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. 2009 Jun 19;75(16):5410–5416. doi: 10.1128/AEM.00116-09

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes▿ †

Gabriele Margos 1,*, Stephanie A Vollmer 1, Muriel Cornet 2, Martine Garnier 2, Volker Fingerle 3, Bettina Wilske 4,§, Antra Bormane 5, Liliana Vitorino 6, Margarida Collares-Pereira 6, Michel Drancourt 7, Klaus Kurtenbach 1,‡
PMCID: PMC2725479  PMID: 19542332

Abstract

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.

Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).

Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.

LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).

In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).

Borrelia samples analyzed.

Borrelia strains used in this study are listed in Table 1.

TABLE 1.

LB species and strains used in this study

Strain Borrelia species Biological source (sample type)a Geographic sourceb Collector Culture collectionc Sourced GenBank accession no.
VS461T B. afzelii Ixodes ricinus Switzerland O. Peter M. Cornet
PKo B. afzelii Human Germany B. Wilske NC_008277
IBS-11 B. afzelii Human Alsace, France B. Jaulhac M. Cornet
IBS-12 B. afzelii Human Alsace, France B. Jaulhac M. Cornet
IBS-13 B. afzelii Human Alsace, France B. Jaulhac M. Cornet
IPT109 B. afzelii I. ricinus Alsace, France CNRB M. Cornet
IPT110 B. afzelii I. ricinus Alsace, France CNRB M. Cornet
IPT118 B. afzelii I. ricinus Auvergne, France CNRB M. Cornet
IPT122 B. afzelii I. ricinus Auvergne, France CNRB M. Cornet
IPT138 B. afzelii I. ricinus Alsace, France CNRB M. Cornet
IPT142 B. afzelii I. ricinus Alsace, France CNRB M. Cornet
IPT152 B. afzelii I. ricinus Limousin, France CNRB M. Cornet
IPT154 B. afzelii I. ricinus Limousin, France CNRB M. Cornet
IPT164 B. afzelii I. ricinus Auvergne, France CNRB M. Cornet
IPT179 B. afzelii I. ricinus Auvergne, France CNRB M. Cornet
20047T B. garinii I. ricinus France J. F. Anderson M. Cornet
PBi B. gariniie Human (CSF) Ingolstadt, Germany B. Wilske V. Fingerle
PFek B. gariniie Human (CSF) Munich, Germany B. Wilske V. Fingerle
PTrob B. gariniie Human (skin) Slovenia B. Wilske V. Fingerle
PRab B. gariniie Human (synovia) Villach, Austria B. Wilske V. Fingerle
POb B. gariniie Human (skin) Munich, Germany B. Wilske V. Fingerle
PFin B. gariniie Human (CSF) Munich, Germany B. Wilske V. Fingerle
PBN B. gariniie Human (CSF) Munich, Germany B. Wilske V. Fingerle
PScf B. gariniie Human (CSF) Munich, Germany B. Wilske V. Fingerle
PHoe B. gariniie Human (CSF) Munich, Germany B. Wilske V. Fingerle
PBaeI B. gariniie Human (CSF) Munich, Germany B. Wilske V. Fingerle
IPT28 B. garinii I. ricinus Alsace, France CNRB M. Cornet
IPT114 B. garinii I. ricinus Alsace, France CNRB M. Cornet
IPT126 B. garinii I. ricinus Alsace, France CNRB M. Cornet
IPT130 B. garinii I. ricinus Alsace, France CNRB M. Cornet
IPT139 B. garinii I. ricinus Alsace, France CNRB M. Cornet
IPT140 B. garinii I. ricinus Alsace, France CNRB M. Cornet
IPT145 B. garinii I. ricinus Limousin, France CNRB M. Cornet
IPT155 B. garinii I. ricinus Limousin, France CNRB M. Cornet
IPT156 B. garinii I. ricinus Auvergne, France CNRB M. Cornet
IPT157 B. garinii I. ricinus Limousin, France CNRB M. Cornet
IPT158 B. garinii I. ricinus Limousin, France CNRB M. Cornet
IPT165 B. garinii I. ricinus Auvergne, France CNRB M. Cornet
IPT167 B. garinii I. ricinus Limousin, France CNRB M. Cornet
IPT168 B. garinii I. ricinus Limousin, France CNRB M. Cornet
IPT169 B. garinii I. ricinus Auvergne, France CNRB M. Cornet
IPT171 B. garinii I. ricinus Auvergne, France CNRB M. Cornet
IPT172 B. garinii I. ricinus Auvergne, France CNRB M. Cornet
IPT178 B. garinii I. ricinus Auvergne, France CNRB M. Cornet
IPT189 B. garinii I. ricinus Normandy, France CNRB M. Cornet
IPT195 B. garinii I. ricinus Normandy, France CNRB M. Cornet
VS116T B. valaisiana I. ricinus Switzerland O. Peter M. Cornet
