Multilocus sequence typing method for identification and genotypic classification of pathogenic <it>Leptospira </it>species

1476-0711-5-28 1476-0711 Research Multilocus sequence typing method for identification and genotypic classification of pathogenic <it>Leptospira </it>species Ahmed Niyaz niyaz.cdfd@gmail.com Devi S Manjulata manju@cdfd.org.in de los Á Valverde M mvalverde@inciensa.sa.cr Vijayachari P vijayacharip@yahoo.com Machang'u S Robert machangu2001@yahoo.com Ellis A William bill.ellis@dardni.gov.uk Hartskeerl A Rudy r.hartskeerl@kit.nl

Pathogen Evolution Group, Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad 500076, India

ISOGEM working group on Spirochetes, The International Society for Genomic and Evolutionary Microbiology (ISOGEM), Sassari, Italy

National Reference Center Leptospirosis. INCIENSA (Costarrican Institute for Research in Nutrition and Health), Costa Rica

Regional Medical Research Centre (RMRC), Port Blair, India

Department of Veterinary Microbiology and Parasitology, Sokoine University of Agriculture, P. O. Box 3019, Morogoro, Tanzania

Veterinary Sciences Division (VSD), The Queen's University of Belfast, Stoney Road, Stormont, Belfast, Northern Ireland, BT4 3SD, UK

WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis, KIT Biomedical Research, KIT (Koninklijk Instituut voor de Tropen/Royal Tropical Institute) Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands

Annals of Clinical Microbiology and Antimicrobials 1476-0711 2006 5 1 28 http://www.ann-clinmicrob.com/content/5/1/28 17121682 10.1186/1476-0711-5-28 12 10 2006 23 11 2006 23 11 2006 2006 Ahmed et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Leptospira are the parasitic bacterial organisms associated with a broad range of mammalian hosts and are responsible for severe cases of human Leptospirosis. The epidemiology of leptospirosis is complex and dynamic. Multiple serovars have been identified, each adapted to one or more animal hosts. Adaptation is a dynamic process that changes the spatial and temporal distribution of serovars and clinical manifestations in different hosts. Serotyping based on repertoire of surface antigens is an ambiguous and artificial system of classification of leptospiral agents. Molecular typing methods for the identification of pathogenic leptospires up to individual genome species level have been highly sought after since the decipherment of whole genome sequences. Only a few resources exist for microbial genotypic data based on individual techniques such as Multiple Locus Sequence Typing (MLST), but unfortunately no such databases are existent for leptospires.

Results

We for the first time report development of a robust MLST method for genotyping of Leptospira. Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a set of 120 strains including 41 reference strains representing different geographical areas and from different sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species.

Conclusion

The main advantages of MLST over other typing methods for leptospires include reproducibility, robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.

Background

Leptospirosis is a zoonotic and an emerging infectious disease caused by the pathogenic Leptospira species and is identified in the recent years as a global public health problem because of its increased mortality and morbidity in different countries. Leptospirosis is frequently misdiagnosed as a result of its protean and non-specific presentation resembling many other febrile diseases, notably viral haemorrhagic fevers such as dengue 1. There is, for certain, an underestimation of the leptospirosis problem due to lack of awareness and under-recognition through a lack of proper use of diagnostic tools.

The common mode of transmission of the infection in humans is either by direct or indirect contact with the urine of infected animals and may lead to potential lethal disease. A unique feature of this organism is to parasitize in a wide variety of wild and domestic animals 2. Traditionally, two species have been identified, i.e. Leptospira interrogans and L. biflexa for pathogenic and non-pathogenic leptospires, respectively. The serovar is the basic identifier, characterized on the basis of serological criteria. To date nearly 300 serovars have been identified under the species L. interrogans alone that have been distributed among 25 different serogroups of antigenically similar serovars 3.

Previously a classification system based on DNA-DNA hybridization studies has been introduced, which now comprises 17 Leptospira species 4567. Among these, 7 species: L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri and L. alexanderi are considered as the main agents of leptospirosis 56. The enormous inventory of serovars, based mainly on an ever-changing surface antigen repertoire, throws an artificial and unreliable scenario of strain diversity. It is therefore difficult to track strains whose molecular identity keeps changing according to the host and the environmental niches they inhabit and cross through.

