The genus Borrelia reloaded

Abstract

The genus Borrelia, originally described by Swellengrebel in 1907, contains tick- or louse-transmitted spirochetes belonging to the relapsing fever (RF) group of spirochetes, the Lyme borreliosis (LB) group of spirochetes and spirochetes that form intermittent clades. In 2014 it was proposed that the genus Borrelia should be separated into two genera; Borrelia Swellengrebel 1907 emend. Adeolu and Gupta 2014 containing RF spirochetes and Borreliella Adeolu and Gupta 2014 containing LB group of spirochetes. In this study we conducted an analysis based on a method that is suitable for bacterial genus demarcation, the percentage of conserved proteins (POCP). We included RF group species, LB group species and two species belonging to intermittent clades, Borrelia turcica Güner et al. 2004 and Candidatus Borrelia tachyglossi Loh et al. 2017. These analyses convincingly showed that all groups of spirochetes belong into one genus and we propose to emend, and re-unite all groups in, the genus Borrelia.

About the Authors:

Gabriele Margos

Contributed equally to this work with: Gabriele Margos, Alex Gofton

Roles Conceptualization, Funding acquisition, Project administration, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliation: Bavarian Health and Food Safety Authority and National Reference Center for Borrelia, Oberschleissheim, Germany

ORCID logo http://orcid.org/0000-0001-5304-9615

Alex Gofton

Contributed equally to this work with: Gabriele Margos, Alex Gofton

Roles Formal analysis, Funding acquisition, Resources, Writing – original draft, Writing – review & editing

Affiliation: Vector & Waterborne Pathogens Research Group, School of Veterinary & Life Sciences, Murdoch University, South St, Murdoch, Australia

Daniel Wibberg

Roles Data curation, Resources, Writing – review & editing

Affiliation: Cebitec, University of Bielefeld, Bielefeld, Germany

Alexandra Dangel

Roles Formal analysis, Resources, Writing – review & editing

Affiliation: Bavarian Health and Food Safety Authority and National Reference Center for Borrelia, Oberschleissheim, Germany

Durdica Marosevic

Roles Formal analysis, Resources, Writing – review & editing

Affiliation: Bavarian Health and Food Safety Authority and National Reference Center for Borrelia, Oberschleissheim, Germany

Siew-May Loh

Roles Funding acquisition, Methodology, Resources, Writing – review & editing

Affiliation: Vector & Waterborne Pathogens Research Group, School of Veterinary & Life Sciences, Murdoch University, South St, Murdoch, Australia

Charlotte Oskam

Roles Conceptualization, Funding acquisition, Resources, Writing – review & editing

Affiliation: Vector & Waterborne Pathogens Research Group, School of Veterinary & Life Sciences, Murdoch University, South St, Murdoch, Australia

ORCID logo http://orcid.org/0000-0001-8886-2120

Volker Fingerle

Roles Conceptualization, Funding acquisition, Resources, Writing – original draft, Writing – review & editing

Affiliation: Bavarian Health and Food Safety Authority and National Reference Center for Borrelia, Oberschleissheim, Germany

Introduction

The spirochete genus Borrelia, named after the French biologist Amédée Borrel, was originally described in 1907 by Swellengrebel [1], with B. anserina (Sakharoff 1891) Bergey et al. 1925 designated as the type species. Since then numerous species and strains have been described, and members of this genus are well recognized as the aetiological agents of Lyme borreliosis (LB) and relapsing fever (RF) in humans. Lyme borreliosis and RF genospecies have long been recognized to have different clinical, biological, and epidemiological characteristics, and phylogenetic data is concordant with this, demonstrating that these two groups are genetically similar yet distinct, and form independent monophyletic sister clades that share a common ancestor [2].

Nevertheless, LB and RF Borrelia share a common set of genetic and biological characteristics that unify these organisms as a group compared to other related spirochetes. Namely, all LB and RF Borrelia species are spirochetes with an obligate parasitic lifestyle, are transmitted between vertebrate hosts by arthropod vectors (ticks and louse), and can be transstadially transmitted within their arthropod vectors. Various vector associations of Borrelia have been found in nature, with the genus Ixodes mainly vectoring LB species while argasid ticks often vector the RF group. However, some members of the RF group are associated with hard ticks of the family Ixodidae (e.g. B. lonestari) [3], with the human body louse Pediculus humanus (B. recurrentis) [4] or the genus Ixodes (e.g. B. miyamotoi) [5]. The genus Ixodes represents an ancient genus of the family Ixodidae sharing numerous original features with argasid ticks. Both, LB and RF spirochetes are dependent on their vertebrate and arthropod hosts for the majority of their nutritional requirements, and share a unique genomic structure comprised of a single highly conserved linear chromosome and numerous extrachromosomal linear and circular plasmids that can be highly variable between strains [6–8].

