRef]
  29. Park, J.W.; Lee, S.H.; Lee, G.S.; Seo, J.J.; Chung, J.K. Epidemiological Characteristics of Field Tick-Borne Pathogens in Gwang-ju Metropolitan Area, South Korea, from 2014 to 2018. Osong Public Health Res. Perspect. 2020, 11, 177–184. [Google Scholar] [CrossRef]
  30. Kim, S.Y.; Kim, T.-K.; Kim, T.Y.; Lee, H.I. Geographical Distribution of Borrelia burgdorferi sensu lato in Ticks Collected from Wild Rodents in the Republic of Korea. Pathogens 2020, 9, 866. [Google Scholar] [CrossRef]
  31. Lee, H.; Lee, S.-H.; Shin, S.; Kwak, D. Molecular Identification of Borrelia spp. from Ticks in Pastures Nearby Livestock Farms in Korea. Insects 2021, 12, 1011. [Google Scholar] [CrossRef]
  32. Limbach, F.X.; Jaulhac, B.; Puechal, X.; Monteil, H.; Kuntz, J.L.; Piemont, Y.; Sibilia, J. Treatment resistant Lyme arthritis caused by Borrelia garinii. Ann. Rheum. Dis. 2001, 60, 284–286. [Google Scholar] [CrossRef]
  33. Rožič, M.; Lah, L.L.; Ružić-Sabljić, E.; Kastrin, A.; Arnež, M. Lyme Neuroborreliosis in Children: Etiology and Comparison of Clinical Findings of Lyme Neuroborreliosis Caused by Borrelia garinii and Borrelia afzelii. Pediatr. Infect. Dis. J. 2019, 38, e279–e284. [Google Scholar] [CrossRef]
  34. Tran, N.; Milewski, M.D. Arthrofibrosis Associated with the Surgical Treatment of Chronic Lyme Arthritis and a Concomitant Medial Meniscal Tear: A Case Report. JBJS Case Connect. 2017, 7, e6. [Google Scholar] [CrossRef]
  35. Stewart, P.E.; Wang, X.; Bueschel, D.M.; Clifton, D.R.; Grimm, D.; Tilly, K.; Carroll, J.A.; Weis, J.J.; Rosa, P.A. Delineating the Requirement for the Borrelia burgdorferi Virulence Factor OspC in the Mammalian Host. Infect. Immun. 2006, 74, 3547–3553. [Google Scholar] [CrossRef]
  36. Lone, A.G.; Bankhead, T. The Borrelia burgdorferi VlsE Lipoprotein Prevents Antibody Binding to an Arthritis-Related Surface Antigen. Cell Rep. 2020, 30, 3663–3670.e5. [Google Scholar] [CrossRef] [PubMed]
  37. Embers, M.E.; Liang, F.T.; Howell, J.K.; Jacobs, M.B.; Purcell, J.E.; Norris, S.J.; Johnson, B.J.B.; Philipp, M.T. Antigenicity and recombination of VlsE, the antigenic variation protein of Borrelia burgdorferi, in rabbits, a host putatively resistant to long-term infection with this spirochete. FEMS Immunol. Med. Microbiol. 2007, 50, 421–429. [Google Scholar] [CrossRef] [PubMed]
  38. Wilske, B.; Busch, U.; Eiffert, H.; Fingerle, V.; Pfister, H.W.; RÖSsler, D.; Preac-Mursic, V. Diversity of OspA and OspC among cerebrospinal fluid isolates of Borrelia burgdorferi sensu lato from patients with neuroborreliosis in Germany. Med. Microbiol. Immunol. 1996, 184, 195–201. [Google Scholar] [CrossRef] [PubMed]
