# The Characteristics of Ubiquitous and UniqueLeptospira Strains from the Collection of Russian Centre for Leptospirosis

Authors: Olga L. Voronina; Marina S. Kunda; Ekaterina I. Aksenova; Natalia N. Ryzhova; Andrey N. Semenov; Evgeny M. Petrov; Lubov V. Didenko; Vladimir G. Lunin; Yuliya V. Ananyina; Alexandr L. Gintsburg
Journal: BioMed Research International
 (2014)

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## Abstract

Background and Aim. Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity ofLeptospira in natural and anthropurgic foci was not clearly defined.Methods. The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS).Results. The most wide spread and prevalentLeptospira species in Russia wereL. interrogans, L. kirschneri, andL. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmentalLeptospirastrain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis ofrrs andrpoB genes and MLST loci for differentLeptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separateLeptospira species.Conclusions. Thus, the natural and anthropurgic foci supported ubiquitousLeptospira species and the pool of genes important for bacterial adaptivity to various conditions.


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## Body

## 1. Introduction

Leptospira is a genus of bacteria that is encountered in all geographical areas, except arctic and arid regions. SomeLeptospira are responsible for leptospirosis, a natural-focus disease. According to the List of Prokaryotic names with Standing in Nomenclature, theLeptospira genus has 21 species [1]. Seven of the species have been established as pathogenic:L. interrogans,L. borgpetersenii,L. kirschneri,L. noguchii,L. santarosai,L. weilii, andL. alexanderi. Two species,L. alstonii andL. kmetyi, are candidates for assignment to the pathogenic group; they are rarely isolated, and the sources of their isolation are not ill humans.L. alstonii was isolated from a frog andL. kmetyi was isolated from soil. Phylogenetic analysis with 16S rRNA gene sequences showed thatL. alstonii andL. kmetyi clustered with the pathogenicLeptospira species [2, 3]. Five species,L. broomii, L. fainei, L. inadai, L. licerasiae, andL. wolffii, are classified as intermediate, and six species,L. biflexa, L. meyeri, L. terpstrae, L. vanthielii, L. wolbachii,andL. yanagawae,are nonpathogenic [4]. One new species,L. idonii, isolated from the environmental water, is candidate for assignment to the nonpathogenic group. This species is placed within the clade of the known saprophytic species of the genusLeptospira on the 16S rRNA gene-based phylogenetic analysis [5].According to WHO guidance [6], the incidence of leptospirosis ranges from about 0.1–1 per 100 000 persons per year in temperate climates to 10–100 per 100 000 in the humid tropics. In the Russian Federation, only 0.01 cases per 100 000 were reported in 2013. During 2012-2013, 506 cases were registered in Russia according to the report of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (http://rospotrebnadzor.ru/). Long-term control of the multiple natural and anthropurgic foci in the USSR has been organized with the participation of the MoH Centre for Leptospirosis, an action which may be responsible for the decrease in incidence. During the registration period, strains ofLeptospira species were isolated, from the animals (maintenance and supplementary hosts of theLeptospira), human patients, and environment, and saved in the Gamaleya Institute Microbial Collection (GIMC).Among the strains ofLeptospira species in GIMC one strain was mysterious and not closely related to anyLeptospira, either pathogenic or saprophytic; it was namedLeptospiraspp. strain Bairam-Ali. Bairam-Ali was isolated from the water of a drainage canal in Turkmenia in 1971. On the basis of phenotypic tests, it was classified as a saprophytic species, although its resistance to the leptospiracidal activity of normal mammal sera and some other features made it closer to the pathogenic leptospires. Thus,Leptospirasp. strain Bairam-Ali was used in a diagnostic system on the basis of macroagglutination reaction. This diagnostic system was different from the original genus-specific microagglutination test, which requires multiple serovars of liveLeptospira and involves a risk of infection. Strain Bairam-Ali is a natural substitute for the microcapsule of synthetic polymer [7] as carrier of antigens similar to pathogenic strains, and it is safe for humans.Only whole genome sequence could help resolve the mystery of the strain Bairam-Ali and clarify its phylogenetic position in theLeptospira genus and relationships with the pathogenicLeptospira species.


## 2. Materials and Methods

### 2.1. Bacterial Strains

Leptospira strains were cultured by the Russian MoH Centre for Leptospirosis laboratory at the N.F. Gamaleya Institute for Epidemiology and Microbiology, Moscow, according to WHO guidance [6]. Fifty-eight strains, including 29 reference strains and 29 isolates from various sources and geographical regions, were analyzed. Twenty-six reference strains were members of seven pathogenic species. By the start of this study, seven reference strains were absent from theLeptospira MLST database [8].


### 2.2. Phenotypic and Serological Characterization of Strain Bairam-Ali

Methods for differentiation of pathogenic versus saprophytic strains and for cross-agglutination-absorption reactions were performed according to WHO guidance [6].


### 2.3. Scanning Electron Microscopy

Samples were prepared as described in detail [9] and analyzed with dual-beam focused ion beam/scanning electron microscope, Quanta 200 3D (FEI Company, USA), in both high and low vacuum, mostly at 5 kV electron beam acceleration [10].


### 2.4. DNA Isolation

DNA for PCR analysis was extracted from the bacterial cultures as described previously [11]. Preparation of genomic DNA for the whole genome sequencing was performed according to [12].


### 2.5.Leptospira Species Identification

The species of isolates were identified by amplification and sequencing of therpoB gene (coding β subunit of bacterial RNA polymerase), according to La Scola et al. [13]. Sequence data forrpoB has been deposited in GenBank, with accession numbers KJ701730–KJ701749.


### 2.6. MLST

MLST for the strains ofL. interrogans andL. kirschneri was performed by use of the original scheme of Thaipadungpanit et al. [14]. After publication of the modified MLST scheme [15], we completed earlier results by usingcaiB gene sequences. For the new isolates and strains of five other species, we used only the modified MLST scheme. Some modifications were inserted in the published protocol. The conditions of the amplification were modified for theglmU,pntA, andsucA genes by raising the melting temperature to 50°C. Also, the MgCl2 concentration was changed to 3.5 mM for theglmU,pntA, andsucA genes. Reference collection strains were used for adaptation of the method to our laboratory and for control of the reproducibility of the results.


### 2.7. PCR Products Sequencing

PCR products were sequenced according to the protocol of BigDye Terminator 3.1 Cycle Sequencing kit for the Genetic Analyzer 3130 of Applied Biosystems/Hitachi.


