BioMed Research International

Volume 2014 (2014), Article ID 649034, 15 pages

http://dx.doi.org/10.1155/2014/649034

Research Article

The Characteristics of Ubiquitous and Unique Leptospira Strains from the Collection of Russian Centre for Leptospirosis

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, and Alexandr L. Gintsburg

N.F. Gamaleya Institute for Epidemiology and Microbiology, Ministry of Health of Russia, Gamaleya Street 18, Moscow 123098, Russia

Received 22 May 2014; Revised 29 July 2014; Accepted 5 August 2014; Published 2 September 2014

Academic Editor: Vassily Lyubetsky

Copyright © 2014 Olga L. Voronina et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 of Leptospira 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 prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. 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 environmental Leptospira strain 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 of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species. Conclusions. Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions.

1. Introduction

Leptospira is a genus of bacteria that is encountered in all geographical areas, except arctic and arid regions. Some Leptospira are responsible for leptospirosis, a natural-focus disease. According to the List of Prokaryotic names with Standing in Nomenclature, the Leptospira 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, and L. alexanderi. Two species, L. alstonii and L. 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 and L. kmetyi was isolated from soil. Phylogenetic analysis with 16S rRNA gene sequences showed that L. alstonii and L. kmetyi clustered with the pathogenic Leptospira species [2, 3]. Five species, L. broomii, L. fainei, L. inadai, L. licerasiae, and L. wolffii, are classified as intermediate, and six species, L. biflexa, L. meyeri, L. terpstrae, L. vanthielii, L. wolbachii, and L. 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 genus Leptospira 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 of Leptospira species were isolated, from the animals (maintenance and supplementary hosts of the Leptospira), human patients, and environment, and saved in the Gamaleya Institute Microbial Collection (GIMC).

Among the strains of Leptospira species in GIMC one strain was mysterious and not closely related to any Leptospira, either pathogenic or saprophytic; it was named Leptospira spp. 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, Leptospira sp. 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 live Leptospira 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 the Leptospira genus and relationships with the pathogenic Leptospira 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 the Leptospira 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 the rpoB gene (coding subunit of bacterial RNA polymerase), according to La Scola et al. [13]. Sequence data for rpoB has been deposited in GenBank, with accession numbers KJ701730–KJ701749.

2.6. MLST

MLST for the strains of L. interrogans and L. 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 using caiB 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 the glmU, pntA, and sucA genes by raising the melting temperature to 50°C. Also, the MgCl₂ concentration was changed to 3.5 mM for the glmU, pntA, and sucA 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 of rpoB and MLST gene sequences was made by use of ClustalW2 [16]. BLAST search was used for species identification; similarity of rpoB 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 of Leptospira spp. MLST database [8]. All new Leptospira strains were submitted in the Leptospira spp. 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 homologues rrs (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 additional Leptospira strains were retrieved. Seventy-five nucleotide sequences represented either complete or partial cds of rrs gene and rpoB 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 from Leptospiraceae, 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 of rrs, rpoB, and MLST gene sequences and analysis nucleotide similarity (in %) were performed by use of ClustalW2 [16].

2.10. Phylogenetic Analysis

Phylogenetic analysis of Leptospira species was performed based on the rrs 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 between Leptospira genotypes 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 of Leptospira spp. 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 of Leptospira spp. 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 of Leptospira from various geographical regions (Table 1, —marker of the reference strains from GIMC).

The analysis of rpoB 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 of Leptospira twenty-four field isolates were related to the three pathogenic species (L. interrogans, L. borgpetersenii, and L. kirschneri). Four cultures were isolated from the commercially available Ellinghausen-McCullough-Johnson-Harris (EMJH) medium: two isolates from this group belonged to the nonpathogenic species L. biflexa and the other two isolates belonged to the intermediate species L. inadai. Thus, the prevailing species among the isolates collected on the territory of Russia comprised L. interrogans, L. borgpetersenii, and L. kirschneri.