  39. Izac, J.R.; Camire, A.C.; Earnhart, C.G.; Embers, M.E.; Funk, R.A.; Breitschwerdt, E.B.; Marconi, R.T. Analysis of the antigenic determinants of the OspC protein of the Lyme disease spirochetes: Evidence that the C10 motif is not immunodominant or required to elicit bactericidal antibody responses. Vaccine 2019, 37, 2401–2407. [Google Scholar] [CrossRef]
  40. Rogovskyy, A.S.; Casselli, T.; Tourand, Y.; Jones, C.R.; Owen, J.P.; Mason, K.L.; Scoles, G.A.; Bankhead, T. Evaluation of the Importance of VlsE Antigenic Variation for the Enzootic Cycle of Borrelia burgdorferi. PLoS ONE 2015, 10, e0124268. [Google Scholar] [CrossRef]
  41. Heikkilä, T.; Seppälä, I.; Saxen, H.; Panelius, J.; Yrjänäinen, H.; Lahdenne, P. Species-Specific Serodiagnosis of Lyme Arthritis and Neuroborreliosis Due to Borrelia burgdorferi Sensu Stricto, B. afzelii, and B.garinii by Using Decorin Binding ProteinA. J. Clin. Microbiol. 2002, 40, 453–460. [Google Scholar] [CrossRef]
  42. Brangulis, K.; Akopjana, I.; Petrovskis, I.; Kazaks, A.; Tars, K. Structural analysis of the outer surface proteins from Borrelia burgdorferi paralogous gene family 54 that are thought to be the key players in the pathogenesis of Lyme disease. J. Struct. Biol. 2020, 210, 107490. [Google Scholar] [CrossRef]
  43. Glöckner, G.; Lehmann, R.; Romualdi, A.; Pradella, S.; Schulte-Spechtel, U.; Schilhabel, M.; Wilske, B.; Sühnel, J.; Platzer, M. Comparative analysis of the Borrelia garinii genome. Nucleic Acids Res. 2004, 32, 6038–6046. [Google Scholar] [CrossRef]
  44. Casjens, S.R.; Mongodin, E.F.; Qiu, W.-G.; Dunn, J.J.; Luft, B.J.; Fraser-Liggett, C.M.; Schutzer, S.E. Whole-Genome Sequences of Two Borrelia afzelii and Two Borrelia garinii Lyme Disease Agent Isolates. J. Bacteriol. 2011, 193, 6995–6996. [Google Scholar] [CrossRef]
  45. Luciani, M.; Krasteva, I.; Schirone, M.; D’Onofrio, F.; Iannetti, L.; Torresi, M.; Di Pancrazio, C.; Perletta, F.; Valentinuzzi, S.; Tittarelli, M.; et al. Adaptive strategies of Listeria monocytogenes: An in-depth analysis of the virulent strain involved in an outbreak in Italy through quantitative proteomics. Int. J. Food Microbiol. 2025, 427, 110951. [Google Scholar] [CrossRef] [PubMed]
  46. Lee, S.H.; Kim, B.J.; Kim, J.H.; Park, K.H.; Yeo, S.J.; Kim, S.J.; Kook, Y.H. Characterization of Borrelia burgdorferi strains isolated from Korea by 16S rDNA sequence analysis and PCR-RFLP analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. Int. J. Syst. Evol. Microbiol. 2000, 50 Pt 2, 857–863. [Google Scholar] [CrossRef] [PubMed]
  47. Cheng, H.; Concepcion, G.T.; Feng, X.; Zhang, H.; Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat. Methods 2021, 18, 170–175. [Google Scholar] [CrossRef] [PubMed]
  48. Chen, Y.; Zhang, Y.; Wang, A.Y.; Gao, M.; Chong, Z. Accurate long-read de novo assembly evaluation with Inspector. Genome Biol. 2021, 22, 312. [Google Scholar] [CrossRef]
  49. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef]