### 2.8. Nucleotide Sequence Analysis

The alignment ofrpoB and MLST gene sequences was made by use of ClustalW2 [16]. BLAST search was used for species identification; similarity ofrpoB gene sequences was more than 98%.Allele numbers for MLST genes were assigned with the help of website MLST Home. Allelic profiles, in the order glmU-pntA-sucA-tpiA-pfkB-mreA-caiB, were used to assign sequence types (STs) to strains. New alleles and ST were controlled and submitted by the curator ofLeptospiraspp. MLST database [8]. All newLeptospira strains were submitted in theLeptospiraspp. MLST database under the identification numbers indicated in Table 1.Table 1
Strains used in genotyping and phylogenetic analysis.
Number
Strain
Species
ST
Our submission ID or GenBank accession number for MLST genes
rpoB GenBank accession number
16S rDNA GenBank accession number
1
*GIMC2029:Li130
L. alexanderi, serogroup Manhao, serovar lichuan
207
337
KJ701742
2
*GIMC2051:Veldrat Bataviae 46
L. borgpetersenii, serogroup Javanica, serovar javanica
143
In MLST Home data base
KJ701733
3
*GIMC2052:Sari
L. borgpetersenii, serogroup Mini, serovar mini
142
In MLST Home data base
KJ701736
4
GIMC2002:101PJ
L. borgpetersenii, serogroup Javanica, serovar poi
146
339
5
GIMC2003:29PJ
L. borgpetersenii, serogroup Javanica, serovar poi
146
340
KJ701734
6
GIMC2004:Yaroslavl 7
L. borgpetersenii, serogroup Javanica, serovar poi
146
341
7
GIMC2005:1217PJ
L. borgpetersenii, serogroup Javanica, serovar javanica
146
342
8
GIMC2006:1622PJ
L. borgpetersenii, serogroup Javanica
146
343
9
GIMC2007:5-I
L. borgpetersenii, serogroup Javanica
146
344
10
*GIMC2049:Castellon 3
L. borgpetersenii, serogroup  Ballum, serovar castellonis
149
In MLST Home data base
KJ701737
11
*GIMC2050:Perepelitsin
L. borgpetersenii, serogroup Tarassovi, serovar tarassovi
153
In MLST Home data base
KJ701735
12
*GIMC2008:Mus 24
L. borgpetersenii, serogroup Sejroe, serovar saxkoebing
155
345
13
*GIMC2048:Naam
L. interrogans, serogroup Icterohaemorrhagiae,  serovar naam
23
In MLST Home data base
KJ701731
14
GIMC2014:AV4
L. interrogans, serovar no data
23
351
15
*GIMC2047:Djasiman
L. interrogans, serogroup Djasiman,  serovar djasiman
11
In MLST Home data base
16
*GIMC2046:RGA
L. interrogans, serogroup Icterohaemorrhagiae,  serovar icterohaemorrhagiae
17
In MLST Home data base
17
GIMC2010:Sv-19
L. interrogans, serogroup Icterohaemorrhagiae
17
347
18
GIMC2009:Rn-2010
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
17
346
19
GIMC2011:Rn-493
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
17
348
20
GIMC2012:Rn-16
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
17
349
21
GIMC2013:Rn-77
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
17
350
22
*GIMC2042:Ezh-1  =  Jez Bratislava
L. interrogans, serogroup Australis,  serovar bratislava
24
In MLST Home data base
23
*GIMC2043:Akiyami A
L. interrogans, serogroup Autumnalis,  serovar autumnalis
27
In MLST Home data base
24
*GIMC2044:Zanoni
L. interrogans, serogroup  Pyrogenes,  serovar zanoni
31
In MLST Home data base
KJ701732
25
*GIMC2045:Hebdomadis
L. interrogans, serogroup  Hebdomadis,  serovar hebdomadis
36
In MLST Home data base
26
GIMC2015:Kashirsky
L. interrogans, serogroup Canicola, serovar canicola
37
352
27
GIMC2016:Mitronov
L. interrogans, serogroup Canicola, serovar canicola
37
353
28
GIMC2017:Bugay
L. interrogans, serogroup Canicola, serovar canicola
37
354
29
GIMC2018:Sobaka 2000
L. interrogans, serogroup Canicola, serovar canicola
37
355
30
GIMC2019:Udalov
L. interrogans, serogroup Canicola, serovar canicola
37
356
31
*GIMC2038:Ballico
L. interrogans, serogroup Australis,  serovar  australis
51
In MLST Home data base
32
*GIMC2039:3705
L. interrogans, serogroup Sejroe, serovar woffi
58
In MLST Home data base
33
*GIMC2040:Szwajizak
L. interrogans, serogroup Mini
73
In MLST Home data base
34
*GIMC2041:Pomona
L. interrogans, serogroup Pomona, serovar pomona
140
In MLST Home data base
35
*GIMC2020:M-20R
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
199
357
KJ701730
36
GIMC2021:Abduloev
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
199
358
37
GIMC2022:CL-11
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
199
359
38
GIMC2030:CL-17
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
206
336
39
*GIMC2023:Vleermuis 3868
L. kirschneri, serogroup Cynopteri, serovar cynopteri
70
360
40
*GIMC2024:Moskva V
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
361
41
GIMC2025:181PG
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
362
KJ701738
42
GIMC2026:617PG
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
363
43
GIMC2027:859PG
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
364
44
GIMC2028:1106PG
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
365
45
*GIMC2037:5621
L. kirschneri, serogroup Pomona, serovar mozdok
117
In MLST Home data base
46
*GIMC2031:HS 26R
L. kirschneri, serogroup Bataviae, serovar djatsi
204
334
KJ701739
47
*GIMC2033:LSU 1945
L. noguchii, serogroup Louisiana, serovar panama
169
In MLST Home data base
KJ701740
48
*GIMC2034:CZ 214 K
L. noguchii, serogroup Panama, serovar panama
171
In MLST Home data base
KJ701741
49
*GIMC2035:Celledoni
L. weilii, serogroup Celledoni, serovar celledoni
185
In MLST Home data base
KJ701746
50
*GIMC2036:Sarmin
L.  weilii, serogroup Sarmin, serovar sarmin
191
In MLST Home data base
51
*GIMC2032:CZ299U
L.  santarosai, serogroup Pomona, serovar tropica
208
338
52
GIMC2001:Bairam-Ali
L.sp., serogroup Bairam-Ali, serovar bairam-ali
KJ676852-KJ676858
KJ701604
KJ701750
53
*GIMC2055:Lyme
L. inadai, serogroup Lyme, serovar lyme
KJ701743
54
GIMC2056:EMJH 86
L. inadai, serovar lyme
KJ701744
55
GIMC2057:Enr 88
L. inadai, serogroup Lyme-Detroit, serovar lyme-detroit
KJ701745
56
*GIMC2060:Sao Paulo
L. yanagawae, serogroup Semaranga, serovar sao paulo
KJ701747
57
GIMC2058:LT-8
L. biflexa, serovar patoc
KJ701748
58
GIMC2059:GR
L.  biflexa, serovar andamana
KJ701749
59
DSM 21527
Turneriella parva
NC_018020
CP002959
CP002959
60
80–412
L. alstoni, serovar pingchang
NZ_AOHD02000041.1
NZ_AOHD02000066.1
61
79601
L. alstoni, serovar sichuan
AY631881
62
L 60
L. alexanderi, serovar manhao 3
NZ_AHMT02000060.1
63
A23
L. alexanderi, serovar manzhuang
AY996803.1
64
A85
L. alexanderi, serovar mengla
DQ991481.1
65
M 6901
L. alexanderi, serovar nanding
AY996804
66
Mus 127
L. borgpetersenii, serovar ballum
EU747302
67
Lely 607
L. borgpetersenii, serogroup Sejroe, serovar hardjo
FJ154586
68
L550
L. borgpetersenii, serovar hardjo-bovis
CP000348.1
69
JB197
L. borgpetersenii, serovar hardjo-bovis
CP000350
70
Lely 607
L. borgpetersenii, serovar hardjo-bovis
EU747305
71
Veldrat Batavia 46
L. borgpetersenii, serovar  javanica
AY887899.1
72
M84
L. borgpetersenii, serovar sejroe
EU747311
73
Perepelitsin
L. borgpetersenii, serovar tarassovi
EU747307
JQ988861.1
74
Whitticombi
L. borgpetersenii, serovar whitticombi
EU747314
75
Mini-CTG
L. borgpetersenii, serovar no data
JQ765635.1
76
Ballico
L. interrogans, serovar australis
FJ154556.1
77
Akiyami A
L. interrogans, serovar autumnalis
AM050580.1
78
Jez-bratislava
L. interrogans, serovar bratislava
EU747300
79
Mallika
L. interrogans, serovar bulgarica
AY996792.1
80
Hond Utrecht IV
L. interrogans,  serovar canicola
FJ154561.1
81
Fiocruz L1-130
L. interrogans, serovar copenhageni
17
AE016823.1
AE016823.1
82
Djasiman
L. interrogans, serovar djasiman
EU747312
FJ154550.1
83
Hardjo_DB33
L. interrogans, serovar hardjo
JQ988854.1
84
RGA
L. interrogans, serovar icterohaemorrhagiae
NR_029361.1
85
Kremastos
L. interrogans, serovar kremastos
AY461868.1
86
IPAV
L.  interrogans,  serovar lai
CP001221
87
LT 398
L. interrogans, serovar manilae
FJ154545.1
88
Hond HC
L. interrogans, serovar medanesis
DQ991471.1
89
Pomona
L. interrogans, serovar pomona
EU747306
AY996800.1
90
Salinem
L. interrogans, serovar pyrogenes
FJ154552.1
91
Vleermuis
L. interrogans, serovar schueffneri
EU747313
92
Szwajizak
L. interrogans, serovar szwajizak
DQ991466.1
93
3705
L. interrogans,  serovar wolffi
EU747308
94
Zanoni
L. interrogans, serovar zanoni
DQ991473
95
Bataviae_DB59
L. interrogans, serovar no data
JQ988841.1
96
Agogo
L. kirschneri, serovar agogo
DQ991476.1
97
Bafani
L. kirschneri, serovar bafani
DQ991477.1
98
1051
L. kirschneri, serovar bim
AY996802.1
99
Butembo
L. kirschneri, serovar butembo
Q991478.1
100
3522 C
L. kirschneri, serovar cynopteri
FJ154546.1
101
Moskva V
L. kirschneri, serovar grippotyphosa
EU747301
102
Kambale
L. kirschneri, serovar kambale
AY461878.1
103
5621
L. kirschneri, serovar pomona
FJ154559.1
104
Wumalasena
L. kirschneri, serovar ratnapura
DQ991479.1
105
Bejo-Iso9
L. kmetyi, serovar malaysia
AHMP02000003
AHMP02000003
NR_041544.1
106
Hook
L. noguchii, serogroup Australis
EU349504
107
Bonito
L. noguchii, serogroup Autumnalis
EU349501
108
Caco
L. noguchii, serogroup Autumnalis
EU349498
109
Cascata
L. noguchii, serogroup Bataviae
EU349502
110
LSU 1945
L. noguchii, serogroup Louisiana
EU349505
111
1348U
L. noguchii,  serovar claytoni
DQ991498.1
112
1996K
L. noguchii, serovar cristobali
DQ991497.1
113
M7
L. noguchii, serovar huallaga
DQ991499.1
114
1011
L. noguchii, serovar nicaragua
EU349499
115
LSU 2580
L. noguchii, serovar orleans
EU349500
116
CZ 214 K
L. noguchii, serovar panama
EU349497
NR_043050.1
117
DB57
L. noguchii, serovar panama
EU349501
JQ988837.1
118
HS 616
L. santarosai, serovar alexi
FJ154585.1
119
Alice
L. santarosai, serovar alice
DQ991493.1
120
MAVJ 401
L. santarosai, serovar arenal
AHMU02000055
121
LT 79
L. santarosai, serovar bakeri
FJ154589.1
122
LT 117
L. santarosai, serovar georgia
AY996805.1
123
CZ320
L. santarosai, serovar kobbe
DQ991495.1
124
1342
L. santarosai, serovar shermani
FJ154576.1
125
CZ390
L. santarosai, serovar weaveri
DQ991496.1
126
CBC613
L. santarosai, serovar no data
ANIH01000040
127
ST188
L. santarosai, serovar no data
AOHA02000081
128
MOR084
L. santarosai, serovar no data
AHON02000051
129
2000030832
L. santarosai, serovar no data
AFJN02000030
130
Celledoni
L. weilii, serovar celledoni
DQ991486.1
131
H27
L. weilii, serovar hekou
DQ991487.1
132
M39090
L. weilii, serovar langati
DQ991488.1
133
WB46
L. weilii, serovar sarmin
U12673.1
134
LT 89-68
L. weilii,  serovar vughia
FJ154590.1
135
5399
L. broomii, serovar hurstbridge
AHMO02000008
AHMO02000008
AHMO02000008.1
136
BUT 6
L. fainei, serovar hurstbridge
AKWZ02000010
AKWZ02000010
NR_043049.1
137
BKID 6
L. fainei, serovar hurstbridge
AY996789.1
138
10
L. inadai, serovar lyme
AHMM02000025
AHMM02000015
AHMM02000015.1
139
VAR 010
L. licerasiae, serovar varillal
AHOO02000005
AHOO02000005
NR_044310.1
140
Khorat-H2
L. wolffii, serovar khorat
AKWX02000020
NZ_AKWX02000004.1
141
Patoc Ames
L. biflexa, serovar patoc
NC_010842
NC_010842
NC_010842
142
Patoc Paris
L. biflexa, serovar patoc
NC_010602
NC_010602
143
CH 11
L. biflexa, serovar andamana
FJ154577.1
144
Veldrat Semarang 173
L. meyeri, serovar semaranga
AF157089.1
145
Went 5
L. meyeri, serovar hardjo
AKXE01000002
AKXE01000001
NZ_AKXE01000007.1
146
LT 11-33 ATCC 700639
L. terpstrae, serovar hualin
AOGW02000006
AOGW02000010
NZ_AOGW02000008.1
147
Waz Holland ATCC 700522
L. vanthielii, serovar holland
AOGY02000070
AOGY02000051
NZ_AOGY02000072.1
148
CDC; ATCC 43284
L. wolbachii, serovar codice
AOGZ02000014
AOGZ02000008
NR_043046.1
149
Sao Paulo ATCC 700523
L. yanagawae, serovar saopaulo
AOGX02000015
AOGX02000024
Z_AOGX02000022.1
150
Eri-1(T) DSM 26084(T) = JCM 18486(T)
L. idonii, serogroup Hebdomadis
AB721966
*The reference strains from GIMC.Relatedness between STs on the base of allelic profiles was analyzed by use of BURST version 1.00 [17, 18].


