BioMed Central Page 1 of 10 (page number not for citation purposes) Annals of Clinical Microbiology and Antimicrobials Open Access Research Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species Niyaz Ahmed* †1,2 , S Manjulata Devi †1 , M de los Á Valverde 3 , P Vijayachari 4 , Robert S Machang'u 5 , William A Ellis 6 and Rudy A Hartskeerl 2,7 Address: 1 Pathogen Evolution Group, Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad 500076, India, 2 ISOGEM working group on Spirochetes, The International Society for Genomic and Evolutionary Microbiology (ISOGEM), Sassari, Italy, 3 National Reference Center Leptospirosis. INCIENSA (Costarrican Institute for Research in Nutrition and Health), Costa Rica, 4 Regional Medical Research Centre (RMRC), Port Blair, India, 5 Department of Veterinary Microbiology and Parasitology, Sokoine University of Agriculture, P. O. Box 3019, Morogoro, Tanzania, 6 Veterinary Sciences Division (VSD), The Queen's University of Belfast, Stoney Road, Stormont, Belfast, Northern Ireland, BT4 3SD, UK and 7 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 Email: Niyaz Ahmed* - niyaz.cdfd@gmail.com; S Manjulata Devi - manju@cdfd.org.in; M de los Á Valverde - mvalverde@inciensa.sa.cr; P Vijayachari - vijayacharip@yahoo.com; Robert S Machang'u - machangu2001@yahoo.com; William A Ellis - bill.ellis@dardni.gov.uk; Rudy A Hartskeerl - r.hartskeerl@kit.nl * Corresponding author †Equal contributors 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. Published: 23 November 2006 Annals of Clinical Microbiology and Antimicrobials 2006, 5:28 doi:10.1186/1476-0711-5- 28 Received: 12 October 2006 Accepted: 23 November 2006 This article is available from: http://www.ann-clinmicrob.com/content/5/1/28 © 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. Annals of Clinical Microbiology and Antimicrobials 2006, 5:28 http://www.ann-clinmicrob.com/content/5/1/28 Page 2 of 10 (page number not for citation purposes) 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 misdiag- nosed as a result of its protean and non-specific presenta- tion resembling many other febrile diseases, notably viral haemorrhagic fevers such as dengue [1]. There is, for cer- tain, 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]. Tradi- tionally, two species have been identified, i.e. Leptospira interrogans and L. biflexa for pathogenic and non-patho- genic 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 [4-7]. Among these, 7 spe- cies: L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri and L. alexanderi are consid- ered as the main agents of leptospirosis [5,6]. The enor- mous 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 cata- loguing of leptospiral agents include restriction endonu- clease assay (REA) [8,9], pulsed field gel electrophoresis (PFGE) [10,11], restriction fragment length polymor- phism (RFLP) [12], arbitrarily primed PCR [13], Variable Number of Tandem Repeats (VNTR) analysis [14] and flu- orescent 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 discrimina- tory power, low reproducibility, ambiguous interpreta- tion 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 bal- ance between technical feasibility and resolution. MLST has been applied to the study of many other bacterial spe- cies like Neisseria meningitides [17], Streptococcus pneumo- niae [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 sophisti- cated genotyping system based on integrated genome analysis approaches to correctly identify and track lept- ospiral strains and is expected to greatly facilitate epidemi- ology 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 ref- erence laboratory at the KIT Biomedical Research Centre at The Royal Tropical Institute, Amsterdam, The Nether- lands (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). 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 Gen- Bank records in the conserved regions flanking the varia- ble internal fragments of the target regions. PCR primers Annals of Clinical Microbiology and Antimicrobials 2006, 5:28 http://www.ann-clinmicrob.com/content/5/1/28 Page 3 of 10 (page number not for citation purposes) 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 Pomona 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 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 Hardjoprajitno Indonesia RB3 INT 11 L. interrogans Icterohaemorrhagiae Lai Lai China RB3 INT 12 L. interrogans Icterohaemorrhagiae Copenhageni M 20 Denmark RB3 INT 13 L. interrogans Grippotyphosa Valbuzzi