Recently, a third group of Borrelia organisms has been described that are associated with reptile and echidna (Tachyglossus aculeatus) hosts, and do not phylogenetically cluster within either the RF or LB clades. Instead these novel borreliae form their own independent lineages which sit as an outgroup to, and shares a most recent common ancestor with, the RF clade [9]. This novel clade currently has two designated species, B. turcica [10] and ‘Candidatus Borrelia tachyglossi’ [9, 11, 12], and several other genetic variants that are yet to be formally taxonomically classified [13]. Known vectors for this group include hard ticks of the genera Amblyomma, Bothriocroton, and Hyalomma [10, 11, 13].

Recently Adeolu and Gupta [14] proposed to divide Borrelia into two genera to reflect the genetic and phenotypic divergence between LB and RF species, however, this proposal remains under debate [15, 16], and has not been widely utilized in the literature. The justification for this proposal was largely based on the identification of conserved signature insertions/deletions (indels) (CSIs) and conserved signature proteins (CSPs) that are differentially present in the LB or RF Borrelia genogroup, as well as average nucleotide identity (ANI) values calculated between whole genomes of 18 Borrelia species including eight LB species and ten RF species. Although it is uncontested that these differences exist between LB and RF Borrelia, we propose that the methodology used to identify these group-specific differences is subjective and has a highly limited power to delineate LB and RF Borrelia into separate genera.

The methodology employed by Adeolu and Gupta [14] reported only “CSIs that are specific for different groups within the Borrelia”, and CSPs only “if either all significant [BLAST] hits were from well-defined group of Borrelia or which involved a large increase in E-values from the last hit belonging to a particular group of Borrelia to the first hit from any other group”. This methodology specifically identifies only CSIs and CSPs that are exclusive only to one Borrelia genogroup, and precludes the detection of CSIs or CSPs that may be shared non-exclusively between both genogroups (i.e. contests the hypothesis that LB and RF belong in different genera). This data presented in isolation misrepresents the extent of genomic divergence between LB and RF Borrelia and fails to consider widespread genomic similarities between these two groups. Additionally, although ANI has previously been used to investigate prokaryote taxonomy [17], a comprehensive review of this method revealed that although ANI can accurately quantify the genetic relationships between strains belonging to the same species, it was not suitable to differentiate prokaryotic genera. This is due to significant overlapping of intergenera ANI and interspecies ANI values [18], which leads to unreliability in the method.

Alternatively, Qin et al. [18] presented a more heuristic method for delineating prokaryotic genera that measures the percentage of conserved proteins (POCP) between whole genome pairs, reasoning that the degree of protein conservation reflect both genetic and phenotypic relatedness more substantially. Qin et al. [18] demonstrated that among 235 prokaryotic species from 97 genera that POCP values had a higher predictive power than ANI to delineate genera. They showed that with few exceptions POCP values of ≥ 50% could be considered a threshold for prokaryotic genus delimitation, pending other genomic factors that influence POCP, such as large differences in genome size.

Here we investigate the validity of the proposed delineation of LB and RF Borrelia into separate genera by performing pairwise analysis of POCP values between 30 Borrelia type strain genomes (where possible), including two new Borrelia genomes from the novel reptile and echidna-associated clade, B. turcica, and ‘Candidatus Borrelia tachyglossi’, which have yet to be analyzed in this context. We also re-examine the CSIs previously used to support the delineation of LB and RF Borrelia in the genomes of B. turcica and ‘Candidatus Borrelia tachyglossi’ to establish whether these molecular markers are useful to establishing the relationship of B. turcica, and ‘Candidatus Borrelia tachyglossi’. Our analyses indicate that insufficient genomic divergence exist between LB and RF Borrelia to consider them separate genera, and that Borrelia CSIs are limited in their ability to unambiguously distinguish the taxonomic identity of B. turcica and ‘Candidatus Borrelia tachyglossi’.