Other than the serological methods, molecular tools that have been employed so far for sub-classification and cataloguing of leptospiral agents include restriction endonuclease assay (REA) 89, pulsed field gel electrophoresis (PFGE) 1011, restriction fragment length polymorphism (RFLP) 12, arbitrarily primed PCR 13, Variable Number of Tandem Repeats (VNTR) analysis 14 and fluorescent amplified fragment length polymorphism (FAFLP) 15. All these techniques however, suffer from certain disadvantages that include requirement of large quantity of pure and high quality DNA, low discriminatory power, low reproducibility, ambiguous interpretation of data and problems associated with transfer of data between different laboratories 14.

MLST is a simple PCR based technique, which makes use of automated DNA sequencers to assign and characterize the alleles present in different target genes. The method allows one to generate sequence data in a low to high-throughput scale, which is unambiguous and suitable for epidemiological and population studies. The selected loci are generally the housekeeping genes, which evolve very slowly over an evolutionary time-scale 16 and hence qualify as highly robust markers of ancient and modern ancestry. The sequencing of multiple loci provides a balance between technical feasibility and resolution. MLST has been applied to the study of many other bacterial species like Neisseria meningitides 17, Streptococcus pneumoniae 18, Yersinia species 19, Campylobacter jejuni 20 and Helicobacter pylori 21.

Our present study is the first attempt to use the MLST, which currently differentiates the species and examines the intra and interspecies relationships of Leptospira. This method in future could be developed as a highly sophisticated genotyping system based on integrated genome analysis approaches to correctly identify and track leptospiral strains and is expected to greatly facilitate epidemiology of leptospirosis apart from deciphering the origins and evolution of leptospires in a global sense.

Methods

Bacterial strains

Bacterial strains (Table 1) were cultured by the WHO reference laboratory at the KIT Biomedical Research Centre at The Royal Tropical Institute, Amsterdam, The Netherlands (all isolates and reference strains labelled RK3) and at the Veterinary Sciences Division (VSD), The Queen's University of Belfast, United Kingdom (reference strains labelled RB3) and the WHO reference centre at Port Blair India (labelled isol 15). A total of 120 strains consisting of 79 isolates and 41 reference strains from different sources and geographical regions were analyzed by MLST. The 41 reference strains included in the study belonged to six Leptospira species (L. interrogans; L. kirschneri; L. noguchii; L. borgpetersenii; L. santarosai and L. alexanderi).

