Policy Document





The original MLST web software was developed by Man-Suen Chan (Oxford University) and this version has been developed by David Aanensen (Imperial College) who is funded by The Wellcome Trust

Multilocus sequence typing (MLST) is an unambiguous procedure for characterising isolates of bacterial species using the sequences of internal fragments of seven house-keeping genes.  Approx. 450-500 bp internal fragments of each gene are used, as these can be accurately sequenced on both strands using an automated DNA sequencer.   For each house-keeping gene, the different sequences present within a bacterial species are assigned as distinct alleles and, for each isolate, the alleles at each of the seven loci define the allelic profile or sequence type (ST).

Each isolate of a species is therefore unambiguously characterised by a series of seven integers which correspond to the alleles at the seven house-keeping loci.

In MLST the number of nucleotide differences between alleles is ignored and sequences are given different allele numbers whether they differ at a single nucleotide site or at many sites.   The rationale is that a single genetic event resulting in a new allele can occur by a point mutation (altering only a single nucleotide site), or by a recombinational replacement (that will often change multiple sites) -  weighting according to the number of nucleotide differences between alleles would imply that the latter allele was more distantly-related to the original allele than the former, which would be true if all nucleotide changes occurred by mutation, but not if the changes occurred by a recombinational replacement.

Most bacterial species have sufficient variation within house-keeping genes to provide many alleles per locus, allowing billions of distinct allelic profiles to be distinguished using seven house-keeping loci.  For example, an average of 30 alleles per locus allows about 20 billion genotypes to be resolved.

MLST is based on the well established principles of multilocus enzyme electrophoresis, but differs in that it assigns alleles at multiple house-keeping loci directly by DNA sequencing, rather than indirectly via the electrophoretic mobility of their gene products.

Maiden, M.C.J., Bygraves, J.A., Feil, E., Morelli, G., Russell, J.E., Urwin, R., Zhang, Q., Zhou, J., Zurth, K., Caugant, D.A., Feavers, I.M., Achtman, M., and Spratt, B.G. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms.Proc. Natl. Acad. Sci. USA, 95, 3140-3145, 1998.

Spratt, B.G.Multilocus sequence typing: molecular typing of bacterial pathogens in an era of rapid DNA sequencing and the Internet. Curr. Opin. Microbiol. 2, 312-316, 1999. 

MLST and strain characterisation

The great advantage of MLST is that sequence data are unambiguous and the allelic profiles of isolates can easily be compared to those in a large central database via the Internet (in contrast to most typing procedures which involve comparing DNA fragment sizes on gels).

A further advantage of MLST is that the allelic profiles of isolates can be obtained from clinical material by PCR amplification of the seven house-keeping loci directly from CSF or blood.  Thus isolates can be precisely characterised even when they cannot be cultured from clinical material.


Enright, M.C., Knox, K., Griffiths, D., Crook, D.W.M., and Spratt, B.G.Multilocus sequence typing of Streptococcus pneumoniae directly from cerebrospinal fluid.Eur. J. Clin. Microbiol. Infect. Dis. 19, 627-630, 2000).

Kriz, P., Kalmusova, J. and Felsberg, J. 2002. Multilocus sequence typing of Neisseria meningitidis directly from cerebrospinal fluid. Epidemiol. Infect. 128: 157-160.

Diggle MA, Bell CM, Clarke SC. .Nucleotide sequence-based typing of meningococci directly from clinical samples. J Med Microbiol. 2003 Jun;52(6):505-508

Some aspects of the MLST procedure can be automated -

Clarke, S.C., Diggle, M.A. and Edwards, G.F. 2001. Semiautomation of multilocus sequence typing for the characterization of clinical isolates of Neisseria meningitidis. J. Clin. Microbiol. 39: 3066-3071.