  50. Walker, B.J.; Abeel, T.; Shea, T.; Priest, M.; Abouelliel, A.; Sakthikumar, S.; Cuomo, C.A.; Zeng, Q.; Wortman, J.; Young, S.K.; et al. Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 2014, 9, e112963. [Google Scholar] [CrossRef]
  51. Simão, F.A.; Waterhouse, R.M.; Ioannidis, P.; Kriventseva, E.V.; Zdobnov, E.M. BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 2015, 31, 3210–3212. [Google Scholar] [CrossRef]
  52. Seemann, T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics 2014, 30, 2068–2069. [Google Scholar] [CrossRef]
  53. Jones, P.; Binns, D.; Chang, H.-Y.; Fraser, M.; Li, W.; McAnulla, C.; McWilliam, H.; Maslen, J.; Mitchell, A.; Nuka, G.; et al. InterProScan 5: Genome-scale protein function classification. Bioinformatics 2014, 30, 1236–1240. [Google Scholar] [CrossRef]
  54. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef] [PubMed]
  55. Huerta-Cepas, J.; Szklarczyk, D.; Forslund, K.; Cook, H.; Heller, D.; Walter, M.C.; Rattei, T.; Mende, D.R.; Sunagawa, S.; Kuhn, M.; et al. EGGNOG 4.5: A hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res. 2016, 44, D286–D293. [Google Scholar] [CrossRef] [PubMed]
  56. Robertson, J.; Nash, J.H.E. MOB-suite: Software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microb. Genom. 2018, 4, e000206. [Google Scholar] [CrossRef] [PubMed]
  57. Afgan, E.; Baker, D.; Batut, B.; van den Beek, M.; Bouvier, D.; Čech, M.; Chilton, J.; Clements, D.; Coraor, N.; Grüning, B.A.; et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018, 46, W537–W544. [Google Scholar] [CrossRef]
  58. Page, A.J.; Cummins, C.A.; Hunt, M.; Wong, V.K.; Reuter, S.; Holden, M.T.G.; Fookes, M.; Falush, D.; Keane, J.A.; Parkhill, J. Roary: Rapid large-scale prokaryote pan genome analysis. Bioinformatics 2015, 31, 3691–3693. [Google Scholar] [CrossRef]
  59. Croucher, N.J.; Page, A.J.; Connor, T.R.; Delaney, A.J.; Keane, J.A.; Bentley, S.D.; Parkhill, J.; Harris, S.R. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Res. 2014, 43, e15. [Google Scholar] [CrossRef]
  60. Nguyen, L.-T.; Schmidt, H.A.; von Haeseler, A.; Minh, B.Q. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol. Biol. Evol. 2014, 32, 268–274. [Google Scholar] [CrossRef]
  61. Kumar, S.; Stecher, G.; Suleski, M.; Sanderford, M.; Sharma, S.; Tamura, K. MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Mol. Biol. Evol. 2024, 41, msae263. [Google Scholar] [CrossRef]
  62. Margos, G.; Gatewood, A.G.; Aanensen, D.M.; Hanincová, K.; Terekhova, D.; Vollmer, S.A.; Cornet, M.; Piesman, J.; Donaghy, M.; Bormane, A.; et al. MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proc. Natl. Acad. Sci. USA 2008, 105, 8730–8735. [Google Scholar] [CrossRef]
  63. National Library of Medicine: National Center for Biotechnology Information. Available online: https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 16 October 2025).