Table 1

Details of leptospiral strains and isolates used for MLST based

Labels

Genome Species

Serogroup

Serovar

Strain

Geographical area

Source

INT 01

L. interrogans

Canicola

Sumneri

Sumner

Malaysia

RB3

INT 02

L. interrogans

Canicola

Portlandvere

MY 1039

Jamaica

RB3

INT 03

L. interrogans

Pomona

Australia

RB3

INT 04

L. interrogans

Pomona

Proechimys

1161 U

Panama

RB3

INT 05

L. interrogans

Pomona

Kenniwicki

LT 1026

USA

RB3

INT 06

L. interrogans

Grippotyphosa

Moskva V

Unknown

RB4

INT 07

L. interrogans

Grippotyphosa

Muelleri

RM 2

Malaysia

RB3

INT 08

L. interrogans

Sejroe

Roumanica

LM 294

Roumania

RB3

INT 09

L. interrogans

Sejroe

Saxkoebing

Mus 24

Denmark

RB3

INT 10

L. interrogans

Sejroe

Hardjoprajitno

Indonesia

RB3

INT 11

L. interrogans

Icterohaemorrhagiae

Lai

China

RB3

INT 12

L. interrogans

Icterohaemorrhagiae

Copenhageni

M 20

Denmark

RB3

INT 13

L. interrogans

Grippotyphosa

Valbuzzi

Australia

RB3

INT 14

L. interrogans

Pyrogenes

Manilae

LT 398

Phillipins

RB3

INT 15

L. interrogans

Australis

Ballico

RK3

INT 16

L. interrogans

Icterohaemorrhagiae

RGA

Germany

RK3

INT 17

L. interrogans

Grippotyphosa

Ratnapura

Field Isolate 1

South Andaman

Isol 15

INT 18

L. interrogans

Icterohaemorrhagiae

Copenhageni

Field Isolate 2

South Andaman

Isol 15

INT 19

L. interrogans

Grippotyphosa

Ratnapura

Field Isolate 3

South Andaman

Isol 15

INT 20

L. interrogans

Grippotyphosa

Ratnapura

Field Isolate 4

South Andaman

Isol 15

INT 21

L. interrogans

Grippotyphosa

Valbuzzi

Field Isolate 5

South Andaman

Isol 15

INT 22

L. interrogans

Icterohaemorrhagiae

Copenhageni

Field Isolate 6

South Andaman

Isol 15

INT 23

L. interrogans

Grippotyphosa

Valbuzzi

Field Isolate 7

North Andaman

Isol 15

INT 24

L. interrogans

Grippotyphosa

Valbuzzi

Field Isolate 8

North Andaman

Isol 15

INT 25

L. interrogans

Grippotyphosa

Ratnapura

Field Isolate 9

South Andaman

Isol 15

INT 26

L. interrogans

Grippotyphosa

Ratnapura

Field Isolate 10

South Andaman

Isol 15

INT 27

L. interrogans

Grippotyphosa

Ratnapura

Field Isolate 11

South Andaman

Isol 15

INT 28

L. interrogans

Grippotyphosa

Unknown

Field Isolate 12

South Andaman

Isol 15

INT 29

L. interrogans

Grippotyphosa

Unknown

Field Isolate 13

South Andaman

Isol 15

INT 30

L. interrogans

Sejroe

Field Isolate 14

South Andaman

Isol 15

INT 31

L. interrogans

Pomona

Unknown

Field Isolate 15

South Andaman

Isol 15

INT 32

L. interrogans

Grippotyphosa

Ratnapura

Field Isolate 16

South Andaman

Isol 15

INT 33

L. interrogans

Australis

Ramisi

Field Isolate 17

South Andaman

Isol 15

INT 34

L. interrogans

Grippotyphosa

Unknown

Field Isolate 18

South Andaman

Isol 15

INT 35

L. interrogans

Grippotyphosa

Valbuzzi

Field Isolate 19

South Andaman

Isol 15

INT 36

L. interrogans

Grippotyphosa

Valbuzzi

Field Isolate 20

South Andaman

Isol 15

INT 37

L. interrogans

Hebdomadis

Goiano

Bovino 131

Brazil

RB3

INT 38

L. interrogans

Canicola*

M12/90

Brazil

Isol

INT 39

L. interrogans

Icterohaemorrhagiae*

Copenhageni*

M9/99

Brazil

Isol

INT 40

L. interrogans

Australis*

Rushan*

L01

Brazil

Isol

INT 41

L. interrogans

Canicola*

L02

Brazil

Isol

INT 42

L. interrogans

Canicola*

L03

Brazil

Isol

INT 43

L. interrogans

Canicola*

L09

Brazil

Isol

INT 44

L. interrogans

Icterohaemorrhagiae*

Copenhageni*

L10

Brazil

Isol

INT 45

L. interrogans

Canicola*

L14

Brazil

Isol

INT 46

L. interrogans

Lyme*

K30B

Isol

INT 47

L. interrogans

Australis*

K9H

Isol

INT 48

L. interrogans

Icterohaemorrhagiae*

Copenhageni*

Isolate 9

Costa Rica

Isol

INT 49

L. interrogans

Unknown*

Isolate 10

Costa Rica

Isol

INT 50

L. interrogans

Australis*

Lora*

1992

Tanzania

Isol

INT 51

L. interrogans

Australis*

Lora*

2324

Tanzania

Isol

INT 52

L. interrogans

Australis*

Lora*

2364

Tanzania

Isol

INT 53

L. interrogans

Australis*

Lora*

2366

Tanzania

Isol

INT 54

L. interrogans

Ballum*

Kenya*

4885

Tanzania

Isol

INT 55

L. interrogans

Ballum*

Kenya*

4883

Tanzania

Isol

KIR 01

L. kirschneri

Canicola

Kuwait

136/2/2

Kuwait

RB3

KIR 02

L. kirschneri

Canicola

Schueffneri

Vleermuis 90 C

Indonesia

RB3

KIR 03

L. kirschneri

Pomona

Mozdok

5621

Soviet Union (Russia)