### 2.9. Nucleotide Sequence Polymorphism

BLAST search was used for retrieving homologuesrrs(16S rRNA-coding), rpoB,and MLST gene sequences from GenBank (http://www.ncbi.nlm.nih.gov/genome/browse/). For comparative sequence analysis and phylogenetic reconstruction, nucleotide sequences of 92 additionalLeptospira strains were retrieved. Seventy-five nucleotide sequences represented either complete or partial cds ofrrs gene andrpoB gene, and 15 sequences represented whole genome sequencing data and genome drafts (Table 1).Turneriella parva have been included in the analysis as an out-group taxon fromLeptospiraceae, for which whole genome sequence data are available (Table 1). Sequences of seven concatenated MLST loci for 201 ST available at the time of analysis were retrieved [8]. The alignments ofrrs, rpoB, and MLST gene sequences and analysis nucleotide similarity (in %) were performed by use of ClustalW2 [16].


### 2.10. Phylogenetic Analysis

Phylogenetic analysis ofLeptospira species was performed based on therrs gene fragment,rpoB gene fragment, and seven concatenated sequences of MLST loci. Phylogenetic trees were constructed by use of neighbor-joining, maximum likelihood, and maximum parsimony methods.Genetic distances betweenLeptospiragenotypes were evaluated by use of Kimura’s two-parameter model [19], which was chosen as an optimal evolution distance model derived from model test based on the Akaike information criterion [20]. The evolutionary history was inferred by using the Maximum Likelihood method based on the general time reversible model [21]. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated by use of the maximum composite likelihood approach and then selecting the topology with superior log likelihood value. A discrete gamma distribution was used to model evolutionary rate differences among sites (six categories (+G, parameter = 0.4818)). Maximum parsimony trees were constructed with an algorithm implemented in MEGA version 6.0 [22]. Bootstrap analyses were performed with 1,000 replicates.


### 2.11. Whole Genome Sequencing

Whole genome sequencing ofLeptospiraspp. strain GIMC2001:Bairam-Ali was performed according to the manufacturer’s guidelines (Roche) for the next generation sequencing (NGS). Two protocols were used for a shotgun-sequencing library preparation: rapid library and pair-end library. The rapid library was made according to the Rapid Library Preparation Method Manual (Roche). The pair-end library was performed according to the 3 kb protocol provided by the manufacturer to aid in scaffold building. The paired-end library insert size was from 1347 to 4364 bp, with an average of 2695 bp.Assembly was performed with 454 Sequencing System Software v.2.7 (Roche), yielding 14 scaffolds, with the largest size being 3 342 467 bp. Gap closure was performed by use of Contig Graph result file generated by GS De Novo Assembler program (Roche). For the oriC region search, Ori-Finder program was used [23].


### 2.12. Gene Annotation

The software Rapid Annotations Subsystems Technology and SEED [24, 25] were used for annotating the genome ofLeptospiraspp. strain GIMC2001:Bairam-Ali.


## 2.1. Bacterial Strains

Leptospira strains were cultured by the Russian MoH Centre for Leptospirosis laboratory at the N.F. Gamaleya Institute for Epidemiology and Microbiology, Moscow, according to WHO guidance [6]. Fifty-eight strains, including 29 reference strains and 29 isolates from various sources and geographical regions, were analyzed. Twenty-six reference strains were members of seven pathogenic species. By the start of this study, seven reference strains were absent from theLeptospira MLST database [8].


## 2.2. Phenotypic and Serological Characterization of Strain Bairam-Ali

Methods for differentiation of pathogenic versus saprophytic strains and for cross-agglutination-absorption reactions were performed according to WHO guidance [6].


## 2.3. Scanning Electron Microscopy

Samples were prepared as described in detail [9] and analyzed with dual-beam focused ion beam/scanning electron microscope, Quanta 200 3D (FEI Company, USA), in both high and low vacuum, mostly at 5 kV electron beam acceleration [10].


## 2.4. DNA Isolation

DNA for PCR analysis was extracted from the bacterial cultures as described previously [11]. Preparation of genomic DNA for the whole genome sequencing was performed according to [12].


## 2.5.Leptospira Species Identification

The species of isolates were identified by amplification and sequencing of therpoB gene (coding β subunit of bacterial RNA polymerase), according to La Scola et al. [13]. Sequence data forrpoB has been deposited in GenBank, with accession numbers KJ701730–KJ701749.


## 2.6. MLST

MLST for the strains ofL. interrogans andL. kirschneri was performed by use of the original scheme of Thaipadungpanit et al. [14]. After publication of the modified MLST scheme [15], we completed earlier results by usingcaiB gene sequences. For the new isolates and strains of five other species, we used only the modified MLST scheme. Some modifications were inserted in the published protocol. The conditions of the amplification were modified for theglmU,pntA, andsucA genes by raising the melting temperature to 50°C. Also, the MgCl2 concentration was changed to 3.5 mM for theglmU,pntA, andsucA genes. Reference collection strains were used for adaptation of the method to our laboratory and for control of the reproducibility of the results.


## 2.7. PCR Products Sequencing

PCR products were sequenced according to the protocol of BigDye Terminator 3.1 Cycle Sequencing kit for the Genetic Analyzer 3130 of Applied Biosystems/Hitachi.