  64. Jolley, K.A.; Bray, J.E.; Maiden, M.C.J. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. 2018, 3, 124. [Google Scholar] [CrossRef]
  65. Jain, C.; Rodriguez-R, L.M.; Phillippy, A.M.; Konstantinidis, K.T.; Aluru, S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat. Commun. 2018, 9, 5114. [Google Scholar] [CrossRef]
  66. Posit Team. RStudio: Integrated Development Environment for R, 2025.05.0.496; Posit Team: Boston, MA, USA, 2025. [Google Scholar]
  67. Wickham, H. ggplot2: Elegant graphics for data analysis. In Use R! Wickham, H., Ed.; Springer Nature Link: Houston, TX, USA, 2016. [Google Scholar]
  68. Wickham, H. Reshaping Data with the reshape Package. J. Stat. Softw. 2007, 21, i12. [Google Scholar] [CrossRef]
  69. Katoh, K.; Standley, D.M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 2013, 30, 772–780. [Google Scholar] [CrossRef] [PubMed]
  70. Sullivan, M.J.; Petty, N.K.; Beatson, S.A. Easyfig: A genome comparison visualizer. Bioinformatics 2011, 27, 1009–1010. [Google Scholar] [CrossRef] [PubMed]
  71. Rudenko, N.; Golovchenko, M.; Horak, A.; Grubhoffer, L.; Mongodin, E.F.; Fraser, C.M.; Qiu, W.; Luft, B.J.; Morgan, R.G.; Casjens, S.R.; et al. Detection of Borrelia garinii in the USA. 2022. preprint. [Google Scholar] [CrossRef]
  72. Bankhead, T.; Chaconas, G. The role of VlsE antigenic variation in the Lyme disease spirochete: Persistence through a mechanism that differs from other pathogens. Mol. Microbiol. 2007, 65, 1547–1558. [Google Scholar] [CrossRef]
  73. Margos, G.; Hofmann, M.; Casjens, S.; Dupraz, M.; Heinzinger, S.; Hartberger, C.; Hepner, S.; Schmeusser, M.; Sing, A.; Fingerle, V.; et al. Genome diversity of Borrelia garinii in marine transmission cycles does not match host associations but reflects the strains evolutionary history. Infect. Genet. Evol. 2023, 115, 105502. [Google Scholar] [CrossRef]
  74. Sun, Y.; Zhang, Y.; Fang, R.; Wang, J.; Yang, X.; Wang, L.; Guo, Y.; Miao, L.; Li, S. Geographical Distribution and Prevalence of Borrelia Genospecies in Eurasian Ticks. Sci. Data 2025, 12, 1530. [Google Scholar] [CrossRef]
  75. Comstedt, P.; Bergström, S.; Olsen, B.; Garpmo, U.; Marjavaara, L.; Mejlon, H.; Barbour, A.G.; Bunikis, J. Migratory passerine birds as reservoirs of Lyme borreliosis in Europe. Emerg. Infect. Dis. 2006, 12, 1087–1102. [Google Scholar] [CrossRef]
  76. Olsen, B.; Jaenson, T.G.T.; Noppa, L.; Bunikis, J.; Bergstrom, S. Lyme borreliosis cycle in seabirds and Ixodes uriae ticks. Nature 1993, 362, 340–342. [Google Scholar] [CrossRef] [PubMed]
  77. Margos, G.; Fingerle, V.; Reynolds, S. Borrelia bavariensis: Vector Switch, Niche Invasion, and Geographical Spread of a Tick-Borne Bacterial Parasite. Front. Ecol. Evol. 2019, 7, e00401. [Google Scholar] [CrossRef]
  78. Comstedt, P.; Jakobsson, T.; Bergström, S. Global ecology and epidemiology of Borrelia garinii spirochetes. Infect. Ecol. Epidemiol. 2011, 1, 9545. [Google Scholar] [CrossRef]
  79. Kurtenbach, K.; Hanincová, K.; Tsao, J.I.; Margos, G.; Fish, D.; Ogden, N.H. Fundamental processes in the evolutionary ecology of Lyme borreliosis. Nat. Rev. Microbiol. 2006, 4, 660–669. [Google Scholar] [CrossRef]
  80. Pedersen, B.N.; Jenkins, A.; Kjelland, V. Tick-borne pathogens in Ixodes ricinus ticks collected from migratory birds in southern Norway. PLoS ONE 2020, 15, e0230579. [Google Scholar] [CrossRef]
  81. Miyamoto, K.; Nakao, M.; Fujita, H.; Sato, F. The ixodid ticks on migratory birds in Japan and the isolation of Lyme disease spirochetes from bird-feeding ticks. Med. Entomol. Zool. 1993, 44, 315–326. [Google Scholar] [CrossRef]
  82. Nakao, M.; Miyamoto, K.; Fukunaga, M. Lyme Disease Spirochetes in Japan: Enzootic Transmission Cycles in Birds, Rodents, And Ixodes Persulcatus Ticks. J. Infect. Dis. 1994, 170, 878–882. [Google Scholar] [CrossRef]
  83. Norris, S.J.; Brangulis, K. Meta-analysis of the Vmp-like sequences of Lyme disease Borrelia: Evidence for the evolution of an elaborate antigenic variation system. Front. Microbiol. 2024, 15, 1469411. [Google Scholar] [CrossRef]
  84. Tilly, K.; Bestor, A.; Rosa, P.A. Lipoprotein succession in Borrelia burgdorferi: Similar but distinct roles for OspC and VlsE at different stages of mammalian infection. Mol. Microbiol. 2013, 89, 216–227. [Google Scholar] [CrossRef]
Pathogens 14 01182 g001
Figure 1. Phylogenetic relationships of Korean B. garinii strains based on (a) ospC gene sequences, (b) multilocus sequence typing (MLST), and (c) core-genome SNPs. Phylogenetic trees were constructed using the maximum-likelihood method in MEGA12 for (a,b) and in IQ-TREE for (c) with 1000 bootstrap replicates. Bootstrap support values are indicated at the nodes, and scale bars represent the number of substitutions per site. Novel strains HN13 and HN18 are highlighted in bold.