RB3

KIR 04

L. kirschneri

Grippotyphosa

Vanderhoedeni

Kipod 179

Israel

RB3

KIR 05

L. kirschneri

Pomona

Tsaratsovo

B 81/7

Bulgaria

RB3

KIR 06

L. kirschneri

Grippotyphosa

Moskva V

Russia

RK3

KIR 07

L. kirschneri

Grippotyphosa

Ratnapura

Wumalasena

Sri Lanka

RK3

KIR 08

L. kirschneri

Icterohaemorrhagiae*

Sokoine*

745

Tanzania

Isol

KIR 09

L. kirschneri

Icterohaemorrhagiae*

Sokoine*

771

Tanzania

Isol

KIR 10

L. kirschneri

Icterohaemorrhagiae*

Mwogolo*

826

Tanzania

Isol

KIR 11

L. kirschneri

Icterohaemorrhagiae*

Mwogolo*

845

Tanzania

Isol

KIR 12

L. kirschneri

Canicola*

Qunjian*

2980

Tanzania

Isol

KIR 13

L. kirschneri

Icterohaemorrhagiae*

Sokoine*

4602

Tanzania

Isol

KIR 14

L. kirschneri

Sejroe*

Ricardi/Saxkoebing*

1499

Isol

KIR 15

L. kirschneri

Sejroe*

Ricardi/Saxkoebing*

1501

Isol

KIR 16

L. kirschneri

Ballum*

Kenya

Njenga

Kenya

RK3

NOG 01

L. noguchii

Pyrogenes

Myocastoris

LSU 1551

USA

RB3

NOG 02

L. noguchii

Louisiana

LSU 1945

USA

RK3

NOG 03

L. noguchii

Panama

CZ214k

Panama

RK3

NOG 04

L. noguchii

Pyrogenes*

Guaratuba *

Isolate 4

Costa Rica

Isol

SAN 01

L. santarosai

Mini

Georgia

LT 117

USA

RB3

SAN 02

L. santarosai

Sejroe

Recreo

380

Nicaragua

RB3

SAN 03

L. santarosai

Pyrogenes

Guaratuba

An 7705

Brazil

RB3

SAN 04

L. santarosai

Pyrogenes

Varela

1019

Nicaragua

RB3

SAN 05

L. santarosai

Grippotyphosa

Canalzonae

CZ188

Panama

RK3

SAN 06

L. santarosai

Bataviae*

Brasiliensis*

An 776

Brazil

Isol

SAN 07

L. santarosai

Sejroe*

Guaricura*

Bov.G

Brazil

Isol

SAN 08

L. santarosai

Sejroe*

Guaricura*

M4/98

Brazil

Isol

SAN 09

L. santarosai

Grippotyphosa*

Bananal*

2ACAP

Brazil

Isol

SAN 10

L. santarosai

Grippotyphosa*

Bananal*

16CAP

Brazil

Isol

SAN 11

L. santarosai

Pyrogenes*

Alexi/Guaratuba/Princestown*

Isolate 1

Costa Rica

Isol

SAN 12

L. santarosai

Sarmin*

Weaveri/Rio*

Isolate 2

Costa Rica

Isol

SAN 13

L. santarosai

Tarassovi*

Rama*

Isolate 3

Costa Rica

Isol

SAN 14

L. santarosai

Tarassovi*

Rama*

Isolate 5

Costa Rica

Isol

SAN 15

L. santarosai

Bataviae*

Claytoni*

Isolate 6

Costa Rica

Isol

SAN 16

L. santarosai

Shermani*

Shermani/Babudieri/Aguaruna*

Isolate 8

Costa Rica

Isol

SAN 17

L. santarosai

unknown*

(putative new serovar)#

Isolate 7

Costa Rica

Isol

SAN 18

L. santarosai

Icterohaemorrhagiae*

Copenhageni*

K13A

Isol

ALE 01

L. alexanderi

Manhao

L60

China

RK3

BOR 01

L. borgpetersenii

Sejroe

Istarica

Bratislava

Slovakia

RB3

BOR 02

L. borgpetersenii

Sejroe

M 84

Denmark

RB3

BOR 03

L. borgpetersenii

Javanica

Dehong

De 10

China

RB3

BOR 04

L. borgpetersenii

Javanica

Veltrat Batavia

Indonesia

RB3

BOR 05

L. borgpetersenii

Javanica

Zhenkang

L 82

China

RB3

BOR 06