Valbuzzi Australia RB3 INT 14 L. interrogans Pyrogenes Manilae LT 398 Phillipins RB3 INT 15 L. interrogans Australis Australis Ballico Ballico RK3 INT 16 L. interrogans Icterohaemorrhagiae 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 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* 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* Canicola* L02 Brazil Isol Annals of Clinical Microbiology and Antimicrobials 2006, 5:28 http://www.ann-clinmicrob.com/content/5/1/28 Page 4 of 10 (page number not for citation purposes) INT 42 L. interrogans Canicola* Canicola* L03 Brazil Isol INT 43 L. interrogans Canicola* Canicola* L09 Brazil Isol INT 44 L. interrogans Icterohaemorrhagiae* Copenhageni* L10 Brazil Isol INT 45 L. interrogans Canicola* Canicola* L14 Brazil Isol INT 46 L. interrogans Lyme* Lyme* K30B UK Isol INT 47 L. interrogans Australis* Australis* K9H UK Isol INT 48 L. interrogans Icterohaemorrhagiae* Copenhageni* Isolate 9 Costa Rica Isol INT 49 L. interrogans Unknown* 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 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 UK Isol KIR 15 L. kirschneri Sejroe* Ricardi/Saxkoebing* 1501 UK 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 Louisiana LSU 1945 USA RK3 NOG 03 L. noguchii Panama 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 Table 1: Details of leptospiral strains and isolates used for MLST based (Continued) Annals of Clinical Microbiology and Antimicrobials 2006, 5:28 http://www.ann-clinmicrob.com/content/5/1/28 Page 5 of 10 (page number not for citation purposes) 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 UK Isol ALE 01 L. alexanderi Manhao Manhao L60 China RK3 BOR 01 L. borgpetersenii Sejroe Istarica Bratislava Slovakia RB3 BOR 02 L. borgpetersenii Sejroe Sejroe M 84 Denmark RB3 BOR 03 L. borgpetersenii Javanica Dehong De 10 China RB3 BOR 04 L. borgpetersenii Javanica Javanica Veltrat Batavia Indonesia RB3 BOR 05 L. borgpetersenii Javanica Zhenkang L 82 China RB3 BOR 06 L. borgpetersenii Javanica Poi Poi Italy RK3 BOR 07 L. borgpetersenii Mini 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 UK Isol BOR 23 L. borgpetersenii Sejroe* Ricardi/Saxkoebing* 1525 UK 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. Table 1: Details of leptospiral strains and isolates used for MLST based (Continued) Annals of Clinical Microbiology and Antimicrobials 2006, 5:28 http://www.ann-clinmicrob.com/content/5/1/28 Page 6 of 10 (page number not for citation purposes) for adk, icdA and secY were based on gene sequences of strains Fiocruz L1-130 and Lai 56601 [22,23] (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 MgCl 2 , 200 μM of dNTP's (MBI Fermentas), 25–50 ng template DNA using Gene Amp 9700 (Applied Biosystems, Foster City, USA) PCR system. Amplification parameters included an initial denatura- tion at 95°C for 5 min followed by 35 cycles of amplifica- tion 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 Biosys- tems, 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 subse- quently 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-synon- ymous to synonymous nucleotide substitutions (d N /d S ). The phylogenetic analysis was performed using concate- nated (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 boot- strap 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 house- keeping 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 per- centage of variable sites being 4.42. Other genes namely LipL32, LipL41, icdA, adk and secY, however, were signifi- cantly 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 substitu- tion (d S ) was much less than 1.0 indicating that these genes are not under positive selection pressure (the selec- tion is against the amino acid change), whereas the rrs2 gene showed d N /d S ratio as 1.369 suggesting a high flexi- bility 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-syn- onymous substitutions were more frequent in all the 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 549 549 Translocase pre- protein secY F-ATGCCGATCATTTTTGCTTC R-CCGTCCCTTAATTTTAGACTTCTTC LipL32 LIC11352 819 1666299–1667117 474 474 Outermenbrane Lipoprotein LipL32 F-ATCTCCGTTGCACTCTTTGC R-ACCATCATCATCATCGTCCA Annals of Clinical Microbiology and Antimicrobials 2006, 5:28 http://www.ann-clinmicrob.com/content/5/1/28 Page 7 of 10 (page number not for citation purposes) genes tested, but highest numbers of 429 and 423 were observed in case of icdA and secY respectively (Table 3). Clustering analysis of Leptospires