Figure 1. Phylogenetic relationships of Korean B. garinii strains based on (a) ospC gene sequences, (b) multilocus sequence typing (MLST), and (c) core-genome SNPs. Phylogenetic trees were constructed using the maximum-likelihood method in MEGA12 for (a,b) and in IQ-TREE for (c) with 1000 bootstrap replicates. Bootstrap support values are indicated at the nodes, and scale bars represent the number of substitutions per site. Novel strains HN13 and HN18 are highlighted in bold.
Pathogens 14 01182 g001
Pathogens 14 01182 g002
Figure 2. Average nucleotide identity (ANI) heatmap of 17 B. garinii chromosome sequences, including B. garinii strains HN13 and HN18 isolated in this study. The color scale indicates ANI values among the genomes, with gray shading representing ANI values below 99%. Novel strains HN13 and HN18 are highlighted with *.
Figure 2. Average nucleotide identity (ANI) heatmap of 17 B. garinii chromosome sequences, including B. garinii strains HN13 and HN18 isolated in this study. The color scale indicates ANI values among the genomes, with gray shading representing ANI values below 99%. Novel strains HN13 and HN18 are highlighted with *.
Pathogens 14 01182 g002
Pathogens 14 01182 g003
Figure 3. Comparison of plasmid p28-3 from B. garinii strains HN13 and HN18 showing the locations of vlsE and vls silent cassettes. Green regions indicate alignments in the same orientation, whereas blue regions indicate alignments in the opposite orientation. The vlsE gene (1091 bp in B. garinii strain HN13 and 1100 bp in HN18) and adjacent vls silent cassettes are located within the lp28-3 plasmid of both strains. The comparison was visualized using Easyfig v.2.2.5 [70].
Figure 3. Comparison of plasmid p28-3 from B. garinii strains HN13 and HN18 showing the locations of vlsE and vls silent cassettes. Green regions indicate alignments in the same orientation, whereas blue regions indicate alignments in the opposite orientation. The vlsE gene (1091 bp in B. garinii strain HN13 and 1100 bp in HN18) and adjacent vls silent cassettes are located within the lp28-3 plasmid of both strains. The comparison was visualized using Easyfig v.2.2.5 [70].
Pathogens 14 01182 g003
Pathogens 14 01182 g004
Figure 4. Phylogenetic analysis of plasmids containing ospC and vls loci in Borrelia garinii. (a) Maximum-likelihood phylogenetic tree of the cp26 plasmid (ospC-containing plasmid) constructed using the full nucleotide sequence of cp26 from available B. garinii strains. (b) Maximum-likelihood phylogenetic tree of plasmids carrying the vls locus, including lp28 and lp38 plasmid types. Trees were generated using MEGA 12 [61] with 1000 bootstrap replicates, and bootstrap values are shown at each node. Branch lengths represent the number of substitutions per site. B. garinii strain 13 and strain 18 from the current study are highlighted in bold.