L. borgpetersenii

Javanica

Poi

Italy

RK3

BOR 07

L. borgpetersenii

Mini

Sari

Italy

RK3

BOR 08

L. borgpetersenii

Ballum*

Kenya*

153

Tanzania

Isol

BOR 09

L. borgpetersenii

Ballum *

Kenya*

159

Tanzania

Isol

BOR 10

L. borgpetersenii

Ballum *

Kenya*

723

Tanzania

Isol

BOR 11

L. borgpetersenii

Ballum *

Kenya*

766

Tanzania

Isol

BOR 12

L. borgpetersenii

Ballum *

Kenya*

1605

Tanzania

Isol

BOR 13

L. borgpetersenii

Ballum *

Kenya*

1610

Tanzania

Isol

BOR 14

L. borgpetersenii

Ballum *

Kenya*

2062

Tanzania

Isol

BOR 15

L. borgpetersenii

Ballum *

Kenya*

2348

Tanzania

Isol

BOR 16

L. borgpetersenii

Ballum *

Kenya*

2447

Tanzania

Isol

BOR 17

L. borgpetersenii

Ballum *

Kenya*

4880

Tanzania

Isol

BOR 18

L. borgpetersenii

Ballum *

Kenya*

4787

Tanzania

Isol

BOR 19

L. borgpetersenii

Hebdomadis*

Kremastos/Hebdomadis*

873

Ireland

Isol

BOR 20

L. borgpetersenii

Hebdomadis*

Kremastos/Hebdomadis*

871

Ireland

Isol

BOR 21

L. borgpetersenii

Sejroe*

Saxkoebing*

1498

Ireland

Isol

BOR 22

L. borgpetersenii

Sejroe*

Ricardi/Saxkoebing*

1522

Isol

BOR 23

L. borgpetersenii

Sejroe*

Ricardi/Saxkoebing*

1525

Isol

BOR 24

L. borgpetersenii

Pomona*

Kunming*

RIM 139

Portugal

Isol

BOR 25

L. borgpetersenii

Pomona*

Kunming*

RIM 201

Portugal

Isol

BOR 26

L. borgpetersenii

Sejroe*

Ricardi/Saxkoebing*

RIM 156

Portugal

Isol

* – Unpublished presumptive classification, # – Unpublished putative new serovar, Isol – Isolates, RB – reference strains from Belfast lab, RK – reference strains from KIT. The numbers 3, 4 and 15 refer to the references describing strains or isolates.

Selection and validation of target genes for MLST

The candidate loci sequences were obtained from the strains L. interrogans Fiocruz L1-130 and L. interrogans Lai 56601 strains from the Leptolist server. Six genes, namely adk (Adenylate Kinase), icdA (Isocitrate dehydrogenase), LipL32 (outer membrane lipoprotein LipL32), rrs2 (16S rRNA), secY (pre-protein translocase SecY protein), and LipL41 (outer membrane Lipoprotein LipL41) (Table 2) were selected for MLST analysis. Many sequences of the rrs2, LipL32 and LipL41 are available in the GenBank 2. PCR primers were designed for these genes based on GenBank records in the conserved regions flanking the variable internal fragments of the target regions. PCR primers for adk, icdA and secY were based on gene sequences of strains Fiocruz L1-130 and Lai 56601 2223 (Table 2). The Primer 3 software 24 was used to design the PCR primers for the amplification of the candidate loci. The PCR amplifications of the different MLST target genes were performed using 1.5 mM MgCl₂, 200 μM of dNTP's (MBI Fermentas), 25–50 ng template DNA using Gene Amp 9700 (Applied Biosystems, Foster City, USA) PCR system.