The modified MLST scheme was applied to the isolates and reference strains representing 7 pathogenic Leptospira species. Twenty-four isolates of pathogenic species belonged to 8 different STs (Table 1). Five STs, common for Russian strains of Leptospira (L. interrogans ST37, 17, and 199; L. kirschneri ST110; and L. borgpetersenii ST146), were identified as having a longtime and ubiquitous distribution in various geographic areas.

Among the strains of Leptospira species available in GIMC, one strain seemed to be mysterious and not closely related to any Leptospira, either pathogenic or saprophytic, or intermediate. It was named Leptospira spp. strain Bairam-Ali, because it was isolated in 1971 in Turkmenia from the water of a drainage canal.

3.1. Morphology of the Leptospira spp. Strain Bairam-Ali

The morphology of the mysterious strain Bairam-Ali is typical of that of the Leptospira 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 of 0.12 μm and length from 9.44 to 10.14 μm, like that of known leptospires ( = 0.15–0.3 μm and = 6,00–20,00 μm) [26].

(a)

(b)

Figure 1: Dual-beam scanning electron microscopy image of L. 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).

3.2. Phenotypic Characterization of the Leptospira spp. Strain Bairam-Ali

Physiological characteristics of Bairam-Ali, demonstrated in Table 2, suggest that strain Bairam-Ali can be classified as saprophytic Leptospira.

Table 2: Physiological characteristics of the Leptospira  spp. 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 pathogenic Leptospira species were resistant too, whereas the saprophytic species L. biflexa was sensitive.

3.3. Serological Characterization of the Leptospira 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 the Leptospira 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 nonpathogenic Leptospira 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 species L. interrogans and L. noguchii.

Table 3: Comparison L. 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

-BA: Leptospira  spp. Bairam-Ali.

Based on rpoB gene sequence, we established that Bairam-Ali is more similar to pathogenic strain L. interrogans Fiocruz-L1-130 (but the level of similarity is only 70.00%). However, by sequence of rDNA genes it is similar to nonpathogenic strains: for gene rrs, coding 16S rRNA, L. meyeri was most closely related (92.74%); for gene rrl, coding 23S rDNA, L. biflexa was more similar (95.00% fragment 1 and 93.00% fragment 2). Sequences of rDNA 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 strain L. biflexa Patoc genome but not in pathogenic L. interrogans Fiocruz-L1-130 and L. borgpetersenii Hardjo-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 of Leptospira; 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 pathogenic Leptospira species. In the strain Bairam-Ali genome, as in the genome of nonpathogenic species L. biflexa [27], genes or orthologs of the major lipoproteins LipL32 and LipL41, which are important immunodominant antigens in pathogenic Leptospira species [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 pathogenic L. 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 (-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-living Leptospira species. 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 the Leptospira genus, we undertook phylogenetic analysis of the Leptospira groups with different pathogenicity.

Based on performed alignments, the percent nucleotide similarities of rrs, rpoB, and MLST gene sequences of analyzed Leptospira 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-coding rrs gene were 1305 bp, aligned sequences of gene rpoB were 493 bp, and aligned sequences of seven MLST tags (glmU-pntA-sucA-tpiA-pfkB-mreA-caiB) were 3105 bp.

Among the investigated Leptospira sequences, gene rpoB showed the highest variability, reaching 40.61%. 16S rRNA-coding gene was more conservative, and general sequence variability of the rrs gene 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 by rrs were less than 1%. Intraspecific differences detected by most polymorphic rpoB 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 the Leptospira genotypes and matched 3% accepted threshold values for prokaryote species divergence [36, 37]. The exception was L. weilii; interspecies differences of L. 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 of Leptospira 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 for L. interrogans (Table 3). At the same time, related levels of sequence similarity also characterized nonpathogenicand pathogenic Leptospira species (67.29–68.95%) and intermediate and pathogenic Leptospira species (70.0–72.8%). Sequence similarity in pairs of Bairam-Ali, to intermediate Leptospira species, to nonpathogenic Leptospira species, and to pathogenic Leptospira species, was 65.8–67.31%, 67.6–69.18%, and 66.27–69.7%, respectively. The level of sequence similarity between Bairam-Ali and Turneriella parva was the lowest (55.3%), which is evidence of Bairam-Ali belonging to the genus Leptospira.