Materials and methods

Strain included in this study

In order to accurately assess Borrelia intra-genus POCP, the proteomes of 30 Borrelia species strains, including n = 17 strains from the LB group, n = 11 from the RF group, and n = 2 from the reptile and echidna-associated group, were retrieved from GenBank (National Center for Biotechnology Information (NCBI), Bethesda (MD), https://www.ncbi.nlm.nih.gov/) or sequenced and assembled from low passage type cultures except ‘Candidatus B. tachyglossi’ which was sequenced from a single tick [12, 19]. A tree summarizing the phylogenetic relationship based on 791 homologous proteins is shown in Fig 1. A full summary of strains used is presented in Table 1. To determine the levels of inter-genera POCP within the order Spirochaetales, an additional 54 proteomes, including n = 8 Brachyspira, n = 21 Leptospira, n = 5 Spirochaeta, and n = 20 Treponema species, were retrieved from GenBank (S1 Table) and included in the POCP analysis.

[Figure omitted. See PDF.]

Fig 1. Phylogenetic reconstruction of Borrelia species based on 791 aligned protein homologs built with the PEPR pipeline and FastTree2 with 100 jackknifed resampling replicates.

All node support values are 100 except where indicated.

https://doi.org/10.1371/journal.pone.0208432.g001

[Figure omitted. See PDF.]

Table 1. Borrelia species and strains included in study.

https://doi.org/10.1371/journal.pone.0208432.t001

Sequence analyses

POCP analysis was performed according to Qin et al. [18] and as described in [20]. Briefly, for each genome pair reciprocal BLASTP [21] was used to identify homologous proteins between genome pairs. Proteins were considered to be conserved if the BLAST matches had an E-value of < 1e-5, >40% sequence identity and >50% of the query sequence in each of the reciprocal searches. The POCP value for a genome pair was then determined as [(C1+C2)/(T1+T2)] x 100, where C1 and C2 are the number of conserved proteins between the genome pair, and T1 and T2 are the total number of proteins in each genome being compared [18]. Scripts used for these analyses are available upon request.

The CSIs presented in Adeolu and Gupta [14] that are differentially present in LB and RF genomes were reinvestigated in the genomes of B. turcica and ‘Candidatus Borrelia tachyglossi’ to establish whether these molecular markers are useful for classifying their taxonomic relationships. To identify CSIs, the conserved amino acid sequences flanking the CSIs were searched against the proteomes of all 30 Borrelia genomes used here using BLASTP [21]. Hits from the matching protein in all 30 Borrelia proteomes were aligned with MUSCLE [22], and visually inspected for the presence of CSIs. The presence of previously defined CSPs in the genomes of B. turcica, and ‘Candidatus Borrelia tachyglossi’ was determined using BLASTP searched as described in Adeolu and Gupta [14].

Results and discussion

In order to determine whether the 50% POCP threshold for genus delineation was appropriate for spirochete taxa, we used pairwise POCP analysis to determine the inter-genera POCP values for 84 spirochete genomes from the genera Borrelia, Brachyspira, Leptospira, Spirochaeta, and Treponema. Among all spirochete genomes investigated inter-genera POCP values ranged between 4.8% and 36.8% (mean 10.1%), indicating a low degree of protein conservation occurs between spirochete genera (S1 Table). These spirochete inter-genera POCP values are at a minimum of 13.2% lower than the 50% value determined by [18], suggesting this value is an appropriate and highly conservative threshold for spirochete genera delineation.

Compared to the low level of protein conservation measured between spirochete genera, POCP values were significantly higher among Borrelia species. Borrelia POCP values were highest among the LB genospecies, which ranged between 81.1–94.4% (mean 90.2%), while POCP values between RF species were generally lower and more variable, ranging between 65.3–93.1% (mean 81.1%) (Figs 2 and 3). Despite sharing a most recent common ancestor with RF Borrelia, B. turcica and ‘Candidatus Borrelia tachyglossi’ genomes shared higher POCP values with many LB Borrelia species compared to RF species, such as B. chilensis (78.9% and 87.7%, respectively), B. americana (77.4% and 82.2%, respectively), B. japonica (77.0% and 82.0%, respectively), and B. mayonii (76.8% and 81.42%, respectively). However, B. turcica and ‘Candidatus Borrelia tachyglossi’ genomes did have higher levels of protein conservation with B. anserina (79.1% and 88.5%, respectively) and B. miyamotoi (‘Candidatus Borrelia tachyglossi’ only: 87.7% POCP) (Figs 2 and 3). Most significantly, all Borrelia pairwise POCP values consistently remained well above the 50% POCP threshold for genus delineations proposed by Qin et al. [18], with a minimum value of 64.8% (B. crocidurae Achema vs. B. chilensis VA1), and a maximum value of 88.8% (B. miyamotoi LB-2001 vs. B. chilensis VA1) (mean value: 73.6%) (Figs 2 and 3).