## 2.8. Nucleotide Sequence Analysis

The alignment ofrpoB and MLST gene sequences was made by use of ClustalW2 [16]. BLAST search was used for species identification; similarity ofrpoB gene sequences was more than 98%.Allele numbers for MLST genes were assigned with the help of website MLST Home. Allelic profiles, in the order glmU-pntA-sucA-tpiA-pfkB-mreA-caiB, were used to assign sequence types (STs) to strains. New alleles and ST were controlled and submitted by the curator ofLeptospiraspp. MLST database [8]. All newLeptospira strains were submitted in theLeptospiraspp. MLST database under the identification numbers indicated in Table 1.Table 1
Strains used in genotyping and phylogenetic analysis.
Number
Strain
Species
ST
Our submission ID or GenBank accession number for MLST genes
rpoB GenBank accession number
16S rDNA GenBank accession number
1
*GIMC2029:Li130
L. alexanderi, serogroup Manhao, serovar lichuan
207
337
KJ701742
2
*GIMC2051:Veldrat Bataviae 46
L. borgpetersenii, serogroup Javanica, serovar javanica
143
In MLST Home data base
KJ701733
3
*GIMC2052:Sari
L. borgpetersenii, serogroup Mini, serovar mini
142
In MLST Home data base
KJ701736
4
GIMC2002:101PJ
L. borgpetersenii, serogroup Javanica, serovar poi
146
339
5
GIMC2003:29PJ
L. borgpetersenii, serogroup Javanica, serovar poi
146
340
KJ701734
6
GIMC2004:Yaroslavl 7
L. borgpetersenii, serogroup Javanica, serovar poi
146
341
7
GIMC2005:1217PJ
L. borgpetersenii, serogroup Javanica, serovar javanica
146
342
8
GIMC2006:1622PJ
L. borgpetersenii, serogroup Javanica
146
343
9
GIMC2007:5-I
L. borgpetersenii, serogroup Javanica
146
344
10
*GIMC2049:Castellon 3
L. borgpetersenii, serogroup  Ballum, serovar castellonis
149
In MLST Home data base
KJ701737
11
*GIMC2050:Perepelitsin
L. borgpetersenii, serogroup Tarassovi, serovar tarassovi
153
In MLST Home data base
KJ701735
12
*GIMC2008:Mus 24
L. borgpetersenii, serogroup Sejroe, serovar saxkoebing
155
345
13
*GIMC2048:Naam
L. interrogans, serogroup Icterohaemorrhagiae,  serovar naam
23
In MLST Home data base
KJ701731
14
GIMC2014:AV4
L. interrogans, serovar no data
23
351
15
*GIMC2047:Djasiman
L. interrogans, serogroup Djasiman,  serovar djasiman
11
In MLST Home data base
16
*GIMC2046:RGA
L. interrogans, serogroup Icterohaemorrhagiae,  serovar icterohaemorrhagiae
17
In MLST Home data base
17
GIMC2010:Sv-19
L. interrogans, serogroup Icterohaemorrhagiae
17
347
18
GIMC2009:Rn-2010
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
17
346
19
GIMC2011:Rn-493
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
17
348
20
GIMC2012:Rn-16
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
17
349
21
GIMC2013:Rn-77
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
17
350
22
*GIMC2042:Ezh-1  =  Jez Bratislava
L. interrogans, serogroup Australis,  serovar bratislava
24
In MLST Home data base
23
*GIMC2043:Akiyami A
L. interrogans, serogroup Autumnalis,  serovar autumnalis
27
In MLST Home data base
24
*GIMC2044:Zanoni
L. interrogans, serogroup  Pyrogenes,  serovar zanoni
31
In MLST Home data base
KJ701732
25
*GIMC2045:Hebdomadis
L. interrogans, serogroup  Hebdomadis,  serovar hebdomadis
36
In MLST Home data base
26
GIMC2015:Kashirsky
L. interrogans, serogroup Canicola, serovar canicola
37
352
27
GIMC2016:Mitronov
L. interrogans, serogroup Canicola, serovar canicola
37
353
28
GIMC2017:Bugay
L. interrogans, serogroup Canicola, serovar canicola
37
354
29
GIMC2018:Sobaka 2000
L. interrogans, serogroup Canicola, serovar canicola
37
355
30
GIMC2019:Udalov
L. interrogans, serogroup Canicola, serovar canicola
37
356
31
*GIMC2038:Ballico
L. interrogans, serogroup Australis,  serovar  australis
51
In MLST Home data base
32
*GIMC2039:3705
L. interrogans, serogroup Sejroe, serovar woffi
58
In MLST Home data base
33
*GIMC2040:Szwajizak
L. interrogans, serogroup Mini
73
In MLST Home data base
34
*GIMC2041:Pomona
L. interrogans, serogroup Pomona, serovar pomona
140
In MLST Home data base
35
*GIMC2020:M-20R
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
199
357
KJ701730
36
GIMC2021:Abduloev
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
199
358
37
GIMC2022:CL-11
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
199
359
38
GIMC2030:CL-17
L. interrogans, serogroup Icterohaemorrhagiae,  serovar copenhageni
206
336
39
*GIMC2023:Vleermuis 3868
L. kirschneri, serogroup Cynopteri, serovar cynopteri
70
360
40
*GIMC2024:Moskva V
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
361
41
GIMC2025:181PG
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
362
KJ701738
42
GIMC2026:617PG
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
363
43
GIMC2027:859PG
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
364
44
GIMC2028:1106PG
L. kirschneri, serogroup Grippotyphosa, serovar grippotyphosa
110
365
45
*GIMC2037:5621
L. kirschneri, serogroup Pomona, serovar mozdok
117
In MLST Home data base
46
*GIMC2031:HS 26R
L. kirschneri, serogroup Bataviae, serovar djatsi
204
334
KJ701739
47
*GIMC2033:LSU 1945
L. noguchii, serogroup Louisiana, serovar panama
169
In MLST Home data base
KJ701740
48
*GIMC2034:CZ 214 K
L. noguchii, serogroup Panama, serovar panama
171
In MLST Home data base
KJ701741
49
*GIMC2035:Celledoni
L. weilii, serogroup Celledoni, serovar celledoni
185
In MLST Home data base
KJ701746
50
*GIMC2036:Sarmin
L.  weilii, serogroup Sarmin, serovar sarmin
191
In MLST Home data base
51
*GIMC2032:CZ299U
L.  santarosai, serogroup Pomona, serovar tropica
208
338
52
GIMC2001:Bairam-Ali
L.sp., serogroup Bairam-Ali, serovar bairam-ali
KJ676852-KJ676858
KJ701604
KJ701750
53
*GIMC2055:Lyme
L. inadai, serogroup Lyme, serovar lyme
KJ701743
54
GIMC2056:EMJH 86
L. inadai, serovar lyme
KJ701744
55
GIMC2057:Enr 88
L. inadai, serogroup Lyme-Detroit, serovar lyme-detroit
KJ701745
56
*GIMC2060:Sao Paulo
L. yanagawae, serogroup Semaranga, serovar sao paulo
KJ701747
57
GIMC2058:LT-8
L. biflexa, serovar patoc
KJ701748
58
GIMC2059:GR
L.  biflexa, serovar andamana
KJ701749
59
DSM 21527
Turneriella parva
NC_018020
CP002959
CP002959
60
80–412
L. alstoni, serovar pingchang
NZ_AOHD02000041.1
NZ_AOHD02000066.1
61
79601
L. alstoni, serovar sichuan
AY631881
62
L 60
L. alexanderi, serovar manhao 3
NZ_AHMT02000060.1
63
A23
L. alexanderi, serovar manzhuang
AY996803.1
64
A85
L. alexanderi, serovar mengla
DQ991481.1
65
M 6901
L. alexanderi, serovar nanding
AY996804
66
Mus 127
L. borgpetersenii, serovar ballum
EU747302
67
Lely 607
L. borgpetersenii, serogroup Sejroe, serovar hardjo
FJ154586
68
L550
L. borgpetersenii, serovar hardjo-bovis
CP000348.1
69
JB197
L. borgpetersenii, serovar hardjo-bovis
CP000350
70
Lely 607
L. borgpetersenii, serovar hardjo-bovis
EU747305
71
Veldrat Batavia 46
L. borgpetersenii, serovar  javanica
AY887899.1
72
M84
L. borgpetersenii, serovar sejroe
EU747311
73
Perepelitsin
L. borgpetersenii, serovar tarassovi
EU747307
JQ988861.1
74
Whitticombi
L. borgpetersenii, serovar whitticombi
EU747314
75
Mini-CTG
L. borgpetersenii, serovar no data
JQ765635.1
76
Ballico
L. interrogans, serovar australis
FJ154556.1
77
Akiyami A
L. interrogans, serovar autumnalis
AM050580.1
78
Jez-bratislava
L. interrogans, serovar bratislava
EU747300
79
Mallika
L. interrogans, serovar bulgarica
AY996792.1
80
Hond Utrecht IV
L. interrogans,  serovar canicola
FJ154561.1
81
Fiocruz L1-130
L. interrogans, serovar copenhageni
17
AE016823.1
AE016823.1
82
Djasiman
L. interrogans, serovar djasiman
EU747312
FJ154550.1
83
Hardjo_DB33
L. interrogans, serovar hardjo
JQ988854.1
84
RGA
L. interrogans, serovar icterohaemorrhagiae
NR_029361.1
85
Kremastos
L. interrogans, serovar kremastos
AY461868.1
86
IPAV
L.  interrogans,  serovar lai
CP001221
87
LT 398
L. interrogans, serovar manilae
FJ154545.1
88
Hond HC
L. interrogans, serovar medanesis
DQ991471.1
89
Pomona
L. interrogans, serovar pomona
EU747306
AY996800.1
90
Salinem
L. interrogans, serovar pyrogenes
FJ154552.1
91
Vleermuis
L. interrogans, serovar schueffneri
EU747313
92
Szwajizak
L. interrogans, serovar szwajizak
DQ991466.1
93
3705
L. interrogans,  serovar wolffi
EU747308
94
Zanoni
L. interrogans, serovar zanoni
DQ991473
95
Bataviae_DB59
L. interrogans, serovar no data
JQ988841.1
96
Agogo
L. kirschneri, serovar agogo
DQ991476.1
97
Bafani
L. kirschneri, serovar bafani
DQ991477.1
98
1051
L. kirschneri, serovar bim
AY996802.1
99
Butembo
L. kirschneri, serovar butembo
Q991478.1
100
3522 C
L. kirschneri, serovar cynopteri
FJ154546.1
101
Moskva V
L. kirschneri, serovar grippotyphosa
EU747301
102
Kambale
L. kirschneri, serovar kambale
AY461878.1
103
5621
L. kirschneri, serovar pomona
FJ154559.1
104
Wumalasena
L. kirschneri, serovar ratnapura
DQ991479.1
105
Bejo-Iso9
L. kmetyi, serovar malaysia
AHMP02000003
AHMP02000003
NR_041544.1
106
Hook
L. noguchii, serogroup Australis
EU349504
107
Bonito
L. noguchii, serogroup Autumnalis
EU349501
108
Caco
L. noguchii, serogroup Autumnalis
EU349498
109
Cascata
L. noguchii, serogroup Bataviae
EU349502
110
LSU 1945
L. noguchii, serogroup Louisiana
EU349505
111
1348U
L. noguchii,  serovar claytoni
DQ991498.1
112
1996K
L. noguchii, serovar cristobali
DQ991497.1
113
M7
L. noguchii, serovar huallaga
DQ991499.1
114
1011
L. noguchii, serovar nicaragua
EU349499
115
LSU 2580
L. noguchii, serovar orleans
EU349500
116
CZ 214 K
L. noguchii, serovar panama
EU349497
NR_043050.1
117
DB57
L. noguchii, serovar panama
EU349501
JQ988837.1
118
HS 616
L. santarosai, serovar alexi
FJ154585.1
119
Alice
L. santarosai, serovar alice
DQ991493.1
120
MAVJ 401
L. santarosai, serovar arenal
AHMU02000055
121
LT 79
L. santarosai, serovar bakeri
FJ154589.1
122
LT 117
L. santarosai, serovar georgia
AY996805.1
123
CZ320
L. santarosai, serovar kobbe
DQ991495.1
124
1342
L. santarosai, serovar shermani
FJ154576.1
125
CZ390
L. santarosai, serovar weaveri
DQ991496.1
126
CBC613
L. santarosai, serovar no data
ANIH01000040
127
ST188
L. santarosai, serovar no data
AOHA02000081
128
MOR084
L. santarosai, serovar no data
AHON02000051
129
2000030832
L. santarosai, serovar no data
AFJN02000030
130
Celledoni
L. weilii, serovar celledoni
DQ991486.1
131
H27
L. weilii, serovar hekou
DQ991487.1
132
M39090
L. weilii, serovar langati
DQ991488.1
133
WB46
L. weilii, serovar sarmin
U12673.1
134
LT 89-68
L. weilii,  serovar vughia
FJ154590.1
135
5399
L. broomii, serovar hurstbridge
AHMO02000008
AHMO02000008
AHMO02000008.1
136
BUT 6
L. fainei, serovar hurstbridge
AKWZ02000010
AKWZ02000010
NR_043049.1
137
BKID 6
L. fainei, serovar hurstbridge
AY996789.1
138
10
L. inadai, serovar lyme
AHMM02000025
AHMM02000015
AHMM02000015.1
139
VAR 010
L. licerasiae, serovar varillal
AHOO02000005
AHOO02000005
NR_044310.1
140
Khorat-H2
L. wolffii, serovar khorat
AKWX02000020
NZ_AKWX02000004.1
141
Patoc Ames
L. biflexa, serovar patoc
NC_010842
NC_010842
NC_010842
142
Patoc Paris
L. biflexa, serovar patoc
NC_010602
NC_010602
143
CH 11
L. biflexa, serovar andamana
FJ154577.1
144
Veldrat Semarang 173
L. meyeri, serovar semaranga
AF157089.1
145
Went 5
L. meyeri, serovar hardjo
AKXE01000002
AKXE01000001
NZ_AKXE01000007.1
146
LT 11-33 ATCC 700639
L. terpstrae, serovar hualin
AOGW02000006
AOGW02000010
NZ_AOGW02000008.1
147
Waz Holland ATCC 700522
L. vanthielii, serovar holland
AOGY02000070
AOGY02000051
NZ_AOGY02000072.1
148
CDC; ATCC 43284
L. wolbachii, serovar codice
AOGZ02000014
AOGZ02000008
NR_043046.1
149
Sao Paulo ATCC 700523
L. yanagawae, serovar saopaulo
AOGX02000015
AOGX02000024
Z_AOGX02000022.1
150
Eri-1(T) DSM 26084(T) = JCM 18486(T)
L. idonii, serogroup Hebdomadis
AB721966
*The reference strains from GIMC.Relatedness between STs on the base of allelic profiles was analyzed by use of BURST version 1.00 [17, 18].