3.6. Leptospira Phylogeny and L. spp. Bairam-Ali Location in the Leptospira 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 pathogenic Leptospira reached 0.159 (MLST data), 0.006 (rrs data), and 0.121 (rpoB data). The most variable species were L. borgpetersenii (0.086 rpoB) and L. noguchii (0.069). Despite the fact that L. interrogans was the most representative species in the analysis (120 MLST genotypes), intraspecies genetic diversity for L. 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 nonpathogenic Leptospira was similar to that of the pathogenic Leptospira species and reached 0.063 (MLST data), 0.007 (rrs data), and 0.139 (rpoB data).

Interestingly, the intermediate group Leptospira had broader genetic diversity than did the nonpathogenic or pathogenic Leptospira (0.167, MLST data; 0.013, rrs data; and 0.188 rpoB data) (Figure 2).

In pathogenic group accessions of each species, excluding L. weilii, clearly distinct phylogenetic clusters, with high bootstrap supporting values, were present.In some data sets, significant subdivision of L. weilii genotypes, combined with a close relationship between L. weilii and L. alexanderi, was found. Possible polyphyletic nature of L. weilii has been described previously, particularly in the highly divergent tpiA locus [15]. According to our data, excluding from analysis glmU and pfkB loci, L. weilii and L. alexanderi genotypes fall into two different but closely related subclusters. This differentiation was found also with rrs gene phylogeny (S4).

Three species in the pathogenic group, L. interrogans, L. kirschneri, and L. 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 and L. kmetyi, fell in the common big cluster of pathogenic Leptospira species. On the MLST tree, L. alstoni formed a sister clade to the L. weilii/L. alexanderi/L. borgpetersenii/L. santarosai group of pathogenic Leptospira species (BI = 70%), whereas L. kmetyi formed a basal branch (BI = 99) (Figure 3). The levels of genetic similarity of L. alstoni and L. kmetyi to pathogenic Leptospira 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 for L. alstoni on the base of 16SrDNA gene and for L. kmetyi on the base of 16SrDNA, gyrB, and rpoB genes analysis [2, 3].

Figure 3: Phylogenetic tree of Leptospira species based on concatenated sequences of seven MLST loci.

The single Leptospira spp. strain, Bairam-Ali, formed a separate distinct branch on dendrogram, with genetic distances comparable to those of distantly related species of different Leptospira groups. 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 between Leptospira 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 other Leptospira species, a result that is consistent with whole genome sequencing data.

It should be noted that the comparative studies of different Leptospira groups based on the rrs gene variability, which added to the analysis the newly described saprophytic L. 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 of Leptospira spp.strain Bairam-Ali.

Thus, according to our results the Leptospira strain Bairam-Ali forms a separate phylogenetic branch of the Leptospira genus and cannot be attributed to any group of pathogenic, intermediate, or saprophytic Leptospira, illustrating its uniqueness.

In conclusion, we found that the natural and anthropurgic foci supported ubiquitous pathogenic, intermediate, and nonpathogenic Leptospira species. They have circulated for a long time, interacting with maintenance and supplementary animal hosts. The relationships between the different Leptospira 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 of Leptospira 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 the Leptospira genotypes, control of the strain’s transmission, and better understanding of the origin and evolution of the Leptospira species.

Abbreviations

L int: | L. interrogans
L kir: | L. kirschneri
L nog: | L. noguchii
L wei: | L. weilii
L ale: | L. alexanderi
L bor: | L. borgpetersenii
L san: | L. santarosai
L als: | L. alstoni
L kme: | L. kmetyi
L lic: | L. licerasiae
L wlf: | L. wolffii
fai: | L. fainei
bro: | L. broomii
L ind: | L. inadai
bif: | L. biflexa
yan: | L. yanagawae
mey: | L. meyeri
ter: | L. terpstrae
van: | L. vanthielii
wlb: | L. wolbachii
LBA: | L. species Bairam-Ali
TURPA: | Turneriella parva
INT: | Intermediate species
NON-PATH: | Nonpathogenic species.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

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