[Figure omitted. See PDF.]

Fig 2. Boxplot of inter- and intra-specific POCP values.

Inter-specific and intra-specific comparisons included Lyme borreliosis (LB) and relapsing-fever species (RF), reptile-associated species (REP) including the echnida-associated species ‘Candidatus B. tachyglossi’. The inter-genera comparison included the members of the genera Borrelia, Brachyspira, Leptospira, Spirochaeta, and Treponema.

https://doi.org/10.1371/journal.pone.0208432.g002

[Figure omitted. See PDF.]

Fig 3. Percentage of conserved proteins (POCP) matrix generated by the method described in [18].

POCP values of species belonging to the LB group, RF group of spirochetes, the reptile-associated species B. turcica and echnida-associated species B. tachyglossi are above the genus threshold of 50%, indicating that all belong into one bacterial genus, Borrelia.

https://doi.org/10.1371/journal.pone.0208432.g003

The original proposal to delineate LB and RF Borrelia was largely based on the occurrence of 53 CSIs that have different forms in LB and RF genogroups [14]. It was subsequently defended by suggesting that novel Borrelia species that group with, or as an outgroup to RF Borrelia would be expected to contain RF-specific CSIs and generally none specific to the LB group [23]. An examination of these 53 CSIs in B. turcica and ‘Candidatus Borrelia tachyglossi’ genomes shows although the majority of CSIs present in these genomes correspond to the RF-specific form of the indels, 9/53 (17.0%) and 11/53 (20.8%) of the CSIs in B. turcica and ‘Candidatus Borrelia tachyglossi’, respectively, correspond to LB-specific forms (Fig 4; Table 2).

[Figure omitted. See PDF.]

Fig 4.

Partial amino acid alignment of (A) a putative lipoprotein (GI: 1195064) and (B) a hypothetical protein (GI: 1194969) showing a CSI in which the form of the indel in ‘Candidatus Borrelia tachyglossi’ and B. turcica matches that in LB species.

https://doi.org/10.1371/journal.pone.0208432.g004

[Figure omitted. See PDF.]

Table 2. Designation of LB and RF-differentiating CSIs in ‘Candidatus Borrelia tachyglossi’ and B. turcica genomes.

https://doi.org/10.1371/journal.pone.0208432.t002

Thus, the results of our analysis using genospecies that were originally defined as belonging to the genus Borrelia showed a very clear pattern. The results demonstrate that LB and RF Borrelia genogroups lack sufficient proteomic differentiation to be classified as different genera according to the POCP threshold determined by Qin et al. [18]; the analysis of inter-genus POCP supported the classification of the five closely related Spirochaetales genera. Therefore, we propose to formally reestablish the genus Borrelia in its original form including species of the LB, RF, and reptile- and echnida-associated genogroups. Additionally, up to 20% of the CSIs identified as having genogroup-specific forms were not concordant with phylogenetic position of B. turcica and ‘Candidatus Borrelia tachyglossi’ as predicted previously [23]. Although categorical molecular markers such as these have been previously used in clarifying prokaryotic taxonomy, here these markers appear to have limited utility in resolving the taxonomic classification of novel Borrelia species.

The reptile- and echidna-associated Borrelia clade to which B. turcica and ‘Candidatus B. tachyglossi’ belong is a very recently described group of Borrelia for which several novel variants have been described based on single- or multi-gene phylogenetic analyses. Although this group clearly shared a more common ancestor with RF Borrelia, the presence of LB-specific CSIs and high protein conservation with LB species suggests this Borrelia may share common genetic and biological characteristics with LB species. Both, PCOP and CSI supported the continuum of Borrelia species between LB and RF which now includes B. turcica and ‘Candidatus B. tachyglossi’. These data suggest that the genus Borrelia in the form it was originally described and is proposed here represents a continuum with RF and LB group species at the extreme ends of the genus, and reptile and echnida-associated, and other Borrelia species (perhaps still to be discovered) sharing a unique mixture of features from both RF and LB groups.