## 2.9. Nucleotide Sequence Polymorphism

BLAST search was used for retrieving homologuesrrs(16S rRNA-coding), rpoB,and MLST gene sequences from GenBank (http://www.ncbi.nlm.nih.gov/genome/browse/). For comparative sequence analysis and phylogenetic reconstruction, nucleotide sequences of 92 additionalLeptospira strains were retrieved. Seventy-five nucleotide sequences represented either complete or partial cds ofrrs gene andrpoB gene, and 15 sequences represented whole genome sequencing data and genome drafts (Table 1).Turneriella parva have been included in the analysis as an out-group taxon fromLeptospiraceae, for which whole genome sequence data are available (Table 1). Sequences of seven concatenated MLST loci for 201 ST available at the time of analysis were retrieved [8]. The alignments ofrrs, rpoB, and MLST gene sequences and analysis nucleotide similarity (in %) were performed by use of ClustalW2 [16].


## 2.10. Phylogenetic Analysis

Phylogenetic analysis ofLeptospira species was performed based on therrs gene fragment,rpoB gene fragment, and seven concatenated sequences of MLST loci. Phylogenetic trees were constructed by use of neighbor-joining, maximum likelihood, and maximum parsimony methods.Genetic distances betweenLeptospiragenotypes were evaluated by use of Kimura’s two-parameter model [19], which was chosen as an optimal evolution distance model derived from model test based on the Akaike information criterion [20]. The evolutionary history was inferred by using the Maximum Likelihood method based on the general time reversible model [21]. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated by use of the maximum composite likelihood approach and then selecting the topology with superior log likelihood value. A discrete gamma distribution was used to model evolutionary rate differences among sites (six categories (+G, parameter = 0.4818)). Maximum parsimony trees were constructed with an algorithm implemented in MEGA version 6.0 [22]. Bootstrap analyses were performed with 1,000 replicates.


## 2.11. Whole Genome Sequencing

Whole genome sequencing ofLeptospiraspp. strain GIMC2001:Bairam-Ali was performed according to the manufacturer’s guidelines (Roche) for the next generation sequencing (NGS). Two protocols were used for a shotgun-sequencing library preparation: rapid library and pair-end library. The rapid library was made according to the Rapid Library Preparation Method Manual (Roche). The pair-end library was performed according to the 3 kb protocol provided by the manufacturer to aid in scaffold building. The paired-end library insert size was from 1347 to 4364 bp, with an average of 2695 bp.Assembly was performed with 454 Sequencing System Software v.2.7 (Roche), yielding 14 scaffolds, with the largest size being 3 342 467 bp. Gap closure was performed by use of Contig Graph result file generated by GS De Novo Assembler program (Roche). For the oriC region search, Ori-Finder program was used [23].


## 2.12. Gene Annotation

The software Rapid Annotations Subsystems Technology and SEED [24, 25] were used for annotating the genome ofLeptospiraspp. strain GIMC2001:Bairam-Ali.


## 3. Results and Discussion

A sample of the collection (GIMC) of the Russian MoH Centre for Leptospirosis was used for the verification the species and strains based on the currently recommended molecular-genetic methods. The sample included 29 reference strains and 29 isolates ofLeptospirafrom various geographical regions (Table 1, *—marker of the reference strains from GIMC).The analysis ofrpoB gene sequences demonstrated that 29 reference strains belonged to seven pathogenic species, one nonpathogenic species (L. yanagawae), and one intermediate species (L. inadai). Among 29 isolates ofLeptospiratwenty-four field isolates were related to the three pathogenic species (L. interrogans,L. borgpetersenii, andL. kirschneri). Four cultures were isolated from the commercially available Ellinghausen-McCullough-Johnson-Harris (EMJH) medium: two isolates from this group belonged to the nonpathogenic speciesL. biflexa and the other two isolates belonged to the intermediate speciesL. inadai. Thus, the prevailing species among the isolates collected on the territory of Russia comprisedL. interrogans,L. borgpetersenii, andL. kirschneri.The modified MLST scheme was applied to the isolates and reference strains representing 7 pathogenicLeptospira species. Twenty-four isolates of pathogenic species belonged to 8 different STs (Table 1). Five STs, common for Russian strains ofLeptospira (L. interrogans ST37, 17, and 199;L. kirschneri ST110; andL. borgpetersenii ST146), were identified as having a longtime and ubiquitous distribution in various geographic areas.Among the strains ofLeptospira species available in GIMC, one strain seemed to be mysterious and not closely related to anyLeptospira, either pathogenic or saprophytic, or intermediate. It was namedLeptospiraspp. strain Bairam-Ali, because it was isolated in 1971 in Turkmenia from the water of a drainage canal.


### 3.1. Morphology of theLeptospira spp. Strain Bairam-Ali

The morphology of the mysterious strain Bairam-Ali is typical of that of theLeptospira genus (Figure 1). Electron microscopy demonstrated that its cells are corkscrew-shaped with end hooks. They are thin and helical, like the cells of all known leptospires. Also, the cells have a diameter (d) of 0.12 μm and length (l) from 9.44 to 10.14 μm, like that of known leptospires (d = 0.15–0.3 μm and l = 6,00–20,00 μm) [26].Dual-beam scanning electron microscopy image ofL.species Bairam-Ali cells. (a) Original image; (b) with measuring the object in the program “Scandium” (green, length 10,14 μm; yellow, length (diameter) 0,12 μm; and green, length 9,44 μm).
(a)
(b)


### 3.2. Phenotypic Characterization of theLeptospira spp. Strain Bairam-Ali

Physiological characteristics of Bairam-Ali, demonstrated in Table2, suggest that strain Bairam-Ali can be classified as saprophyticLeptospira.Table 2
Physiological characteristics of theLeptospiraspp. strain Bairam-Ali.
Strain
Growth at temp (°C) of
Growth in the presence of
Lipase activity
Hemolytic activity against sheep erythrocytes
11
30
37
8-Azaguanine 225 pg/mL
Bairam-Ali
+
+
+
+
+
−For pathogenicity experiment, six four-week-old male golden Syrian hamsters were inoculated subcutaneously with 108 cells of strain Bairam-Ali in 1 mL of PBS. No hamster died from infection even at such a high bacterial dose.Leptospira cells were not detected during following bacterioscopic and bacteriological examination of the hamsters’ viscera. This is an additional evidence of saprophytic quality of Bairam-Ali.On the other hand, Bairam-Ali was resistant to the bactericidal (leptospiracidal) activity of the normal serum of human and of some other mammal animals. The most of pathogenicLeptospira species were resistant too, whereas the saprophytic speciesL. biflexa was sensitive.


### 3.3. Serological Characterization of theLeptospira spp. Strain Bairam-Ali

The strain Bairam-Ali had no antigenic affinity with 18 different serovars represented by reference strains. So the conclusion about the original serotype of Bairam-Ali was made. The serovar of Bairam-Ali was named bairam-ali. It formed the separated serogroup Bairam-Ali.