Table 2

Details of gene loci and the corresponding primer sequences used for MLST analysis

Gene

Locus

Gene size (bp)

Co-ordinates

PCR product size (bp)

Size of polymorphic sequence (bp)

Function

Primer sequences

adk

LIC12852

564

3458298–3458861

531

430

Adenylate Kinase

F-GGGCTGGAAAAGGTACACAA

R-ACGCAAGCTCCTTTTGAATC

icdA

LIC13244

1197

3979829–3981025

674

557

Isocitarate Dehydrogenase

F-GGGACGAGATGACCAGGAT

R-TTTTTTGAGATCCGCAGCTTT

LipL41

LIC12966

1068

3603575–3604642

520

518

Outermenbrane Lipoprotein LipL41

F-TAGGAAATTGCGCAGCTACA

R-GCATCGAGAGGAATTAACATCA

rrs2

LIC11508

1512

1862433–1863944

541

452

16S ribosomal RNA

F-CATGCAAGTCAAGCGGAGTA

R-AGTTGAGCCCGCAGTTTTC

secY

LIC12853

1383

3458869–3460251

549

Translocase pre-protein secY

F-ATGCCGATCATTTTTGCTTC

R-CCGTCCCTTAATTTTAGACTTCTTC

LipL32

LIC11352

819

1666299–1667117

474

Outermenbrane Lipoprotein LipL32

F-ATCTCCGTTGCACTCTTTGC

R-ACCATCATCATCATCGTCCA

Amplification parameters included an initial denaturation at 95°C for 5 min followed by 35 cycles of amplification comprising of denaturation (94°C for 30 sec), annealing (58°C for 30 sec) and primer extension (72°C for 1 min) steps and a final extension of 7 min at 72°C. All the amplified fragments were checked on 1.5% or 2% agarose gel with ethidium bromide staining and the amplicons were sequenced in both the directions using Big Dye Terminator cycle sequencing Kit (Applied Biosystems, Foster City, USA) on ABI 3100 DNA sequencers (Applied Biosystems, Foster City, USA).

MLST data analysis

The electropherograms were viewed by using Chromas Lite version 2.01 (Technelysium Pty Ltd, Australia) and the resulting DNA sequences corresponding to both the forward and reverse reads were aligned using the Seqscape software (Applied Biosystems, Foster City, USA). Low quality nucleotide sequences were trimmed from the ends while comparing with the reference sequence of the Fiocruz strain and all the processed sequences were subsequently aligned by Clustal X 25. The Sequence Type Analysis and Recombinational Test (START) programme 26 was used to determine Guanine-Cytosine content, number of polymorphic sites and the ratio of non-synonymous to synonymous nucleotide substitutions (d_N/d_S). The phylogenetic analysis was performed using concatenated (2980bp) sequences in the order adk, icdA, LipL32, LipL41, rrs2 and secY for each strain using MEGA 3.1 27 and the consensus tree was drawn based on 1000 bootstrap replicates with Kimura 2 parameter.