In our study we included as many type strains as possible, as type strains are the representatives of the species and can be obtained from microbial culture collections. However, for two of the species belonging to the LB group of spirochetes, genomic data of the type strains were not available to us. As a surrogate we used genomic data available for closely related strains of these species, i.e. PKo for B. afzelii and A14S for B. spielmanii. Previous data on multilocus sequence typing have shown that these two isolates are closely related to the type strain of the respective genospecies and fall into the same phylogenetic cluster [24, 25].

We consider that Borreliella bavariensis (Margos et al. 2013) Adeolu and Gupta 2015, Borreliella burgdorferi (Johnson et al. 1984) Adeolu and Gupta 2015, Borreliella carolinensis (Rudenko et. al 2011) Adeolu and Gupta 2015, Borreliella garinii (Baranton et al. 1992) Adeolu and Gupta 2015, Borreliella japonica (Kawabata et al. 1994) Adeolu and Gupta 2015, Borreliella kurtenbachii (Margos et al. 2014) Adeolu and Gupta 2015, Borreliella sinica (Masuzawa et al. 2001) Adeolu and Gupta 2015, Borreliella spielmanii (Richter et al. 2006) Adeolu and Gupta 2015 should be more appropriately placed in the genus Borrelia as Borrelia bavariensis Margos et al. 2013, Borrelia burgdorferi Johnson et al. 1984, Borrelia carolinensis Rudenko et. al 2011, Borrelia garinii Baranton et al. 1992, Borrelia japonica Kawabata et al. 1994, Borrelia kurtenbachii Margos et al. 2014, Borrelia sinica Masuzawa et al. 2001, Borrelia spielmanii Richter et al. 2006 which we consider to be the correct name of the taxon. Borreliella bavariensis (Margos et al. 2013) Adeolu and Gupta 2015, Borreliella carolinensis (Rudenko et. al 2011) Adeolu and Gupta 2015, Borreliella garinii (Baranton et al. 1992) Adeolu and Gupta 2015, Borreliella japonica (Kawabata et al. 1994) Adeolu and Gupta 2015, Borreliella kurtenbachii (Margos et al. 2014) Adeolu and Gupta 2015, Borreliella sinica (Masuzawa et al. 2001) Adeolu and Gupta 2015, Borreliella spielmanii (Richter et al. 2006) Adeolu and Gupta 2015 should be considered to be synonyms.

Conclusion

The data presented in this study very clearly demonstrate that all groups investigated, i.e. RF group spirochetes, LB group spirochetes, reptile- and echnida-associated Borrelia species belong to the same genus as values for POCP were consistently above the proposed threshold for genus delimitation. We propose to re-establish the genus Borrelia in its original form.

Emended description of the genus Borrelia (Swellengrebel 1907) (approved lists 1980)

Organisms are helical (0.2–0.3 μm by 10–35 μm). Periplasmic flagella overlap in the central region of the cell. Cells are flexible and motile with rotational and forward/backwards movement. Organisms are host-associated and microaerophilic. They are vectored by argasid ticks, prostriate ixodid ticks, metastriate ixodid ticks and the human body louse. The genome is fragmented into a linear main chromosome, and linear or circular plasmids. The G/C content of the genomic DNA is 27–32 (mol%).

Members of this genus are the causative agents of relapsing fever, Lyme borreliosis or of unknown pathogenicity.

The type species is Borrelia anserina (Sakharoff 1891) Bergey et al. 1925(Approved Lists 1980).

Description of Borrelia afzelii Canica et al 1994

The description is the same as in Canica et al 1994. DNA-DNA hybridization, RFLP of the rrs gene as well as reactivity of monoclonal antibodies differentiates B. afzelii from other Borrelia species [26, 27]. B. afzelii strains are also distinguishable from all other LB species by using Multilocus sequence analysis [24].

Type strain VS461T (= DSM 10508T = CIP 103469T)

Description of Borrelia lusitaniae (Le Fleche et al. 1997, sp. nov.)

The description is the same as given in Le Fleche et al. 1997.

Type strain PotiB2 T (= CIP-105366 T, = DSM-107168T)

Supporting information

[Figure omitted. See PDF.]

S1 Table. Additional proteomes from the order Spirochaetales included in this study.