### 3.4. The Whole Genome Sequence Analysis

The whole genome sequence could help to resolve the mystery of the strain Bairam-Ali and clarify its phylogenetic position in theLeptospira genus.According to the NGS data the whole Bairam-Ali genome is 4,4 Mb, with GC % 34,74 and two chromosomes. The first chromosome is 4 059 463 bp and the second is 325 649 bp. Plasmid, typical of nonpathogenicLeptospira species, was absent from the Bairam-Ali genome. On the base of genome size and GC content, demonstrated in Table 3, Bairam-Ali is more similar to the pathogenic speciesL. interrogans andL. noguchii.Table 3
ComparisonL. spp. Bairam-Ali strain genome characteristics with most closely related Leptospira species.
Leptospira strain or species
Genome size, Mbp
GC%
L-BA*
4,4
34,7
L. interrogans
4,56 ± 0,32
35,22 ± 0,27
L. noguchii
4,76 ± 0,16
35,63 ± 0,27
L. terpstrae
4,09
38,2
L. meyeri
4,15
38,05
L. vanthielii
4,23
38,9
L. biflexa
3,95
38,9
*
L-BA: Leptospiraspp. Bairam-Ali.Based onrpoBgene sequence, we established that Bairam-Ali is more similar to pathogenic strainL. interrogansFiocruz-L1-130 (but the level of similarity is only 70.00%). However, by sequence ofrDNAgenes it is similar to nonpathogenic strains: for generrs, coding 16S rRNA,L. meyeri was most closely related (92.74%); for generrl, coding 23S rDNA,L. biflexa was more similar (95.00% fragment 1 and 93.00% fragment 2). Sequences ofrDNA genes of Bairam-Ali were deposited in GenBank, accession numbers KJ701750, KJ701751, and KJ701752.These data suggest dual phenotypic characteristics of the strain Bairam-Ali. In spite of having differences from both pathogenic and nonpathogenic species, many of the functional categories that are involved in essential housekeeping functions are represented in its core gene [27, 28]. Thus, basic groups of proteins involved in cell metabolism, survival, environmental adaptability, and potential pathogenic factors were present. Enzyme complexes participating in implementation of genetic information, in particular in DNA replication, were identified in strain Bairam-Ali: chromosomal replication initiator protein DNA, single-stranded DNA-binding protein; all subunits of DNA polymerase III, DNA polymerase I, and DNA polymerase IV; DNA gyrase; ligase; and helicase [29]. Also, the large groups of enzymes that take part in DNA reparation [29], for example, excinuclease ABC subunits A–C and exodeoxyribonuclease (III, V, and VII), and proteins of postreplicative mismatch repair system (Mutator S and Mutator L) were present. Archaeal DNA polymerase I gene was detected in the Bairam-Ali genome; this gene is a member of Family B and bacterial DNA polymerase II. The same gene was present in nonpathogenic strainL. biflexa Patoc genome but not in pathogenicL. interrogansFiocruz-L1-130 andL. borgpeterseniiHardjo-bovis-L550 genomes [27, 30]. The sequences of DNA metabolome have been deposited in GenBank, with accession numbers KJ701710–KJ701729.According to Bourret et al. [31], enteric bacteria usually have about 50 genes coding for structural and functional proteins involved in motility. Motility is a distinctive feature ofLeptospira; forty-seven different proteins provide bacterial locomotion [32]. In the Bairam-Ali genome, the genes for proteins of the basic parts of periplasmic flagella were detected: basal-body (Flg B–D, FlgF, and FlgG), hook (FlgE, FlgK, FlgL, FliE, FliD, and fliK), and four copies of FlaA (sheath protein of filament). The genes for proteins of flagellar rings L (FlgH), MS (FliF), and P (FlgI); biosynthesis protein (FlhA, FlhB (2), FlhF, FliL, and Fli Q-S); motors (MotB (3), MotA (2), FliG (3), FliM, and FliN); flagellar assembly factor (FliW and FliH), cell division (BolA (2), FtsA, FtsH (2), FtsI, FtsK, FtsW, and FtsZ), and gliding motility (GldF and GldG) proteins also were registered. Strain Bairam-Ali was found to have more than ten genes for the regulation of flagellum genes transcription and for signal transduction to flagella motor. Sequences of the flagellum genes have been deposited in GenBank, accession numbers KJ701653–KJ701709.It should be noted that the important structural element, that is, numerous groups of predicted lipoproteins (Lip), which may be either surface-exposed or located in the periplasm, is present in both saprophytic and pathogenicLeptospiraspecies. In the strain Bairam-Ali genome, as in the genome of nonpathogenic speciesL. biflexa [27], genes or orthologs of the major lipoproteins LipL32 and LipL41, which are important immunodominant antigens in pathogenicLeptospiraspecies [33, 34], were not identified. Nevertheless, in the genome of Bairam-Ali, we detected six genes of LipL45 (GenBank accession numbers KJ701647–KJ701652), which are processed to a peripheral membrane associated with the outer membrane complex. According to Matsunaga et al. [35] expression of P31, derived from the carboxy terminus of LipL45, is upregulated in stationary phase cultures; thus it may have a membrane-stabilizing function. Also, in a hamster model infected with pathogenicL. kirschneri, antibodies to LipL45 [35] were produced, suggesting its involvement in pathogenesis.Further analysis of the genome of Bairam-Ali revealed the presence of genes for more than 40 different classes of proteins, ensuring its natural resistance to a wide range of antibiotics (β-lactams, tetracyclines, glycopeptides (vancomycin), and polymyxin); efflux system and resistance to heavy metals (Czc A–C and arsenical-resistance proteins); and possible abortive infection phage-resistance protein. The broad resistance characteristics of strain Bairam-Ali are consistent with it being a natural reservoir for storage and possible transmission of these properties to other free-livingLeptospiraspecies.Sequences of the resistome genes and their proteins have been deposited in GenBank, accession numbers KJ701605–KJ701646.


### 3.5. Sequence Polymorphism

To determine the place of the original strain Bairam-Ali in theLeptospira genus, we undertook phylogenetic analysis of theLeptospira groups with different pathogenicity.Based on performed alignments, the percent nucleotide similarities ofrrs, rpoB, and MLST gene sequences of analyzedLeptospira genotypes were determined; see Supplementary Material available online at http://dx.doi.org/10.1155/2014/649034 (S1, S2, and S3). According to the obtained data, aligned sequences of 16S rRNA-codingrrs gene were 1305 bp, aligned sequences of generpoB were 493 bp, and aligned sequences of seven MLST tags (glmU-pntA-sucA-tpiA-pfkB-mreA-caiB) were 3105 bp.Among the investigatedLeptospira sequences, generpoB showed the highest variability, reaching 40.61%. 16S rRNA-coding gene was more conservative, and general sequence variability of therrsgene across all strains and species reached 11.52%. Sequence variability of seven MLST tags reached 34.88%.Detectable intraspecific sequence polymorphism for studied loci also was different. Intraspecific differences detected byrrswere less than 1%. Intraspecific differences detected by most polymorphicrpoB genes ranged from 0.61% (L. kirschneri) to 11.63% (L. borgpetersenii), with, in some cases, overlapping of intraspecific and interspecific values (S1, S2, and S3).Notably, the intraspecific differences detected by seven MLST tags resolved all of theLeptospira genotypes and matched 3% accepted threshold values for prokaryote species divergence [36, 37]. The exception wasL. weilii; interspecies differences ofL. weilii strains revealed by seven MLST tags reached 5.77% (S1, S2, and S3).Despite the differences in resolution ability of studied loci, all of them confirmed the unique nature ofLeptospira spp. strain Bairam-Ali.rrs,rpoB, and MLST tags obtained for Bairam-Ali had low similarity with other sequences. Maximum similarity of Bairam-Ali MLST tags (69.7%) was detected forL. interrogans (Table 3). At the same time, related levels of sequence similarity also characterized nonpathogenicand pathogenicLeptospira species (67.29–68.95%) and intermediate and pathogenicLeptospira species (70.0–72.8%). Sequence similarity in pairs of Bairam-Ali, to intermediateLeptospira species, to nonpathogenicLeptospiraspecies, and to pathogenicLeptospira species, was 65.8–67.31%, 67.6–69.18%, and 66.27–69.7%, respectively. The level of sequence similarity between Bairam-Ali andTurneriella parvawas the lowest (55.3%), which is evidence of Bairam-Ali belonging to the genusLeptospira.