Figure 4. Phylogenetic analysis of plasmids containing ospC and vls loci in Borrelia garinii. (a) Maximum-likelihood phylogenetic tree of the cp26 plasmid (ospC-containing plasmid) constructed using the full nucleotide sequence of cp26 from available B. garinii strains. (b) Maximum-likelihood phylogenetic tree of plasmids carrying the vls locus, including lp28 and lp38 plasmid types. Trees were generated using MEGA 12 [61] with 1000 bootstrap replicates, and bootstrap values are shown at each node. Branch lengths represent the number of substitutions per site. B. garinii strain 13 and strain 18 from the current study are highlighted in bold.
Pathogens 14 01182 g004
Table 1. Genome assembly statistics and gene contents of three Borrelia garinii strains isolated in South Korea.
Table 1. Genome assembly statistics and gene contents of three Borrelia garinii strains isolated in South Korea.
B. garinii Strain HN13B. garinii Strain HN18B. garinii Strain 935 *
ContigsSize (bp)GC (%)Depth (x)Size (bp)GC (%)Depth (x)Size (bp)GC (%)Depth (x)
Chromosome906,42928.4381906,08328.41251918,13628.4-
lp5452,48626.9214159,23726.64813---
lp3625,21923.6149824,71623.6428640,67423.9-
cp32-629,08329.1119529,46628.866936,85028.5-
lp32-1026,63025.571032,15626.73690---
lp28-315,33333.839128,20331.4108866,30924.8-
lp28-440,50825.4103333,58925.12574---
lp28-7---28,22532.24472---
cp2627,01425.9155226,97325.984640,09025.4-
lp1717,94022.9158222,60224.2641429,57123.2-
lp54_lp32-10------67,10926.6-
* Genome sequence information was obtained from GenBank (accession no.: GCF_000714705.1).
Table 2. Plasmid profiles of 17 B. garinii strains analyzed in this study.
Table 2. Plasmid profiles of 17 B. garinii strains analyzed in this study.
StrainsLinear-Form PlasmidsCircular-Form PlasmidsOther *Total
lp54lp36lp32lp28lp25lp17cp32cp26cp9
HN13lp54lp36lp32-10lp28-3
lp28-4		-lp17cp32-6cp26--8
HN18lp54lp36lp32-10lp28-3
lp28-4
lp28-7		-lp17cp32-6cp26--9
935-lp36-lp28-3-lp17cp32-6cp26-lp54_lp32-106
BgVirlp54------cp26--2
20047lp54lp36lp32-10lp28-3
lp28-4
lp28-7		-lp17cp32-3
cp32-6		cp26--10
CIP103362lp54lp36lp32-10lp28-4
lp28-7		-lp17cp32-3
cp32-6		cp26--9
PBesp_lp54p_lp36p_lp32-5
p_lp32-9
p_lp32-10		p_lp28-3
p_lp28-7		p_lp25p_lp17-p_cp26p_cp9-11
NMJW1----------0
SZ----------0
17-63N1lp54--lp28-9lp25-p_cp32-3
p_cp32-6		p_cp26-lp17_32-10
Plasmid		8
NG-Z6lp54--lp28-2
lp28-3		lp25lp17-cp26cp9Plasmid8
FNG-1Z1lp54lp36lp32-10lp28-9lp25lp17-cp26--7
17-29Z1lp54lp36lp32-10lp28-9lp25lp17-cp26--7
17-54Z3lp54-lp32-10lp28-3lp25lp17-cp26-Plasmid7
17-59N1lp54lp36lp32-10lp28-9lp25lp17-cp26--7
17-58N4lp54lp36lp32-10lp28-9lp25lp17-cp26--7
FNG-2Z14lp54-lp32-10lp28-3lp25lp17-cp26--6
* This column includes unnamed, unclassified, or combined plasmids in which more than two plasmid contigs were merged.
Table 3. Distribution of some of virulence-related genes identified on plasmids of 17 Borrelia garinii strains.