A distance matrix of POCP values is given for genera included and shows that the within genus percentages are generally higher than 50% except for Treponema.

https://doi.org/10.1371/journal.pone.0208432.s001

(XLSX)

Acknowledgments

The authors acknowledge Cecilia Hizo-Teufel, Christine Hartberger und Sylvia Stockmeier for excellent technical support. The sequencing service was provided by the Norwegian Sequencing Centre (http://www.sequencing.uio.no), a national technology platform hosted by the University of Oslo and supported by the “Functional Genomics” and “Infrastructure” programs of the Research Council of Norway and the Southeastern Regional Health Authorities. The authors gratefully acknowledge the services of DSMZ.

Citation: Margos G, Gofton A, Wibberg D, Dangel A, Marosevic D, Loh S-M, et al. (2018) The genus Borrelia reloaded. PLoS ONE 13(12): e0208432. https://doi.org/10.1371/journal.pone.0208432

References

1. Skerman VBD, McGowan V, Sneath PHA. Approved List of Bacterial Names. Washington (DC): ASM Press; 1980.

2. Takano A, Goka K, Une Y, Shimada Y, Fujita H, Shiino T, et al. Isolation and characterization of a novel Borrelia group of tick-borne borreliae from imported reptiles and their associated ticks. Environ Microbiol. 2010;12(1):134–46. pmid:19758349.

3. Burkot TR, Mullen GR, Anderson R, Schneider BS, Happ CM, Zeidner NS. Borrelia lonestari DNA in adult Amblyomma americanum ticks, Alabama. Emerg Infect Dis. 2001;7(3):471–3. Epub 2001/06/01. pmid:11384533; PubMed Central PMCID: PMC2631781.

4. Cutler SJ, Moss J, Fukunaga M, Wright DJ, Fekade D, Warrell D. Borrelia recurrentis characterization and comparison with relapsing-fever, Lyme-associated, and other Borrelia spp. Int J Syst Bacteriol. 1997;47(4):958–68. pmid:9336893.

5. Fukunaga M, Koreki Y. The flagellin gene of Borrelia miyamotoi sp. nov. and its phylogenetic relationship among Borrelia species. FEMS Microbiol Lett. 1995;134(2–3):255–8. pmid:8586277.

6. Casjens SR, Mongodin EF, Qiu WG, Luft BJ, Schutzer SE, Gilcrease EB, et al. Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids. PloS ONE. 2012;7(3):e33280. Epub 2012/03/21. pmid:22432010; PubMed Central PMCID: PMC3303823.

7. Marosevic D, Margos G, Wallich R, Wieser A, Sing A, Fingerle V. First insights in the variability of Borrelia recurrentis genomes. PLoS neglected tropical diseases. 2017;11(9):e0005865. Epub 2017/09/14. pmid:28902847; PubMed Central PMCID: PMC5612729.

8. Miller SC, Porcella SF, Raffel SJ, Schwan TG, Barbour AG. Large linear plasmids of Borrelia species that cause relapsing fever. J Bacteriol. 2013;195(16):3629–39. Epub 2013/06/12. pmid:23749977; PubMed Central PMCID: PMC3754566.

9. Loh SM, Gillett A, Ryan U, Irwin P, Oskam C. Molecular characterization of 'Candidatus Borrelia tachyglossi' (family Spirochaetaceae) in echidna ticks, Bothriocroton concolor. Int J Syst Evol Microbiol. 2017;67(4):1075–80. Epub 2017/05/06. pmid:28475032; PubMed Central PMCID: PMC5817193.

10. Guner ES, Watanabe M, Hashimoto N, Kadosaka T, Kawamura Y, Ezaki T, et al. Borrelia turcica sp. nov., isolated from the hard tick Hyalomma aegyptium in Turkey. Int J Syst Evol Microbiol. 2004;54(Pt 5):1649–52. pmid:15388724.

11. Loh SM, Gofton AW, Lo N, Gillett A, Ryan UM, Irwin PJ, et al. Novel Borrelia species detected in echidna ticks, Bothriocroton concolor, in Australia. Parasit Vectors. 2016;9(1):339. Epub 2016/06/16. pmid:27301754; PubMed Central PMCID: PMC4908759.

12. Gofton AW, Margos G, Fingerle V, Hepner S, Loh SM, Ryan U, et al. Genome-wide analysis of Borrelia turcica and 'Candidatus Borrelia tachyglossi' shows relapsing fever-like genomes with unique genomic links to Lyme disease Borrelia. Infect Genet Evol. 2018;66:72–81. Epub 2018/09/22. pmid:30240834.