### 3.6.Leptospira Phylogeny andL. spp. Bairam-Ali Location in theLeptospira Genus

The most prominent phylogenetic information was obtained through concatenated sequences of seven MLST tags. All genotypes were divided into three species groups: pathogenic, nonpathogenic, and intermediate.T. parva formed a distinct basal out-group branch.The genetic diversity of pathogenicLeptospira reached 0.159 (MLST data), 0.006 (rrs data), and 0.121 (rpoB data). The most variable species wereL. borgpetersenii(0.086rpoB) andL. noguchii (0.069). Despite the fact thatL. interrogans was the most representative species in the analysis (120 MLST genotypes), intraspecies genetic diversity forL. interrogans was only 0.002 (Figure 2).Figure 2
The number of base substitutions per site from averaging over all sequence pairs within each group is shown. Analyses were conducted using the Kimura 2-parameter model.The genetic diversity of nonpathogenicLeptospirawas similar to that of the pathogenicLeptospiraspecies and reached 0.063 (MLST data), 0.007 (rrs data), and 0.139 (rpoB data).Interestingly, the intermediate groupLeptospira had broader genetic diversity than did the nonpathogenic or pathogenicLeptospira (0.167, MLST data; 0.013,rrsdata; and 0.188rpoBdata) (Figure 2).In pathogenic group accessions of each species, excludingL. weilii, clearly distinct phylogenetic clusters, with high bootstrap supporting values, were present.In some data sets, significant subdivision ofL. weilii genotypes, combined with a close relationship betweenL. weilii andL. alexanderi,was found.Possible polyphyletic nature ofL. weilii has been described previously, particularly in the highly divergenttpiA locus [15]. According to our data, excluding from analysisglmU andpfkB loci,L. weilii andL. alexanderigenotypes fall into two different but closely related subclusters. This differentiation was found also withrrs gene phylogeny (S4).Three species in the pathogenic group,L. interrogans, L. kirschneri,andL. noguchii, formed one close genetic subgroup (BI = 99%, bootstrap index). The strains of these species are often the cause of the human leptospirosis. Four species,L. weilii, L. alexanderi, L. borgpetersenii, and L. santarosai,formed another phylogenetic subgroup (BI = 98%) (Figure 3). The strains of these species are usually isolated in natural foci. The candidate pathogenic species,L. alstoni andL. kmetyi, fell in the common big cluster of pathogenicLeptospira species. On the MLST tree,L. alstoniformed a sister clade to theL. weilii/L. alexanderi/L. borgpetersenii/L. santarosaigroup of pathogenicLeptospira species (BI = 70%), whereasL. kmetyiformed a basal branch (BI = 99) (Figure 3). The levels of genetic similarity ofL. alstoni andL. kmetyito pathogenicLeptospira species (0.913 and 0.926) were much higher than to nonpathogenic (0.13 and 0.09) or intermediate species (0.58 and 0.54). Our data confirm the results of previous characterization forL. alstoni on the base of16SrDNA gene and forL. kmetyi on the base of16SrDNA, gyrB, andrpoB genes analysis [2, 3].Figure 3
Phylogenetic tree ofLeptospira species based on concatenated sequences of seven MLST loci.The singleLeptospira spp. strain, Bairam-Ali, formed a separate distinct branch on dendrogram, with genetic distances comparable to those of distantly related species of differentLeptospiragroups. For example, differences between pathogenic and nonpathogenic, pathogenic and intermediate, and intermediate and nonpathogenic groups were 1.208, 0.873, and 0.845, respectively (Figure 2), whereas the differences betweenLeptospira spp. Bairam-Ali and pathogenic, intermediate, and nonpathogenic species were 1.232, 0.803, and 0.727, respectively; this result points to significant differences between Bairam-Ali and otherLeptospira species, a result that is consistent with whole genome sequencing data.It should be noted that the comparative studies of differentLeptospiragroups based on therrs gene variability, which added to the analysis the newly described saprophyticL. idonii strain Eri-1T [5] and recently approved 16S rDNA sequences of leptospires from the Peruvian Amazon, previously termed “clade C” [38, 39], did not change separate phylogenetic position ofLeptospira spp.strain Bairam-Ali.Thus, according to our results theLeptospira strain Bairam-Ali forms a separate phylogenetic branch of theLeptospira genus and cannot be attributed to any group of pathogenic, intermediate, or saprophyticLeptospira, illustrating its uniqueness.In conclusion, we found that the natural and anthropurgic foci supported ubiquitous pathogenic, intermediate, and nonpathogenicLeptospira species. They have circulated for a long time, interacting with maintenance and supplementary animal hosts. The relationships between the differentLeptospira strains provide for horizontal gene transfer and create in the bacterial population the pool of genes important for adaptivity to various conditions. Modern molecular genetic methods are suitable for investigation ofLeptospira and control of the pathogenic species, which are the causal agent of leptospirosis. Also, the taxonomic position of the new strains with unexpected properties can be founded on the base of these methods. Further investigation of the genetic diversity in the natural and anthropurgic foci is required for the identification of theLeptospiragenotypes, control of the strain’s transmission, and better understanding of the origin and evolution of theLeptospira species.


## 3.1. Morphology of theLeptospira spp. Strain Bairam-Ali

The morphology of the mysterious strain Bairam-Ali is typical of that of theLeptospira genus (Figure 1). Electron microscopy demonstrated that its cells are corkscrew-shaped with end hooks. They are thin and helical, like the cells of all known leptospires. Also, the cells have a diameter (d) of 0.12 μm and length (l) from 9.44 to 10.14 μm, like that of known leptospires (d = 0.15–0.3 μm and l = 6,00–20,00 μm) [26].Dual-beam scanning electron microscopy image ofL.species Bairam-Ali cells. (a) Original image; (b) with measuring the object in the program “Scandium” (green, length 10,14 μm; yellow, length (diameter) 0,12 μm; and green, length 9,44 μm).
(a)
(b)


## 3.2. Phenotypic Characterization of theLeptospira spp. Strain Bairam-Ali

Physiological characteristics of Bairam-Ali, demonstrated in Table2, suggest that strain Bairam-Ali can be classified as saprophyticLeptospira.Table 2
Physiological characteristics of theLeptospiraspp. strain Bairam-Ali.
Strain
Growth at temp (°C) of
Growth in the presence of
Lipase activity
Hemolytic activity against sheep erythrocytes
11
30
37
8-Azaguanine 225 pg/mL
Bairam-Ali
+
+
+
+
+
−For pathogenicity experiment, six four-week-old male golden Syrian hamsters were inoculated subcutaneously with 108 cells of strain Bairam-Ali in 1 mL of PBS. No hamster died from infection even at such a high bacterial dose.Leptospira cells were not detected during following bacterioscopic and bacteriological examination of the hamsters’ viscera. This is an additional evidence of saprophytic quality of Bairam-Ali.On the other hand, Bairam-Ali was resistant to the bactericidal (leptospiracidal) activity of the normal serum of human and of some other mammal animals. The most of pathogenicLeptospira species were resistant too, whereas the saprophytic speciesL. biflexa was sensitive.


## 3.3. Serological Characterization of theLeptospira spp. Strain Bairam-Ali

The strain Bairam-Ali had no antigenic affinity with 18 different serovars represented by reference strains. So the conclusion about the original serotype of Bairam-Ali was made. The serovar of Bairam-Ali was named bairam-ali. It formed the separated serogroup Bairam-Ali.


## 3.4. The Whole Genome Sequence Analysis

The whole genome sequence could help to resolve the mystery of the strain Bairam-Ali and clarify its phylogenetic position in theLeptospira genus.According to the NGS data the whole Bairam-Ali genome is 4,4 Mb, with GC % 34,74 and two chromosomes. The first chromosome is 4 059 463 bp and the second is 325 649 bp. Plasmid, typical of nonpathogenicLeptospira species, was absent from the Bairam-Ali genome. On the base of genome size and GC content, demonstrated in Table 3, Bairam-Ali is more similar to the pathogenic speciesL. interrogans andL. noguchii.Table 3
ComparisonL. spp. Bairam-Ali strain genome characteristics with most closely related Leptospira species.
Leptospira strain or species
Genome size, Mbp
GC%
L-BA*
4,4
34,7
L. interrogans
4,56 ± 0,32
35,22 ± 0,27
L. noguchii
4,76 ± 0,16
35,63 ± 0,27
L. terpstrae
4,09
38,2
L. meyeri
4,15
38,05
L. vanthielii
4,23
38,9
L. biflexa
3,95
38,9
*
L-BA: Leptospiraspp. Bairam-Ali.Based onrpoBgene sequence, we established that Bairam-Ali is more similar to pathogenic strainL. interrogansFiocruz-L1-130 (but the level of similarity is only 70.00%). However, by sequence ofrDNAgenes it is similar to nonpathogenic strains: for generrs, coding 16S rRNA,L. meyeri was most closely related (92.74%); for generrl, coding 23S rDNA,L. biflexa was more similar (95.00% fragment 1 and 93.00% fragment 2). Sequences ofrDNA genes of Bairam-Ali were deposited in GenBank, accession numbers KJ701750, KJ701751, and KJ701752.These data suggest dual phenotypic characteristics of the strain Bairam-Ali. In spite of having differences from both pathogenic and nonpathogenic species, many of the functional categories that are involved in essential housekeeping functions are represented in its core gene [27, 28]. Thus, basic groups of proteins involved in cell metabolism, survival, environmental adaptability, and potential pathogenic factors were present. Enzyme complexes participating in implementation of genetic information, in particular in DNA replication, were identified in strain Bairam-Ali: chromosomal replication initiator protein DNA, single-stranded DNA-binding protein; all subunits of DNA polymerase III, DNA polymerase I, and DNA polymerase IV; DNA gyrase; ligase; and helicase [29]. Also, the large groups of enzymes that take part in DNA reparation [29], for example, excinuclease ABC subunits A–C and exodeoxyribonuclease (III, V, and VII), and proteins of postreplicative mismatch repair system (Mutator S and Mutator L) were present. Archaeal DNA polymerase I gene was detected in the Bairam-Ali genome; this gene is a member of Family B and bacterial DNA polymerase II. The same gene was present in nonpathogenic strainL. biflexa Patoc genome but not in pathogenicL. interrogansFiocruz-L1-130 andL. borgpeterseniiHardjo-bovis-L550 genomes [27, 30]. The sequences of DNA metabolome have been deposited in GenBank, with accession numbers KJ701710–KJ701729.According to Bourret et al. [31], enteric bacteria usually have about 50 genes coding for structural and functional proteins involved in motility. Motility is a distinctive feature ofLeptospira; forty-seven different proteins provide bacterial locomotion [32]. In the Bairam-Ali genome, the genes for proteins of the basic parts of periplasmic flagella were detected: basal-body (Flg B–D, FlgF, and FlgG), hook (FlgE, FlgK, FlgL, FliE, FliD, and fliK), and four copies of FlaA (sheath protein of filament). The genes for proteins of flagellar rings L (FlgH), MS (FliF), and P (FlgI); biosynthesis protein (FlhA, FlhB (2), FlhF, FliL, and Fli Q-S); motors (MotB (3), MotA (2), FliG (3), FliM, and FliN); flagellar assembly factor (FliW and FliH), cell division (BolA (2), FtsA, FtsH (2), FtsI, FtsK, FtsW, and FtsZ), and gliding motility (GldF and GldG) proteins also were registered. Strain Bairam-Ali was found to have more than ten genes for the regulation of flagellum genes transcription and for signal transduction to flagella motor. Sequences of the flagellum genes have been deposited in GenBank, accession numbers KJ701653–KJ701709.It should be noted that the important structural element, that is, numerous groups of predicted lipoproteins (Lip), which may be either surface-exposed or located in the periplasm, is present in both saprophytic and pathogenicLeptospiraspecies. In the strain Bairam-Ali genome, as in the genome of nonpathogenic speciesL. biflexa [27], genes or orthologs of the major lipoproteins LipL32 and LipL41, which are important immunodominant antigens in pathogenicLeptospiraspecies [33, 34], were not identified. Nevertheless, in the genome of Bairam-Ali, we detected six genes of LipL45 (GenBank accession numbers KJ701647–KJ701652), which are processed to a peripheral membrane associated with the outer membrane complex. According to Matsunaga et al. [35] expression of P31, derived from the carboxy terminus of LipL45, is upregulated in stationary phase cultures; thus it may have a membrane-stabilizing function. Also, in a hamster model infected with pathogenicL. kirschneri, antibodies to LipL45 [35] were produced, suggesting its involvement in pathogenesis.Further analysis of the genome of Bairam-Ali revealed the presence of genes for more than 40 different classes of proteins, ensuring its natural resistance to a wide range of antibiotics (β-lactams, tetracyclines, glycopeptides (vancomycin), and polymyxin); efflux system and resistance to heavy metals (Czc A–C and arsenical-resistance proteins); and possible abortive infection phage-resistance protein. The broad resistance characteristics of strain Bairam-Ali are consistent with it being a natural reservoir for storage and possible transmission of these properties to other free-livingLeptospiraspecies.Sequences of the resistome genes and their proteins have been deposited in GenBank, accession numbers KJ701605–KJ701646.