IPT29 B. valaisiana I. ricinus Meuse, France CNRB M. Cornet
IPT31 B. valaisiana I. ricinus Meuse, France CNRB M. Cornet
IPT33 B. valaisiana I. ricinus Meuse, France CNRB M. Cornet
IPT47 B. valaisiana I. ricinus Alsace, France CNRB M. Cornet
IPT85 B. valaisiana I. ricinus Alsace, France CNRB M. Cornet
IPT102 B. valaisiana I. ricinus Auvergne, France CNRB M. Cornet
IPT111 B. valaisiana I. ricinus Alsace, France CNRB M. Cornet
IPT121 B. valaisiana I. ricinus Alsace, France CNRB M. Cornet
IPT144 B. valaisiana I. ricinus Limousin, France CNRB M. Cornet
IPT153 B. valaisiana I. ricinus Limousin, France CNRB M. Cornet
IPT163 B. valaisiana I. ricinus Auvergne, France CNRB M. Cornet
IPT174 B. valaisiana I. ricinus Auvergne, France CNRB M. Cornet
IPT177 B. valaisiana I. ricinus Limousin, France CNRB M. Cornet
IPT184 B. valaisiana I. ricinus Limousin, France CNRB M. Cornet
IPT186 B. valaisiana I. ricinus Limousin, France CNRB M. Cornet
IPT187 B. valaisiana I. ricinus Limousin, France CNRB M. Cornet
IPT188 B. valaisiana I. ricinus Normandy, France CNRB M. Cornet
IPT2 B. burgdorferi I. ricinus Alsace, France CNRB M. Cornet
IPT19 B. burgdorferi I. ricinus Alsace, France CNRB M. Cornet
IPT23 B. burgdorferi I. ricinus Alsace, France CNRB M. Cornet
IPT39 B. burgdorferi I. ricinus Alsace, France CNRB M. Cornet
IPT58 B. burgdorferi I. ricinus Alsace, France CNRB M. Cornet
IPT69 B. burgdorferi I. ricinus Alsace, France CNRB M. Cornet
IPT135 B. burgdorferi I. ricinus Auvergne, France CNRB M. Cornet
IPT137 B. burgdorferi I. ricinus Alsace, France CNRB M. Cornet
IPT190 B. burgdorferi I. ricinus Normandy, France CNRB M. Cornet
IPT191 B. burgdorferi I. ricinus Normandy, France CNRB M. Cornet
IPT193 B. burgdorferi I. ricinus Normandy, France CNRB M. Cornet
IPT198 B. burgdorferi I. ricinus Normandy, France CNRB M. Cornet
NE49 B. burgdorferi I. ricinus Switzerland L. Gern M. Cornet
Z41293 B. burgdorferi I. ricinus Germany M. Wittenbrink M. Cornet
Z41493 B. burgdorferi I. ricinus Germany M. Wittenbrink M. Cornet
B31T B. burgdorferi I. scapularis Shelter Island, NY, USA A. Barbour NC_001318
PoHL1 B. lusitaniae Human Lisbon, Portugal M. Collares-Pereira M. Collares-Pereira
PoTiBL37 B. lusitaniae I. ricinus Mafra, Portugal S. Baptista M. Collares-Pereira
PoTiBGr41 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr82 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr130 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr131 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr136 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr143 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr209 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr211 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr213 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr288 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr293 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
PoTiBGr409 B. lusitaniae I. ricinus Grândola, Portugal A. Quaresma M. Collares-Pereira
CA128 B. bissettii I. spinipalpis California, USA R. S. Lane CDC M. Schriefer
gom93-283 B. bissettii I. spinipalpis Colorado, USA G. O. Maupin CDC M. Schriefer
gom93-299 B. bissettii I. spinipalpis Colorado, USA G. O. Maupin CDC M. Schriefer
A14S B. spielmanii Human The Netherlands GPID 28635
DAH B. hermsii Human Washington, DC, USA NC_010673
91E135 B. turicatae Ornithodoros turicatae USA NC_008710
Ly B. duttonii Human Tanzania M. Drancourt
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a

CSF, cerebrospinal fluid.

b

USA, United States of America.

c

CNRB, Centre National de Référence des Borrelia, Institute Pasteur, Paris, France; CDC, Centers for Disease Control and Prevention, Fort Collins, CO.

d

The strains were provided by M. Cornet, V. Fingerle, M. Collares-Pereira, M. Schriefer, and M. Drancourt.

e

In this study, we suggest that these strains, assigned previously to B. garinii, be made a new species, Borrelia bavariensis sp. nov.

DNA extraction, primers, and PCR conditions.

Genomic DNA was extracted and purified from cultured isolates as described earlier (10, 30, 48). The loci analyzed comprised the “housekeeping” genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA), the 5S-23S IGS, ospA, and ospC. Primers and PCR conditions have been described in detail previously (14, 25, 30, 34). Two new outer primers (clpA1237F and clpA2218R) and a new inner forward primer (clpA1258F) were designed for clpA: clpA1237F, 5′-AAAGATAGATTTCTTCCAGAC-3′; clpA2218R, 5′-GAATTTCATCTATTAAAAGCTTTC-3′; and clpA1258F, 5′-AAAGCTTTTGATATTTTAGATG-3′. PCR conditions remained the same as published previously (30).

Sequence analyses.

Sequence analyses were performed as described elsewhere (30), and further information can be found in supplemental material. Values of the pairwise genetic distances enabled us to determine the threshold levels of sequence divergence between species using strains B31, Z41293, and NE49 (37, 40).

MLSA and phylogeny.

MLSA and phylogenetic analyses were performed as described previously (30). MEGA 4 (21) was used for sequence alignments, and MrBayes 3.1.2 software (19) was used for phylogenetic analyses. Only the phylogenetic trees generated using MLSA were rooted. B. lusitaniae sequences were omitted from ospA and ospC analyses because of insufficient sequence overlap for phylogenetic analyses (48). For detailed information, see supplemental material.

In this study, a novel MLSA scheme has been used to study the phylogenetic relationships within and among the most common species of LB group spirochetes, i.e., Borrelia burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii (Table 1). Species defined by other methods formed discrete clusters in phylogenetic trees of concatenated housekeeping genes (Fig. 1). Strains from different species did not share any alleles, suggesting that horizontal gene transfer at these chromosomal loci did not occur between species of LB group spirochetes (see Table S2 in the supplemental material). B. garinii represented the most diverse species in this analysis. However, OspA serotype 4 strains (PBi and PBi-like strains) formed a fairly uniform group that was distinct from all other B. garinii strains (Fig. 1) (see Tables S2 and S3 in the supplemental material). Our study suggests that these strains, previously assigned to B. garinii, are sufficiently divergent genetically and ecologically to raise them to species status.

FIG. 1.

FIG. 1.

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Rooted Bayesian phylogenetic inference of concatenated housekeeping gene sequences of LB group spirochetes. Posterior probability values of clades are provided. Previously assigned Borrelia species are color coded as follows: red, B. burgdorferi (Bb) sensu stricto; blue, B. afzelii (Ba); green, B. garinii (Bg) or B. bavariensis sp. nov.; yellow, B. valaisiana (Bv); purple, B. bissettii (Bbis); sky blue, B. spielmanii (Bsp); and pink, B. lusitaniae (Bl). The tree was rooted with sequences of the relapsing fever spirochetes Borrelia duttonii, B. hermsii, and B. turicatae. The branch length of the outgroup is not according to scale as indicated by the slashes. The bar labeled 0.1 depicts 10% divergence.

For LB group spirochetes, new species have been delineated recently using a different MLSA scheme which showed agreement with whole DNA-DNA hybridization (37, 40). To compare the genetic distances obtained by our MLSA scheme with previously reported values, we included type strains of B. burgdorferi, B. afzelii, B. garinii, and B. valaisiana. The pairwise genetic distances between the type strains ranged from 0.06 to 0.08 (see Table S4 in the supplemental material), a similar range as described previously (37, 40). B. burgdorferi strains Z41293 and NE49 have been used to define species threshold levels (pairwise genetic distance for strains B31/Z41293 = 0.021) for the MLSA scheme developed by Richter and colleagues (37, 40). Using these strains, we determined the species threshold level for our MLSA system to be 0.0170 (see Table S4 in the supplemental material).

The pairwise genetic distance of the concatenated housekeeping genes between B. garinii type strain 20047 and PBi-like strains was 0.0200, which is much higher than the genetic distance of strain 20047 to other bird-transmitted B. garinii isolates. This genetic distance in combination with the ecological differences (see below) would justify raising OspA serotype 4 strains to species status. We propose the name Borrelia bavariensis sp. nov., because it was first found in Bavaria, Germany.

In the Bayesian phylogenetic tree (Fig. 1) based on the concatenated sequences of the eight housekeeping genes, strain PBi and related OspA serotype 4 strains formed a distinct subclade, being basal to the remaining B. garinii strains in 100% of the trees. This was found regardless of the tree building method used (Fig. 1) (see Fig. S5 in the supplemental material). In phylogenetic analysis of the 5S-23S IGS, strain PBi and related OspA serotype 4 strains (with the exception of strain PHoe) also formed a distinct cluster within the B. garinii clade (see Fig. S2 in the supplemental material).

In our study, the housekeeping genes showed different evolutionary pathways compared with the plasmid-located genes ospA and ospC (see Fig. S3 and S4 in the supplemental material), which is consistent with reports on plasmid exchange and/or genetic recombination (2, 26, 39, 49, 50). While in the tree generated using ospC sequences, strains from all species were dispersed into different clusters, including mixed-species clusters (see Fig. S4 in the supplemental material), in the ospA tree both B. garinii and B. valaisiana strains were split into two separate clusters (see Fig. S3 in the supplemental material). The division of B. valaisiana strains into two separate groups using OspA protein sequences has been reported previously, and it has been proposed that their genes evolved from two distinct ancestors (50).

The evolutionary relationships among the LB group spirochetes included in this study support the hypothesis that bird specialization has been acquired at least twice independently (i.e., in B. valaisiana and in B. garinii). B. valaisiana and B. garinii were found to form distinct sequence clusters in phylogenetic trees of MLSA sequences (Fig. 1). However, they are, with the exception of OspA serotype 4 strains, very similar ecologically, in that they occur sympatrically and utilize the same spectrum of tick vectors and vertebrate hosts, i.e., birds (16, 22, 24, 45). It is likely that B. valaisiana and B. garinii evolved allopatrically, which may explain their pronounced genetic distance. Strains assigned to B. garinii by other methods, on the other hand, represent at least two ecotypes (rodent-associated ecotype versus bird-associated ecotype), which are congruent with distinct clusters revealed by MLSA. Although a previous study suggested that OspA serotype 4 strains represent a recently emerged clonal lineage within B. garinii (28), such strains (PBi and related strains) were found to be at the base of the B. garinii clade in the MLSA tree. This suggests that specialization of OspA serotype 4 strains to rodents is a more ancient trait and that genetic elements of B. garinii that allow its transmission by birds were acquired more recently. A more recent adaptation of B. garinii to birds as reservoir hosts could also explain the present-day sympatric distribution of B. valaisiana and B. garinii. It also suggests that adaptation to avian hosts evolved at least twice independently in the LB group of spirochetes (i.e., in B. valaisiana and B. garinii). Phylogenetic studies of other bird-associated LB species, such as B. turdi, may provide more information on the evolution of host specialization.

The consistency in species clustering and the fact that trees can be rooted using sequences of relapsing fever spirochetes highlights the suitability of MLSA based on housekeeping genes for evolutionary studies of LB group spirochetes. LB spirochetes comprise distinct ecotypes that are broadly defined by their spectrum of vertebrate hosts (22, 24). Ecotypes of LB group spirochetes can, therefore, be determined more easily than for free-living bacteria. Raising sufficiently divergent sequence clusters of LB group spirochetes, which correspond to ecotypes, to bacterial species status would have the advantage of being ecologically, epidemiologically, and clinically predictive.

Nucleotide sequence accession numbers.

The sequences of the housekeeping genes have been submitted to the MLST/MLSA website hosted at Imperial College London (http://www.mlst.net) and can be accessed via the strain name or sequence type. The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers are as follows: for 5S-23S IGS, FJ546482 to FJ546547 and GQ178225 to GQ178231; for ospA, FJ546596 to FJ546657 and GQ178232 to GQ178244; and for ospC, FJ546548 to FJ546595, GQ178223, and GQ178224.

Supplementary Material

[Supplemental material]
supp_75_16_5410__index.html (2.6KB, html)

Acknowledgments

This work was supported by The Wellcome Trust, London, United Kingdom (grant 074322/Z/04/Z).

We thank D. Davis for help in using “Aquila” for MrBayes analyses, Marty Schriefer and Andrias Hoigaard, CDC, Fort Collins, CO, for providing B. bissettii DNA, L. Hurst for suggestions on sequence alignments, E. Feil for helpful discussions, and B. Spratt and D. Aanensen for curating the MLST website (http://www.mlst.net).

Footnotes

▿

Published ahead of print on 19 June 2009.

†

Supplemental material for this article may be found at http://aem.asm.org/.

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