13. Takano A, Fujita H, Kadosaka T, Konnai S, Tajima T, Watanabe H, et al. Characterization of reptile-associated Borrelia sp. in the vector tick, Amblyomma geoemydae, and its association with Lyme disease and relapsing fever Borrelia spp. Environmental microbiology reports. 2011;3(5):632–7. Epub 2011/10/01. pmid:23761345.

14. Adeolu M, Gupta RS. A phylogenomic and molecular marker based proposal for the division of the genus Borrelia into two genera: the emended genus Borrelia containing only the members of the relapsing fever Borrelia, and the genus Borreliella gen. nov. containing the members of the Lyme disease Borrelia (Borrelia burgdorferi sensu lato complex). Antonie van Leeuwenhoek. 2014;105(6):1049–72. Epub 2014/04/20. pmid:24744012.

15. Margos G, Marosevic D, Cutler S, Derdakova M, Diuk-Wasser M, Emler S, et al. There is inadequate evidence to support the division of the genus Borrelia. Int J Syst Evol Microbiol. 2017;67(4):1081–4. Epub 2016/12/09. pmid:27930271.

16. Stevenson B, Fingerle V, Wormser GP, Margos G. Public health and patient safety concerns merit retention of Lyme borreliosis-associated spirochetes within the genus Borrelia, and rejection of the genus novum Borreliella. Ticks Tick Borne Dis. 2018. Epub 2018/08/30. 30154058. pmid:30154058

17. Konstantinidis KT, Tiedje JM. Genomic insights that advance the species definition for prokaryotes. Proc Natl Acad Sci U S A. 2005;102(7):2567–72. pmid:15701695.

18. Qin Q-L, Xie B-B, Zhang X-Y, Chen X-L, Zhou B-C, Zhou J, et al. A Proposed Genus Boundary for the Prokaryotes Based on Genomic Insights. Journal of Bacteriology. 2014;196(12):2210–5. pmid:24706738

19. Becker NS, Margos G, Blum H, Krebs S, Graf A, Lane RS, et al. Recurrent evolution of host and vector association in bacteria of the Borrelia burgdorferi sensu lato species complex. BMC Genomics. 2016;17(1):734. Epub 2016/09/17. pmid:27632983; PubMed Central PMCID: PMC5025617.

20. Adamek M, Alanjary M, Sales-Ortells H, Goodfellow M, Bull AT, Winkler A, et al. Comparative genomics reveals phylogenetic distribution patterns of secondary metabolites in Amycolatopsis species. BMC Genomics. 2018;19(1):426. Epub 2018/06/03. pmid:29859036; PubMed Central PMCID: PMC5984834.

21. Gish W, States DJ. Identification of protein coding regions by database similarity search. Nat Genet. 1993;3(3):266–72. Epub 1993/03/01. pmid:8485583.

22. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004;5:113. Epub 2004/08/21. pmid:15318951; PubMed Central PMCID: PMC517706.

23. Barbour AG, Adeolu M, Gupta RS. Division of the genus Borrelia into two genera (corresponding to Lyme disease and relapsing fever groups) reflects their genetic and phenotypic distinctiveness and will lead to a better understanding of these two groups of microbes (Margos et al. (2016) There is inadequate evidence to support the division of the genus Borrelia. Int. J. Syst. Evol. Microbiol. ). Int J Syst Evol Microbiol. 2017;67(6):2058–67. Epub 2017/02/01. 10.1099/ijsem.0.001815. pmid:28141502

24. Margos G, Vollmer SA, Cornet M, Garnier M, Fingerle V, Wilske B, et al. A new Borrelia species defined by Multilocus Sequence Analysis of Housekeeping Genes. Appl Environ Microbiol. 2009;75(16):5410–6. pmid:19542332

25. Richter D, Postic D, Sertour N, Livey I, Matuschka FR, Baranton G. Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov. Int J Syst Evol Microbiol. 2006;56(Pt 4):873–81. pmid:16585709.

26. Baranton G, Postic D, Saint Girons I, Boerlin P, Piffaretti JC, Assous M, et al. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int J Syst Bacteriol. 1992;42(3):378–83. pmid:1380285.

27. Canica MM, Nato F, du Merle L, Mazie JC, Baranton G, Postic D. Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis. Scand J Infect Dis. 1993;25(4):441–8. pmid:8248743.