## 3.5. Sequence Polymorphism

To determine the place of the original strain Bairam-Ali in theLeptospira genus, we undertook phylogenetic analysis of theLeptospira groups with different pathogenicity.Based on performed alignments, the percent nucleotide similarities ofrrs, rpoB, and MLST gene sequences of analyzedLeptospira genotypes were determined; see Supplementary Material available online at http://dx.doi.org/10.1155/2014/649034 (S1, S2, and S3). According to the obtained data, aligned sequences of 16S rRNA-codingrrs gene were 1305 bp, aligned sequences of generpoB were 493 bp, and aligned sequences of seven MLST tags (glmU-pntA-sucA-tpiA-pfkB-mreA-caiB) were 3105 bp.Among the investigatedLeptospira sequences, generpoB showed the highest variability, reaching 40.61%. 16S rRNA-coding gene was more conservative, and general sequence variability of therrsgene across all strains and species reached 11.52%. Sequence variability of seven MLST tags reached 34.88%.Detectable intraspecific sequence polymorphism for studied loci also was different. Intraspecific differences detected byrrswere less than 1%. Intraspecific differences detected by most polymorphicrpoB genes ranged from 0.61% (L. kirschneri) to 11.63% (L. borgpetersenii), with, in some cases, overlapping of intraspecific and interspecific values (S1, S2, and S3).Notably, the intraspecific differences detected by seven MLST tags resolved all of theLeptospira genotypes and matched 3% accepted threshold values for prokaryote species divergence [36, 37]. The exception wasL. weilii; interspecies differences ofL. weilii strains revealed by seven MLST tags reached 5.77% (S1, S2, and S3).Despite the differences in resolution ability of studied loci, all of them confirmed the unique nature ofLeptospira spp. strain Bairam-Ali.rrs,rpoB, and MLST tags obtained for Bairam-Ali had low similarity with other sequences. Maximum similarity of Bairam-Ali MLST tags (69.7%) was detected forL. interrogans (Table 3). At the same time, related levels of sequence similarity also characterized nonpathogenicand pathogenicLeptospira species (67.29–68.95%) and intermediate and pathogenicLeptospira species (70.0–72.8%). Sequence similarity in pairs of Bairam-Ali, to intermediateLeptospira species, to nonpathogenicLeptospiraspecies, and to pathogenicLeptospira species, was 65.8–67.31%, 67.6–69.18%, and 66.27–69.7%, respectively. The level of sequence similarity between Bairam-Ali andTurneriella parvawas the lowest (55.3%), which is evidence of Bairam-Ali belonging to the genusLeptospira.


## 3.6.Leptospira Phylogeny andL. spp. Bairam-Ali Location in theLeptospira Genus

The most prominent phylogenetic information was obtained through concatenated sequences of seven MLST tags. All genotypes were divided into three species groups: pathogenic, nonpathogenic, and intermediate.T. parva formed a distinct basal out-group branch.The genetic diversity of pathogenicLeptospira reached 0.159 (MLST data), 0.006 (rrs data), and 0.121 (rpoB data). The most variable species wereL. borgpetersenii(0.086rpoB) andL. noguchii (0.069). Despite the fact thatL. interrogans was the most representative species in the analysis (120 MLST genotypes), intraspecies genetic diversity forL. interrogans was only 0.002 (Figure 2).Figure 2
The number of base substitutions per site from averaging over all sequence pairs within each group is shown. Analyses were conducted using the Kimura 2-parameter model.The genetic diversity of nonpathogenicLeptospirawas similar to that of the pathogenicLeptospiraspecies and reached 0.063 (MLST data), 0.007 (rrs data), and 0.139 (rpoB data).Interestingly, the intermediate groupLeptospira had broader genetic diversity than did the nonpathogenic or pathogenicLeptospira (0.167, MLST data; 0.013,rrsdata; and 0.188rpoBdata) (Figure 2).In pathogenic group accessions of each species, excludingL. weilii, clearly distinct phylogenetic clusters, with high bootstrap supporting values, were present.In some data sets, significant subdivision ofL. weilii genotypes, combined with a close relationship betweenL. weilii andL. alexanderi,was found.Possible polyphyletic nature ofL. weilii has been described previously, particularly in the highly divergenttpiA locus [15]. According to our data, excluding from analysisglmU andpfkB loci,L. weilii andL. alexanderigenotypes fall into two different but closely related subclusters. This differentiation was found also withrrs gene phylogeny (S4).Three species in the pathogenic group,L. interrogans, L. kirschneri,andL. noguchii, formed one close genetic subgroup (BI = 99%, bootstrap index). The strains of these species are often the cause of the human leptospirosis. Four species,L. weilii, L. alexanderi, L. borgpetersenii, and L. santarosai,formed another phylogenetic subgroup (BI = 98%) (Figure 3). The strains of these species are usually isolated in natural foci. The candidate pathogenic species,L. alstoni andL. kmetyi, fell in the common big cluster of pathogenicLeptospira species. On the MLST tree,L. alstoniformed a sister clade to theL. weilii/L. alexanderi/L. borgpetersenii/L. santarosaigroup of pathogenicLeptospira species (BI = 70%), whereasL. kmetyiformed a basal branch (BI = 99) (Figure 3). The levels of genetic similarity ofL. alstoni andL. kmetyito pathogenicLeptospira species (0.913 and 0.926) were much higher than to nonpathogenic (0.13 and 0.09) or intermediate species (0.58 and 0.54). Our data confirm the results of previous characterization forL. alstoni on the base of16SrDNA gene and forL. kmetyi on the base of16SrDNA, gyrB, andrpoB genes analysis [2, 3].Figure 3
Phylogenetic tree ofLeptospira species based on concatenated sequences of seven MLST loci.The singleLeptospira spp. strain, Bairam-Ali, formed a separate distinct branch on dendrogram, with genetic distances comparable to those of distantly related species of differentLeptospiragroups. For example, differences between pathogenic and nonpathogenic, pathogenic and intermediate, and intermediate and nonpathogenic groups were 1.208, 0.873, and 0.845, respectively (Figure 2), whereas the differences betweenLeptospira spp. Bairam-Ali and pathogenic, intermediate, and nonpathogenic species were 1.232, 0.803, and 0.727, respectively; this result points to significant differences between Bairam-Ali and otherLeptospira species, a result that is consistent with whole genome sequencing data.It should be noted that the comparative studies of differentLeptospiragroups based on therrs gene variability, which added to the analysis the newly described saprophyticL. idonii strain Eri-1T [5] and recently approved 16S rDNA sequences of leptospires from the Peruvian Amazon, previously termed “clade C” [38, 39], did not change separate phylogenetic position ofLeptospira spp.strain Bairam-Ali.Thus, according to our results theLeptospira strain Bairam-Ali forms a separate phylogenetic branch of theLeptospira genus and cannot be attributed to any group of pathogenic, intermediate, or saprophyticLeptospira, illustrating its uniqueness.In conclusion, we found that the natural and anthropurgic foci supported ubiquitous pathogenic, intermediate, and nonpathogenicLeptospira species. They have circulated for a long time, interacting with maintenance and supplementary animal hosts. The relationships between the differentLeptospira strains provide for horizontal gene transfer and create in the bacterial population the pool of genes important for adaptivity to various conditions. Modern molecular genetic methods are suitable for investigation ofLeptospira and control of the pathogenic species, which are the causal agent of leptospirosis. Also, the taxonomic position of the new strains with unexpected properties can be founded on the base of these methods. Further investigation of the genetic diversity in the natural and anthropurgic foci is required for the identification of theLeptospiragenotypes, control of the strain’s transmission, and better understanding of the origin and evolution of theLeptospira species.



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*Source: 649034-2014-09-02.xml*