based on MLST The neighbor-joining tree was constructed for representa- tive isolates based on a 'super locus' of 2980bp compris- ing 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 differ- ent 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). MLST analysis also clearly identified each of the field iso- lates 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 sero- logical 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. inter- rogans member belonging to serovar Muelleri of the sero- group 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 can- didate 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 sev- eral drawbacks. Methods like PFGE, RFLP, and REA need large quantity of purified DNA, present tedious method- ology, have low discriminatory levels, are hard to interpret the data, suffer from lack of reproducibility, require spe- cialized equipment such as counter clamped homoge- nous 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 laborato- ries 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 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 40 117 100 329 27.2 0.039 icd1 40.9 51 127 126 429 22.8 0.017 LipL32 46.46 36 54 112 362 11.3 0.091 LipL41 42.88 52 109 123 393 21.04 0.055 rrs2 51.92 29 20 112 338 4.42 1.369 secY 39.16 49 158 126 423 28.7 0.019 Annals of Clinical Microbiology and Antimicrobials 2006, 5:28 http://www.ann-clinmicrob.com/content/5/1/28 Page 8 of 10 (page number not for citation purposes) (Figure 1). This method is more suitable in identifying the species of leptospires as indicated by the clustering pat- terns up to species level (Figure 1). The tree generated gives an idea on the phylogenetic organization of the Lept- ospira. 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 spe- cies. 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 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)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). * Unpublished presumptive serological classification. 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FI 3 ) L . i n t e r r o g a n s G r i p p o t y p h o s a R a t n a p u r a ( F I 4 ) L . i n t e r r o g a n s G r i p p o t y p h o s a V a l b u z z i ( F I 8 ) L. i n t er r og an s G r i pp ot y ph o s a R at n a p ur a ( F I 1 1 ) L . i n t e r r o g a n s G r i p p o t y p h o s a V a l b u z z i ( F I 1 9 ) L . i n t e r r o ga ns G r i p po t y p ho s a ( F I 1 3) L . i n t e r r o g a n s G r i p p o t y p h o s a R a t n a p u r a ( F I 1 6 ) L . i n t e r r o g a n s G r i p p o t y p h o s a V a l b u z z i ( F I 7 ) L . i n t e r r o g a n s G r i p p o t y p h o s a ( F I 1 8 ) L . i n t e r r o g a n s G r i p p o t y p h o s a V a l b u z z i ( F I 5 ) L . i n t e r r o g a n s G r i p p o t y p h o s a R a t n a p u r a ( F I 9 ) L . i n t e r r o g an s G r i p p o t y p h o s a R a t n a p u r a ( F I 1 0 ) L . i n t e r r o g a n s G r i p p o t y p h o s a V a l b u z z i ( F I 2 0 ) L . i n t e r r o g a n s G r i p p o t y p h o s a M u e l l e r i R M 2 L . i 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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 profil- ing. This will help establish population genetic structure of this pathogen with diverse host range and under differ- ent ecological conditions and will provide a scope for gen- otype-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 electron- ically portable to facilitate easy access, extensive compari- sons, remote access and retrieval in sets. Competing interests The author(s) declare that they have no competing inter- ests. 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 charac- terizations of the isolates, extracted DNA from isolates and reference strains and provided geographic and epide- miological data. Acknowledgements We thank Prof. Seyed E. Hasnain, University of Hyderabad, India for discus- sions 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 pro- viding some of the isolates and staff of the WHO/FAO/OIE Leptospirosis Reference Centre, KIT Biomedical Research for technical and material sup- port 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. 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Molecular typing methods for the identification of pathogenic. 1 of 10 (page number not for citation purposes) Annals of Clinical Microbiology and Antimicrobials Open Access Research Multilocus sequence typing method for identification and genotypic classification. 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