Leptospira spp.

Introduction
The Leptospira MLST database currently contains information on over 200 isolates. Of these, around two thirds have been isolated from patients with leptospirosis in Thailand between the years 2000 to 2005, and one third are reference strains from the collection maintained by the WHO/FAO/OIE Collaborating Center for Reference & Research on Leptospirosis, Australia. Also included are a small number of isolates cultured from rodents in northeast Thailand during 2004. Species have been confirmed for non-reference isolates by sequencing of the near full-length of the 16s rRNA gene. Two species are currently represented in this collection: L. interrogans and L. kirschneri. Together, these species represent 96% of clinical and rodent isolates from Thailand (94% and 2%, respectively). The remaining 4% of clinical isolates in Thailand are L. borgpetersenii; the primers described here do not give a full allelic profile and are not currently suitable for evaluation of this species. The database will be expanded by the addition of further clinical and reference isolates, and primers will be developed for L. borgpetersenii and other species of Leptospira.

A recent study has provided evidence that a sustained outbreak of leptospirosis in northeast Thailand which began in 1999 and peaked in 2000 was due to a single successful clone of L. interrogans (Thaipadungpanit J, Wuthiekanun V, Chierakul W, Smythe LD, Petkanchanapong W, et al. (2007) A Dominant Clone of Leptospira interrogans Associated with an Outbreak of Human Leptospirosis in Thailand. PLoS Negl Trop Dis 1(1): e56. doi:10.1371/journal.pntd.0000056). It is envisaged that the allelic profiles of isolates associated with outbreaks occurring across the world will be defined and maintained in this database, and that this will provide a resource to the scientific community interested in molecular epidemiology and disease pathogenesis.

Acknowledging the use of the MLST database in your publications.
Please acknowledge the use of this site in your publications as follows: 'We acknowledge the use of the Leptospira MLST database which is located at Imperial College London and is funded by the Wellcome Trust'.
 
 
Obtaining an allelic profile and comparing your strains with those in our database
The allelic profile of a leptospira strain is obtained by sequencing internal fragments of seven house-keeping genes.
The primers for the amplification and sequencing of these gene fragments can be obtained here .
The sequences must be obtained on both strands, and they must be 100% accurate, since even a single error may convert a known allele into a novel allele.
The sequences have to be trimmed so that they correspond exactly to the region that we use to define the alleles.

By selecting the multiple locus and allelic profile query, followed by submit. You can cut and paste your seven sequences into the corresponding boxes and submit them.

The software will check that the sequences are the correct length and that they do not contain any unrecognised characters. A check is also made to see if the submitted sequence is at least 70% similar to another allele at that locus (in case you have cut and pasted a sequence into the wrong box).
After submitting the seven sequences, you will obtain the allelic profile of your isolate and details of any leptospira isolates that are identical to the one you submitted. You can also search for isolates that have allelic profiles that are similar to yours. For example, isolates that have at least 4/7, 5/7 or 6/7 matches to the submitted allelic profile.
Further details about strains that are identical, or similar, to the submitted strain can be obtained by clicking on the strain names.
There are also options to assign the allele at a single locus, or to enter an allelic profile and find isolates in the database that match or nearly match this profile, or to browse the database (e.g. to look at the details of all strains of a particular serotype) and for advanced querying.
 
Is it really a Leptospira?
If a query allele is more than one or two percent different from any known allele, the strain may not be a L. interrogans or L. kirschneri. For example, if the strain is non-typeable, and has novel alleles at six or seven loci, and these differ by several percent from the alleles in the MLST database, it is likely that the strain is a closely related species.  The more likely scenario is that some PCR reactions fail to give an amplification product. For example, L. borgpetersenii fails to amplify at 5 or 6 loci using the primers given here. The serovar will guide the interpretation; this can be further supported as necessary by sequencing of the 16s RNA gene.  
 

The seven loci and the primers and conditions used for PCR
The leptospira MLST scheme uses internal fragments of the following seven house-keeping genes:-
glmU(UDP-N-acetylglucosamine pyrophosphorylase)
pntA(NAD(P) transhydrogenase subunit alpha)
sucA(2-oxoglutarate dehydrogenase decarboxylase component)
fadD (Probable long-chain-fatty-acid--CoA ligase)
tpiA(Triosephosphate isomerase)
pfkB(Ribokinase)
mreA(Rod shape-determining protein rodA)

The primer pairs used for the PCR amplification of internal fragments of these genes are:-
glmU-up,  5'-GGAAGGGCACCCGTATGAA and
glmU-dn,  5'-TCCCTGAGCGTTTTGATTT
pntA-up, 5'- TGCCGATCCTACAACATTA and
pntA-dn, 5'- AAGAAGCAAGATCCACAACTAC
sucA-up, 5'- AGAAGAGGCCGGTTATCATCAG and
sucA-dn, 5'- CTTCCGGGTCGTCTCCATTTA
fadD-up, 5'- AGTATGGCGTATCTTCCTCCTT and
fadD-dn, 5'- TTCCCACTGTAATTTCTCCTAA
tpiA-up, 5'- AAGCCGTTTTCCTAGCACATTC and
tpiA-dn, 5'- AGGCGCCTACAAAAAGACCAGA
pfkB-up, 5'- CCGAAGATAAGGGGCATACC and
pfkB-dn, 5'- CAAGCTAAAACCGTGAGTGATT
mreA-up, 5'- GTAAAAGCGGCCAACCTAACAC and
mreA-dn, 5'- ACGATCCCAGACGCAAGTAA

PCR amplification is carried out on chromosomal DNA using an extension time of 50 seconds, and an annealing temperature of 50 °C (fadD and glmU) or 52°C (mreA, pfkB, pntA, sucA, and tpiA), with Qiagen Taq polymerase. As the same primers are used for amplification and sequencing, it is important that only a single DNA fragment is amplified in the initial PCR. This may involve some optimisation of the annealing temperature. The DNA fragments are purified and sequencing reactions are carried out, in each direction, using the primers that were used for the initial PCR amplification. The samples are applied to an automated DNA sequencer.