Table 3. Distribution of some of virulence-related genes identified on plasmids of 17 Borrelia garinii strains.
ospAospBospCdbpAdbpBvlsE/vls Silent
Cassettes		PFam54/60 (Bbcrasp-1
Domain) **
HN13lp54lp54cp26-lp54lp28-3/lp28-3lp54, lp36, lp32-10, lp28-4
HN18lp54-cp26lp54lp54lp28-3/lp28-3, lp28-4lp54; lp36, lp32-10; lp28-4
935lp54_lp32-10 *lp54_lp32-10 *cp26-lp54_lp32-10 *-/-lp54_lp32-10 *; lp36, lp28-3
BgVirlp54lp54cp26lp54lp54-/-lp54
20047lp54-cp26lp54lp54lp36/lp36, lp28-3, lp28-4lp54, lp36, lp32-10, lp28-4,
CIP103362lp54-cp26lp54lp54lp36/lp36, lp28-4lp54, lp36, lp32-10, lp28-4,
PBesp_lp54-p_cp26-p_lp54p_lp28-3p_lp54, p_lp32-10, p_lp28-3, p_lp28-5, p_lp25
NMJW1No plasmid
SZNo plasmid
17-63N1lp54-p_cp26lp54lp54lp28-9, plasmidlp54, p_cp32-3, lp28-9, lp25, plasmid
NG-Z6lp54-cp26-lp54lp28-3lp54, lp25, plasmid
FNG-1Z1lp54-cp26lp54lp54lp28-9lp54, lp36, lp32-10, lp25
17-29Z1lp54-cp26lp54lp54lp28-9lp54, lp36, lp32-10, lp25
17-54Z3lp54-cp26lp54lp54lp28-3lp54, lp32-10, lp28-3, lp25
17-59N1lp54-cp26lp54lp54lp28-9lp54, lp36, lp32-10, lp25
17-58N4lp54-cp26lp54lp54lp28-9lp54, lp36, lp32-10, lp25
FNG-2Z14lp54-cp26lp54lp54lp28-3lp54, lp32-10, lp28-3, lp25
* The virulence-associated genes were annotated in B. garinii strain 935 based on in silico analysis of the fused plasmid lp54_lp32-10. ** Pfam54/60: Borrelia Bbcrasp-1-domain-containing protein.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Kang, H.; Choi, Y.-J.; Park, J.-Y.; Lee, K.; Jang, W.-J. Comparative Genomics of Two Newly Sequenced Rodent-Derived and One Previously Reported Tick-Derived Borrelia garinii Strains from South Korea Reveals Plasmid Variation and Virulence Gene Diversity. Pathogens 2025, 14, 1182. https://doi.org/10.3390/pathogens14111182

AMA Style

Kang H, Choi Y-J, Park J-Y, Lee K, Jang W-J. Comparative Genomics of Two Newly Sequenced Rodent-Derived and One Previously Reported Tick-Derived Borrelia garinii Strains from South Korea Reveals Plasmid Variation and Virulence Gene Diversity. Pathogens. 2025; 14(11):1182. https://doi.org/10.3390/pathogens14111182

Chicago/Turabian Style

Kang, Hyungsuk, Yeon-Joo Choi, Ji-Young Park, Kwangjun Lee, and Won-Jong Jang. 2025. "Comparative Genomics of Two Newly Sequenced Rodent-Derived and One Previously Reported Tick-Derived Borrelia garinii Strains from South Korea Reveals Plasmid Variation and Virulence Gene Diversity" Pathogens 14, no. 11: 1182. https://doi.org/10.3390/pathogens14111182

APA Style

Kang, H., Choi, Y.-J., Park, J.-Y., Lee, K., & Jang, W.-J. (2025). Comparative Genomics of Two Newly Sequenced Rodent-Derived and One Previously Reported Tick-Derived Borrelia garinii Strains from South Korea Reveals Plasmid Variation and Virulence Gene Diversity. Pathogens, 14(11), 1182. https://doi.org/10.3390/pathogens14111182

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop