1
Division of the genus Borrelia into two genera (corresponding to Lyme disease and relapsing fever
2
groups) reflects their genetic and phenotypic distinctiveness and will lead to a better understanding of
3
these two groups of microbes (Margos et al. (2016) There is inadequate evidence to support the
4
division of the genus Borrelia. Int. J. Syst. Evol. Microbiol. doi: 10.1099/ijsem.0.001717)
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6
Alan G. Barbour1, Mobolaji Adeolu2 and Radhey S. Gupta2*
7
1
8
Biology, University of California Irvine, Irvine, California, USA
9
2
10
Departments of Medicine, Microbiology & Molecular Genetics, and Ecology & Evolutionary
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario,
Canada.
11
12
* Corresponding Author
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Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario,
14
Canada. Email: [email protected]
15
16
17
The members of the family Borreliaceae include species that are the causative agents of Lyme
18
disease (LD) and others that are the agents of tick- and louse-borne relapsing fever (RF) [1-3]. Recent
19
phylogenetic and comparative analyses of protein sequences from Borreliaceae genomes have
20
provided compelling evidence that the Borreliaceae species in fact comprise two genetically distinct
21
groups and that they can be reliably distinguished on numerous independent molecular characteristics
22
[4]. Based on these phylogenetic/genomic studies, as well as supporting phenotypic characteristics,
23
Adeolu and Gupta [4] proposed the division of the genus Borrelia into two genera. In that proposal an
24
emended genus Borrelia retained all the species known to cause the clinically and epidemiologically
25
distinctive disorder of RF, while all agents of LD, as well as closely-related species not known to cause
26
human disease, were placed into a new genus, Borreliella [4]. This latter group has been widely
27
referred to as "Borrelia burgdorferi sensu lato", which has functioned as a short-hand for
28
distinguishing this increasingly numerous but cohesive group from all other Borrelia species. Although
29
the family Borreliaceae also contains the species Cristispira pectinis with a validly published name
30
[5,6], there is no cultured isolate or sequence information available for this species. Thus, for all
31
practical purposes, the family Borreliaceae was originally comprised of only of a single genus,
32
Borrelia, which is now split into two genera, reflecting the long standing recognition of two groups
1
33
within it, i.e "B. burgdorferi sensu lato" (also termed the “Lyme disease group of spirochetes” or
34
“Lyme disease Borrelia”) and the RF clade.
35
Margos et al. [7] in a Letter to the Editor in the IJSEM criticized the splitting the family
36
Borreliaceae into two genera on three major grounds: (i) The evidence based on genomic
37
characteristics distinguishing the two proposed groups was preliminary. (ii) Some Borreliaceae species
38
do not adhere to the typical phenotypic patterns of the LD and RF groups, thus blurring the distinction
39
between the groups of disease agents. (iii) This division would have various “adverse consequences”.
40
While recognizing that a name change for a medically-important microbe is never to be taken lightly,
41
we respectfully disagree with the conclusions of Margos et al. [7] and discuss point-by-point the issues
42
raised in their letter. We show that much of the criticism of splitting the family Borreliaceae into two
43
genera is unfounded.
44
45
A. Genomic evidence for the cohesiveness of the family Borreliaceae and for the presence of two
46
distinct groups within this family
47
The phylogram presented in Figure 1 of Borreliaceae species for which there are genome
48
sequences, summarizes and updates the evidence for two cohesive groups/genera within the family.
49
The tree is based on concatenated sequences of 762 proteins comprising the core proteins in the
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genomes of Borreliaceae species. The tree is very similar in topology and branch support to the earlier
51
tree that was based on 25 conserved proteins from fewer species [4]. As can be seen, the Borreliaceae
52
species form two distinct clades, which are resolved with high (~100%) statistical support,
53
corresponding to the RF and the LD group of species. Although all Borreliaceae species studied to
54
date have a similar G+C nucleotide contents of ~29% for their chromosomes, 5 B. burgdorferi sensu
55
lato species had significantly higher CG dinucleotide contents for a given G+C content than 8 RF
56
group species, including B. miyamotoi and B. anserina [2].
57
Figure 1 also summarizes and maps the different molecular markers that were identified
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previously [4], namely, conserved signature insertions/deletions (CSIs) and conserved signature
59
proteins (CSPs), which are either specifically characteristic of all Borreliaceae species or uniquely
60
characteristic of one or the other of the two observed clades. Margos et al. [7] in their critique did not
61
acknowledge the greater than 100 molecular markers that were described for the family Borreliaceae
62
and they also overlooked 38 other described CSIs (Table 4 of Adeolu and Gupta [4]article), which
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distinguish the LD and the RF groups of Borreliaceae species.
64
65
A gauge of the utility of a categorization protocol is whether the distinguishing criteria (i.e., the
“rules”) hold as new organisms are characterized and then become candidates for taxonomic
2
66
classification. Since the publication of the paper describing the proposed split of the genus Borrelia,
67
genome sequences for 5 additional Borreliaceae species (viz. B. coriaceae, B. finlandensis, B.
68
japonica, B. mayonii and B. chilensis) became available. B. coriaceae groups with the RF clade, and
69
the other four species group in the LD clade (Figure 1). We examined the distribution (i.e.
70
presence/absence) of different described molecular markers in these species. Significantly, all of the
71
described CSIs and CSPs specific for the family Borreliaceae as a whole are present in all 5 of these
72
species. Likewise, all of the CSIs and CSPs for the RF group are present in B. coriaceae, but they are
73
lacking in B. finlandensis, B. japonica, B. mayonii and B. chilensis. The complementary pattern is
74
observed for the distribution of molecular markers that are specific for the LD group of Borreliaceae
75
species. Sequence information for one representative updated CSI each for the LD and RF group of
76
species is presented in Figure 2. Based on these results, the described molecular markers are not only
77
specific for the two groups of Borreliaceae species, but they continue to exhibit a high degree of
78
predictive ability to be found in any new species belonging or related to these groups.
79
Margos et al.[7] also criticised the Adeolu and Gupta [4] article for excluding information for a
80
newly-described but relatively uncharacterized group of Borreliaceae species that use reptiles as
81
reservoir hosts. However, this reading of the article is incorrect. The 16S rRNA phylogram (Figure 2)
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in the Adeolu and Gupta [4] article in fact includes B. turcica, which constitutes the species from this
83
group with validly published name and its position was indicated to be indeterminate based on the
84
presented results. However, in the phylogenetic tree presented by Margos et al. [7], which includes B.
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turcica and other related reptile-associated strains of Borreliaceae without taxonomic standing, these
86
species/strains exhibit the same pattern of branching as described by Takano et al. [8], which was
87
acknowledged by Adeolu and Gupta [4]. It should be noted though that in other recent studies [9,10],
88
the position of the REP-associated Borrelia species with respect to its branching position was found to
89
be variable. Thus, further studies based on whole genome data are needed to clarify the position of B.
90
turcica and the ‘reptile group’ of strains.
91
But let us prospectively consider how more sequence information, as it becomes available, for
92
this newly-identified group of Borreliaceae spirochetes (or, for that manner, any new species related to
93
this family) might affect the proposed division for this family. Any new species belonging to the
94
family Borreliaceae could exhibit one of the three branching patterns noted below. The distribution
95
patterns of the described signatures for three possible branching patterns involving a new species can
96
be predicted as follows:
97
1.
The species branches either within the LD group or as an outgroup of this clade. In this
98
case, it is predicted that the sequenced strain (as seen for B. finlandensis, B. japonica, B.
3
99
mayonii and B. chilensis) will contain either some or all of the signatures specific for the LD
100
group, but generally none for the RF group, indicating that the strain is related to the LD group
101
of species. The addition of B. finlandensis, B. japonica, B. mayonii and B. chilensis are
102
examples of this branching pattern (Figure 1).
103
2.
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clade, such a strain (as seen with B. coriaceae) is expected to contain either some or all of the
105
signatures for the RF clade, but generally none for the LD group. The addition of B. coriaceae
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is an example of this branching pattern (Figure 1).
107
3.
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in phylogenetic trees. While harboring at least some of the signatures for the family
109
Borreliaceae it does not contain any of the signatures specific for the LD or the RF clades. In
110
this case, further comparative genomic studies on this group of species/strains need be carried
111
out to identify molecular markers specific for this clade. Depending upon the other genetic and
112
phenotypic characteristics of these species/strains, consideration that they may form a separate
113
genus within the family Borreliaceae would be warranted.
The new species/strain branches either within the RF group or as an outgroup of this
The sequenced species/strain forms outgroup of both the LD and RF groups of species
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115
Based upon the distribution pattern of the described signatures in any new species, it should be
116
possible to establish, in all cases, whether the newly-described species is a part of the described LD or
117
RF groups, or, alternatively, it forms a clade that is distinct from these two groups. Thus, in contrast to
118
the assertion of Margos et al. [7] that, as new Borreliaceae species are discovered the distinction
119
between the two groups will become increasingly blurred, we predict that the molecular markers
120
described here will prove instrumental in unambiguously establishing the relationship of newly
121
identified species to either the RF or the LD groups of species and that this can provide insights into
122
the biology of these organisms. The notion that there is a smooth continuum of species out there--with
123
all possible permutations in current existence, waiting only to be discovered--does not reflect the
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general experience of evolutionary biology.
125
Margos et al. [7] also questioned the signature value of a few of the CSPs, because they are
126
lacking in an isolated species. However, the loss of a gene in a particular species, which is otherwise
127
uniquely shared by all other members of the group, does not significantly diminish its value as a
128
signature or diagnostic characteristic for the group. Additionally, they also critiqued the Adeolu and
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Gupta [4] article on the grounds that a number of CSPs that distinguish between the two groups of
130
Borreliaceae are of unknown function at the time of annotation and, for that reason, are of limited
131
utility as useful signatures. However, this contention appears to reflect a misunderstanding of the
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earlier work on CSPs for other groups of prokaryotes, including Alphaproteobacteria, Actinobacteria,
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the PVC superphylum, Halobacteria, and Chlamydiae [11-15]. Most of these CSPs were of unknown
134
function at the time they were first characterized, however further studies have shown them to be
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highly reliable molecular markers for these groups, and biochemical studies on them are leading to
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discovery of novel and important functions that were hitherto unrecognized [16-20]. Thus, not
137
knowing the function of a conserved protein that is uniquely shared by a given group of species should
138
not be a cause for criticism. Rather, it should be recognized as an opportunity and a challenge, as
139
indicated below, to understand its function and why it is uniquely present in one group of organisms
140
but not another [21].
141
Margos et al. [7] present a figure showing high synteny between the chromosomes of B.
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burgdorferi and both an example from the LD group and an example from the RF group (A similar
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analysis had earlier been published by one of us [2]. However, this was principally a demonstration of
144
conservation of gene order and indirect evidence of the rarity of chromosome rearrangements, such as
145
inversions and large duplications, in these taxa. This is plausibly attributable to the absence or rarity of
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insertion sequences, transposable elements, or lysogenic phages in the chromosome or, alternatively, to
147
imposed constraints from the linear organization of their chromosomes [22]. Moreover, the analysis by
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Margos et al. [7] excludes the multiple plasmids that are present in different Borreliaceae species
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which make up approximately one-third of overall genetic content. One of the distinguishing features
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of the RF group of species, inlcuding B. anserina (GenBank accession number CP014325), is the
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presence of large linear plasmids (megaplasmid) of 90-190 kb, which have limited synteny and
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similarity with any single plasmid of a LD group species [23]. The chromosomes and megaplasmids in
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the RF clade appear to be co-evolving [2].
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More informative than chromosome gene order for inferring evolutionary relationships are
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genome-wide pairwise comparisons of genes and proteins that species have in common. Adeolu and
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Gupta [4] previously reported pairwise comparisons of the genomic average nucleotide identity (ANI)
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values between species from the RF group and those in the LD group. ANI is a widely accepted
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criterion for determining the genomic relatedness of species at species and genus levels [24-26]. The
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ANI analysis showed that the species from the RF clade are distinct from those in the LD clade and the
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differences in ANI values between the two groups support their placement into two distinct genera [4].
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In Figure 3, we present here an average amino acid (AAI) similarity matrix for all Borreliaceae species
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for which genomes have been sequenced and are publicly available. The Borreliaceae species again
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form two distinct groups, corresponding to the two proposed genera.
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In conclusion, all the different lines of evidences based on genome sequence data strongly
support the existence of two distinct and cohesive groups within the family Borreliaceae.
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B. Phenotypic traits distinguishing the relapsing fever group and the Lyme disease group of
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spirochetes
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Here, we review the informative phenotypic traits of "Borrelia burgdorferi sensu lato", which
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either singly or in combination, serve to distinguish this clade from the original members of the genus
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Borrelia (i.e. the RF clade), which mostly comprise the several different species that cause relapsing
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fever but also include the long-recognized agents of avian spirochetosis (B. anserina) and bovine
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borreliosis (B. theileri), as well the more recently described disease agents, like B. miyamotoi. Margos
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et al. [7] contend that the genus Borrelia prior to the proposed split was “cohesive” due to their “shared
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spirochaetal morphology” and “common vector-borne style”, but then list exceptions to this broad
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statement.
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An obvious difference between the two clades, and one of undoubted medical and public health
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relevance, is that one cluster includes all the known species that cause Lyme disease and the other
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cluster includes all the known species that cause relapsing fever [1-3]. The differences in the diseases
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that occur in humans have their counterparts in natural and experimental infections in mammalian
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hosts [3,27-30]. In comparison to infections by the RF group of species (including B. anserina and B.
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miyamotoi), infections caused LD group species tend to be chronic and with higher persistent densities
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of spirochetes in tissues rather than the blood [31-33]. During RF in humans and experimental animal
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models, spirochetes in unconcentrated blood are usually detectable by light microscopy of thin blood
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smears. In fact, this is still the most common method for laboratory diagnosis of relapsing fever
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[34,35].
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The newly-described LD agent B. mayonii was reported to reach higher densities in the blood
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of infected patients than was observed during infections with B. burgdorferi [36], but Pritt et al.’s
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estimated cell densities for B. mayonii in the blood of 104-105 per milliliter are at least two orders of
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magnitude lower than what is commonly observed during relapsing fever in humans and animal
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models. Moreover, contrary to the reported findings in some human cases, B. mayonii spirochetes were
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not detected by Dolan et al. [37] in the blood of experimentally-infected mice while they persistently
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had spirochetes in skin tissue and remained infectious for ticks. This pattern of few or no spirochetes in
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the blood while they persist in the tissues fits the phenotypes of other LD species. In our opinion,
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Margos et al.'s [7] statement that “B. mayonii produced high spirochetal blood densities, akin to that
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seen following infection with species of the RF group” is not an accurate interpretation of the
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literature. Contrary to Margos et al.’s [7] assertion the “symptomology [sic] that differentiates [human
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infection from] RF spirochaetes from [human infection from] the LB group of spirochaetes” has not
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been further “blurred”.
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Two vector-related traits that stand out for differentiating between the two groups of
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spirochetes are the location of the spirochetes in an unfed tick at the time that the blood meal
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commences [38,39], and the maintenance of the spirochete in nature by transovarial transmission [40-
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42].
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The vectors for the RF group of species represent a variety of arthropods, including the body
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louse, argasid (or soft) ticks, and the two major types, prostriate and metastriate, of ixodid (or hard)
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ticks [3,41,43]. Except for the special case of B. recurrentis, which is not transmitted by its louse
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vector through saliva [39], all the other RF group species that have been investigated to this point are
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disseminated from the midgut to the salivary glands before the subsequent molt and the next blood
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meal. This is of medical and ecological importance, because it means transmission of the pathogen to
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the host can begin soon after feeding begins. These include species that are transmitted by either
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argasid, prostriate, or metastriate ticks: B. duttonii in Ornithodoros moubata [44], B. anserina in Argas
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spp. [45], B. theileri in Rhipicephalus (Boophilus) microplus [46], B. crocidurae in Ornithodoros spp.
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[47], B. hermsii in O. hermsi [48], B. turicatae in O. turicata [49,50], and B. miyamotoi in Ixodes
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scapularis (personal communication, Linda Bockenstedt, Yale University). We note that this
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characteristic of the RF clade holds even for Borrelia spp. transmitted by hard ticks, which are slow-
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feeding in contrast to the rapid-feeding soft ticks, such as Ornithodoros and Argas spp.
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This characteristic contrasts with the findings for B. burgdorferi sensu lato, for which the only
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known competent vectors are certain species of the prostriate genus Ixodes. In their consensus
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summary of a workshop on the relationship between the pathogen and its arthropod vector, Burgdorfer
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et al. [51] wrote that “...the Lyme disease spirochete in its Ixodes vectors remains in the midgut, where
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it aggregates near the microvillar brush border and in the intercellular spaces of the gut epithelium”.
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Subsequent studies have confirmed this (reviewed in [38,39]). Previous reports of dissemination of a
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Lyme disease agent in unfed ticks were before B. miyamotoi was discovered and may have been
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misidentification of B. miyamotoi as B. burgdorferi or other Lyme disease agent by non-specific stains
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or cross-reactive polyclonal antisera.
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Most species in the RF group feature transovarial transmission in their tick vectors; this
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phenomenon has not been documented in any member of B. burgdorferi sensu lato to date [40-42].
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Transovarial transmission has been shown for RF group species transmitted by hard ticks as well as
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soft ticks. These include B. theileri in the metastriate tick R. microplus [46], B. miyamotoi in the
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prostriate I. scapularis [52], and B. lonestari in the metastriate Amblyomma americanum [53]. Older
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reports of transovarial transmission of B. burgdorferi sensu lato in Ixodes spp. ticks are attributable to
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misidentification of B. miyamotoi for a Lyme disease species [42]. Transovarial maintenance is of
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medical and ecological importance, because it confers the capacity for larval ticks to transmit the
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pathogen to humans and other vertebrates.
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We end this survey with a consideration of morphologic differences that were observed
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between the “Lyme disease spirochetes” and other Borrelia spp. In their 1984 paper Schmid et al. [54]
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noted that “the Lyme disease spirochetes differ from Borrelia spp. morphologically”. Johnson et al.
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[55] and Hovind-Hougen [56] distinguished between the new spirochetes and known Borrelia spp. in
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the number of insertion points for flagella they had at their ends. While ultrastructural morphology is
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infrequently used at present in descriptions of bacteria, a literature review reveals that even as
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additional species were added to the B. burgdorferi sensu lato cluster, the original observation of
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comparatively fewer flagella in “Lyme disease spirochetes” than in other Borrelia spp. has generally
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held up. The most commonly reported values for flagella at one end of cells of B. burgdorferi sensu
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lato is 7-8, within a range of 4-12, for the following species: B. burgdorferi [3,57-60]; B. afzelii and B.
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garinii [58-60]; B. sinica [61]; and B. japonica [62].
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Except for B. anserina, which was reported to have 7-8 flagella at an end [59], all other
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examples of RF group species that have been adequately examined by ultrastructural criteria have had
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an estimated 15-30 flagella at one end. These include B. recurrentis [57,59], B. hermsii [63,64], B.
249
microti [59,65], and B. persica [66]. B. recurrentis was reported to have 8 flagella at each end, but the
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insertion points are not well visualized in the figure that was provided [67].
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In conclusion, the phenotypic differences between the RF and LD clades, including the diseases
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that members cause, and the ecological and medical significances of these differences are greater than
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what Margos et al.[7] claim in their letter.
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C. Possible adverse consequences of the reclassification of members of the Family Borreliaceae
Margos et al. [7] write of “adverse consequences of splitting the genus Borrelia”. There is only
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limited space in this venue to address these undocumented assertions. Suffice it to say that there are
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counter-arguments about the "adverse consequences" of continued dependence on "Borrelia
259
burgdorferi sensu lato" as a genus proxy for an expanding group of species, only a few of which are
260
truly associated with human disease.
261
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But we first anticipate another potential criticism: allusion to the attempted split of the
medically-important genus Chlamydia into two genera, Chlamydia and Chlamydophila [68], as an
8
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argument against splitting Borreliaceae species. But the analogy between the taxonomic circumstances
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for these two sets of pathogens can be taken only so far. The evidence in favor of this split of
265
Chlamydia was comparatively weak [69]. The two proposed genera exhibit a high level of similarity in
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their 16S rRNA gene sequences and no consistent phenotypic differences [70,71].
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In another, more recent example of name change for a medically-important organism, the
268
species Clostridium difficile was transferred to a new genus, Clostridioides, based primarily on its
269
branching in a separate clade in the 16S rRNA tree and some protein trees from that corresponding to
270
the type species of the genus Clostridium butyricum [72,73]. The only other molecular evidence used
271
in support of the reclassification of C. difficile was a number of CSIs that Gupta and Gao [74]
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previously described for the “Clostridium sensu stricto” clade that were absent in C. difficile.
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In comparison to the Chlamydia and Clostridium proposals, the case for the existence of two
274
cohesive groups within the genus Borrelia is much stronger. From the presented evidence, it is clear
275
that the members of the family Borreliaceae comprise two distinct groups differing from each other in
276
numerous genetic and genomic characteristics, phenotypic characteristics, and in the diseases they
277
cause in humans and other animals. As stated above, prior to the proposal for the split, the genus
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Borrelia was functionally treated as different two groups, with the LD group pervasively referred to as
279
“B. burgdorferi sensu lato” or “Lyme disease Borrelia” to distinguish those species from the RF agents
280
and other Borrelia species [1,2,75]. Thus, the proposal for splitting the family Borreliaceae into two
281
groups only formalizes the existence of two de facto cohesive groups within this family, which were
282
already widely recognized [1,2,75].
283
Before addressing specific concerns, we also highlight some benefits of a clarification of
284
phylogenetic relationships for better understanding the microbiology and ecology of these pathogens.
285
Extensive work on these sequence-based characteristics markers on other groups of prokaryotes have
286
shown that these markers possess high degree of predictive ability to be identified in other group
287
members, for which no sequence information may be available at the time (reviewed in [76,77]. With
288
the more explicit division of the Borreliaceae species into two genus level groups, attention can be
289
focused on unique genetic and molecular characteristics that differentiate the two groups of microbes
290
[78]; the described characteristics in other groups of bacteria have been found to play important and
291
generally essential roles [79]. For Borreliaceae species, some of the CSIs that distinguish between the
292
RF and LD groups are in proteins, such as the flagellar motor switch protein FliM, chemotaxis proteins
293
CheY and CheW, and cell shape determining protein MreB [4], that may be determinants of the
294
aforementioned morphological differences between Borreliaceae species.
9
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Margos et al.[7] write that “splitting the genus does not provide any assistance as far as clinical
296
evaluation is concerned” and further that “[it] does not help end user communities including in clinical
297
medical practice, public health, or those studying the ecology of the bacteria”. They more
298
provocatively write that this “division complicates an already complicated situation which will serve
299
only to lead to further confusion among scientists, clinicians, public health authorities and the general
300
public.” These claims are not developed or documented in the Letter. Many journal readers, especially
301
the majority who are not specialists in the field of LD, may be mystified about what the "complicated
302
situation" is. One could reasonably make the counter-argument that timely diagnosis and treatment of
303
RF, a Neglected Tropical Disease (http://journals.plos.org/plosntds) and an infection with a higher
304
mortality rate than LD [35], is hindered by the “confusion” from the plethora of Borrelia species
305
names and the mistaken assumption that if it is a "Borrelia", it means Lyme disease.
306
That said, we acknowledge the argument for retaining the genus name Borrelia for the LD
307
group of species, if for no other reason than the most commonly-known species in the genus is B.
308
burgdorferi. However, B. anserina is the type species of the genus Borrelia and is part of the RF clade
309
(Figure 1). Hence, according to the rules governing the description of any new genus, the genus name
310
Borrelia must be retained for the RF clade [1,80,81]. The splitting of a genus into subgenera is also
311
avoided in prokaryotic systematics and none appear to have been proposed for over 35 years. Thus, the
312
only option available was to place the LD group of Borreliaceae species into a new genus to indicate
313
their distinctness from the RF group of species. The similarity of the name Borreliella to the original
314
name of the genus Borrelia, makes it unlikely that the species with the new names (e.g. Borreliella
315
burgdorferi) could be confused with an unrelated species. We note that it remains “B. burgdorferi” in
316
abbreviation for the scientific and medical literature and as a stand-alone complete name (as in "E.
317
coli") in news media and for the general public. Furthermore, the name Borreliaceae for the family
318
allows researchers, clinicians, and epidemiologists working in the LD area to continue using the term
319
“borreliosis", e.g. “Lyme borreliosis” and “neuroborreliosis”, to describe the disease and its
320
manifestations.
321
Finally, we recognize the inconvenience and disruptions that for a while follow a taxonomic
322
name change in a medically-important group of microbes, but ultimately medical progress and public
323
health advancement are best served by openness to scientific findings. Thus, the division of
324
Borreliaceae species into two distinct genera, which reflects the underlying genetic and phenotypic
325
diversity of this group, should prove greatly advantageous for progress of biomedical and ecological
326
studies on these microbes in the long run.
327
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Acknowledgements: We thank Jorge Benach, Sven Bergström, George Chaconas, Michel Drancourt,
329
Job Lopez, Richard Marconi, Tara Moriarty, Steven Norris, Vartul Sangal, Tom Schwan and Iain
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Sutcliffe for reading earlier versions of the manuscript and for comments and suggestions they
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provided. We thank Seema Alnazar for assistance in updating information for the CSIs.
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Figure legends
549
family Borreliaceae or distinguish the two phylogenetically distinct clades corresponding to the Lyme
550
disease (LD) and relapsing fever (RF) Borreliaceae species. The molecular markers are conserved
551
signature insertions/deletions (CSIs) and conserved signature proteins (CSPs). The specificities of the
552
molecular markers are mapped on a phylogenetic tree based on 762 conserved proteins comprising the
553
core proteins in the genomes of the Borreliaceae species. The tree was rooted using sequences from
554
Treponema denticoloa and Treponema caldarium. The * indicates CSIs which distinguish the two
555
groups of Borreliaceae species in proteins that are uniquely found in this family. The Tables referred
556
to in this figure are from Adeolu & Gupta [4].
Figure 1. Phylogram and summary of the molecular markers that are specific for the members of the
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558
Figure 2. Updated information for conserved signature indels in (A) glucose-6-phosphate isomerase
559
and (B) GTP binding protein which are specific for the Lyme disease (LD) and Relapsing fever (RF)
560
clades. Sequence information for the following four species (viz. B. coriaceae, B. finlandensis, B.
561
japonica, B. mayonii and B. chilensis), which have become available since the Adeolu and Gupta [4]
562
publication has been added to the sequence alignments and the new species are found to contain the
563
CSIs specific for the LD or the RF clade as predicted.
564
565
Figure 3. An average amino acid identity (AAI) matrix showing the average proportion of identical
566
aligned amino acid positions in 762 conserved core proteins widely distributed among the sequenced
567
members of the family Borreliaceae. Individual cells in the matrix are shaded in a continuous gradient
568
ranging from white at the lowest AAI values (0.69) to dark gray at the highest AAI values (0.98).
569
570
18