Results

Diversity among the candidate loci analyzed

The 5' parts of rrs2, LipL32, LipL41 and the 3' part of secY were considered for the analysis based on abundance of nucleotide substitution positions found in these regions. The sizes of the fragments analyzed for the selected housekeeping genes ranged between 430bp (adk) and 557bp (icdA). The positions of these MLST loci were scattered throughout the chromosome I of L. interrogans Fiocruz L1-130 (Table 2). Clustal X programme was used to align all the individual sequences separately and we observed that there were no large insertions and deletions in the selected region. According to our analysis the rrs2 gene was found to be highly conserved among all the isolates with the percentage of variable sites being 4.42. Other genes namely LipL32, LipL41, icdA, adk and secY, however, were significantly diverse with the percentages of variable sites being 11.3, 21.04, 22.8, 27.2 and 28.7 respectively. The locus with highest diversity was icdA with 51 different alleles found among the set of 120 different isolates studied. The ratio of non-synonymous (d_N) to synonymous substitution (d_S) was much less than 1.0 indicating that these genes are not under positive selection pressure (the selection is against the amino acid change), whereas the rrs2 gene showed d_N/d_Sratio as 1.369 suggesting a high flexibility for amino acid changes. The percentage of G + C content in these loci ranged from 39.16 (secY) to 51.92 (rrs2) (Table 3). The synonymous substitution which, plays a role in the divergence of strains was more frequent in icdA and secY with 126 different synonymous sites. When compared to synonymous substitutions, non-synonymous substitutions were more frequent in all the genes tested, but highest numbers of 429 and 423 were observed in case of icdA and secY respectively (Table 3).

Table 3

Allelic diversity parameters observed for the six target genes used for MLST analysis of leptospires

Gene

G+C%

No. of alleles

Polymorphic sites

Synonymous sites

Non-synonymous sites

% of variable nucleotide sites

d _N /d _S ratio

adk

41.55

117

100

329

27.2

0.039

icd1

40.9

127

126

429

22.8

0.017

LipL32

46.46

112

362

11.3

0.091

LipL41

42.88

109

123

393

21.04

0.055

rrs2

51.92

112

338

4.42

1.369

secY

39.16

158

126

423

28.7

0.019

Clustering analysis of Leptospires based on MLST

The neighbor-joining tree was constructed for representative isolates based on a 'super locus' of 2980bp comprising concatenated sequence of all the six loci. For this, the genes were fused in the order – adk, icdA, LipL32, LipL41, rrs2 and secY. The phylogenetic tree generated five different clusters where L. interrogans (56 samples), L. noguchii (4 samples), L. kirschneri (16 samples), L. santarosai (18 samples), L. alexanderi (1 sample), L. borgpetersenii (26 samples) separated according to their genome species (Figure 1).

Figure 1

Genetic relatedness among Leptospira isolates based on the concatenated sequences of the six housekeeping and candidate gene loci analyzed (see table 1 for detailed information on isolates/strains)

Genetic relatedness among Leptospira isolates based on the concatenated sequences of the six housekeeping and candidate gene loci analyzed (see table 1 for detailed information on isolates/strains). * Unpublished presumptive serological classification.

MLST analysis also clearly identified each of the field isolates up to the species level and in general, classification based on these observations corroborated with previous taxonomic status of these isolates determined either by serological criteria or by genomic methods such as FAFLP (data not shown). There are two isolates for which serological classification seemed to be in contrast to MLST identification, i.e. INT 46, L. interrogans serovar Lyme and SAN 18, L. santarosai serovar Copenhageni. It should be noted that in these cases serovar designation is based on preliminary serological analysis, which may be incorrect. L. alexanderi was found to be genomically highly similar to L. santarosai and clustered accordingly. This could therefore be a subspecies of L. santarosai.

L. interrogans isolate SAN 17 from Costa Rica, indicated as putative new serovar (Table 1) along with another L. interrogans member belonging to serovar Muelleri of the serogroup Grippotyphosa, formed an isolated branch under the L. interrogans cluster arguing for a separate taxonomic status, possibly another subspecies of L. interrogans.

Discussion

The present study was a first attempt in the development of MLST for Leptospira species; the main objective being the selection of the housekeeping and candidate genes that are species specific, stable and evolve slowly. The availability of the complete sequence of L. interrogans Lai 56601 and Fiocruz L1-130 helped us in selecting the candidate loci. Genetically diverse group of strains was used for the study to evaluate the sequence diversity among the tested housekeeping genes. The six genes selected and studied here appear to be distinctly resolving to reveal a wide variety of genotypes among the isolates analyzed. This indicates a significant heterogeneity and sequence variation at each locus (Table 3).

The six loci selected were found to be suitable for MLST typing as they can be amplified and sequenced in all the isolates irrespective of species as these loci are unlinked on the L. interrogans chromosome I and exhibit a modest degree of sequence diversity and resolution. A total of 585 polymorphic sites were observed in the 'super locus' of 2980bp. Non-synonymous sites were more abundant as compared to synonymous sites (Table 3) indicating that the amino acid sequence variability possibly represents acclimatization to the specific host and environmental restrictions 2.

Several molecular tools that have been so far described for the characterization of Leptospira are associated with several drawbacks. Methods like PFGE, RFLP, and REA need large quantity of purified DNA, present tedious methodology, have low discriminatory levels, are hard to interpret the data, suffer from lack of reproducibility, require specialized equipment such as counter clamped homogenous electric field electrophoresis systems and give poor data transfer. The VNTR or MLVA technique described by Majed et al 14 and Slack et al 28 are more specific to L. interrogans. MLST overcomes all these disadvantages as this technique is simple, and easy to standardize on an automated DNA sequencer that is more widely available in most of the laboratories and above all the sequence data generated are unambiguous, specific and explicit. The main advantage of MLST is the transfer of data that can be shared and compared between different laboratories easily through the Internet. To date, a large number of organisms have been typed by MLST, which proved to be a highly discriminatory technique 29. MLST analysis on Leptospira strains showed that the similar serovars and the serogroups of different species are not clustered together (Figure 1). This method is more suitable in identifying the species of leptospires as indicated by the clustering patterns up to species level (Figure 1). The tree generated gives an idea on the phylogenetic organization of the Leptospira. The L. interrogans seems to be like a clonal branch as the isolates are more closely related and emerge from L. kirschneri indicating that they have evolved from this species. The L. interrogans and the L. kirschneri emerge from L. noguchii branch indicating it as a monophyletic group 2. Due to the greater sequence diversity observed in all the six genes except rrs2, the dendrogram generated could differentiate effectively the L. interrogans, L. kirschneri, L. noguchii, L. santarosai and L. borgpetersenii.

Conclusion

With this new technique of MLST, we believe the issues related to ever-increasing serotype diversity would be effectively addressed via high throughput genome profiling. This will help establish population genetic structure of this pathogen with diverse host range and under different ecological conditions and will provide a scope for genotype-phenotype correlation to be established. Analyses based on the allelic profiles generated by our method may be successfully used to gain insights into the evolution and phylogeographic affinities of leptospires as it has been done for many other organisms. Large-scale, global genotyping, therefore, largely constitutes the essential mandate of studying leptospirosis in different hosts at the population level. Such approaches always generate extremely valuable information that can be translated into a wealth of databases to search for strain specific markers for epidemiology or to construct evolutionary history of the strains for a particular epidemiological catchment area. This task becomes greatly simplified if the genotypic data are categorized, stacked, archived and made electronically portable to facilitate easy access, extensive comparisons, remote access and retrieval in sets.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

NA and SMD carried out all the experiments related to primer designing, DNA sequencing and phylogenetic analyses and wrote the manuscript. NA and RAH designed the study and edited the manuscript. MDLAV, RSM, PV and WAE performed isolations of Leptospira. WAE and RAH performed serological and (other) molecular characterizations of the isolates, extracted DNA from isolates and reference strains and provided geographic and epidemiological data.

Acknowledgements

We thank Prof. Seyed E. Hasnain, University of Hyderabad, India for discussions and helpful suggestions. We thank three anonymous experts who served as referees for this work and their constructive suggestions have helped the manuscript a great deal to become worth publication. We also thank S. A. Vasconcello from the Univesidada de São Paulo, Brazil for providing some of the isolates and staff of the WHO/FAO/OIE Leptospirosis Reference Centre, KIT Biomedical Research for technical and material support in the (provisional) typing of Leptospira isolates. NA would like to thank Dept. of Biotechnology, Govt. of India for the financial support in terms of core grants to CDFD. Authors also acknowledge the financial support of the European Union (Lepto and dengue Project, INCO-Dev ICA4-CT-2001-10086 and RATZOOMAN Project, INCO-Dev ICA4-CT-2002-10056).

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