Jefferies J, Clarke S, Diggle M, Smith A, Dowson C, Mitchell T. .Automated Pneumococcal MLST Using Liquid-Handling Robotics and a Capillary DNA Sequencer. Mol Biotechnol. 2003 Jul;24(3):303-8

MLST and Population Biology

The sequences of seven loci, from many hundreds of isolates of a bacterial species, provide data that can be used to address the population and evolutionary biology of the species.  For example:-

Feil, E.J., Maiden, M.C.J., Achtman, M., and Spratt, B.G. The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis.Mol. Biol. Evol. 16, 1496-1502, 1999.

Holmes, E.C., Urwin, R. and Maiden, M.C.J.  The influence of recombination on the population structure and evolution of the human pathogen Neisseria meningitidis. Mol. Biol. Evol. 16:741-749, 1999.

Feil, E.J., Maynard Smith, J., Enright, M.C. and Spratt, B.G.Estimating recombinational parameters in Streptococcus pneumoniae from multilocus sequence typing data.Genetics, 154, 1439-1450, 2000.

Feil, E.J., Holmes, E.C., Bessen, D.E., Chan, M.-S., Day, N.P.J., Enright, M.C., Goldstein, R., Hood, D.W., Kalia, A., Moore, C.E., Zhou, J. and Spratt, B.G.    Recombination within natural populations of pathogenic bacteria: short-term consequences and long-term phylogenetic consequences.  Proc. Natl. Acad. Sci. USA, 98, 182-187, 2001.

Feil, E.J., Cooper JE, Grundmann H, Robinson DA, Enright MC, Berendt T, Peacock SJ, Smith JM, Murphy M, Spratt BG, Moore CE, Day NP.
B.G.    How clonal is Staphylococcus aureus?  J Bacteriol. 2003 Jun;185(11):3307-16

MLST Schemes

Neisseria meningitidis (the original description used six loci; Click here for THE current seven locus scheme.

Maiden, M.C., Bygraves, J.A., Feil, E., Morelli, G., Russell, J.E., Urwin, R., Zhang, Q., Zhou, J., Zurth, K., Caugant, D.A., Feavers, I.M., Achtman, M. and Spratt, B.G. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA. 95: 3140-3145.

Streptococcus pneumoniae

Enright, M.C. and Spratt, B.G. 1998. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144: 3049-3060.

Staphylococcus aureus

Enright, M.C., Day, N.P., Davies, C.E., Peacock, S.J. and Spratt, B.G. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38: 1008-1015.

Streptococcus pyogenes

Enright, M.C., Spratt, B.G., Kalia, A., Cross, J.H. and Bessen, D.E. 2001. Multilocus sequence typing of Streptococcus pyogenes and the relationships between emm type and clone. Infect. Immun. 69: 2416-2427.

Campylobacter jejuni

Dingle, K.E., Colles, F.M., Wareing, D.R., Ure, R., Fox, A.J., Bolton, F.E., Bootsma, H.J., Willems, R.J., Urwin, R. and Maiden, M.C. 2001. .Multilocus sequence typing system for Campylobacter jejuni. J. Clin. Microbiol. 39: 14-23.

Haemophilus influenzae

Meats E., Feil EJ, Stringer S, Cody AJ, Goldstein R, Kroll JS, Popovic T, Spratt BG 2003 .Characterization of encapsulated and noncapsulated Haemophilus influenzae and determination of phylogenetic relationships by multilocus sequence typing. J Clin Microbiol 2003 Apr;41(4):1623-36

Burkholderia pseudomallei

Godoy D, Randle G, Simpson AJ, Aanensen DM, Pitt TL, Kinoshita R, Spratt BG. 2003 .Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei. J Clin Microbiol 2003 May;41(5):2068-79

Streptococcus suis

King, S.J., Leigh, J.A., Heath, P.J., Luque, I., Tarradas, C., Dowson, C.G. and Whatmore, A.M. 2002. Development of a multilocus sequence typing scheme for the pig pathogen Streptococcus suis: identification of virulent clones and potential capsular serotype exchange. J. Clin. Microbiol. 40: 3671-3680.

Enterococcus faecium

Homan, W.L., Tribe, D., Poznanski, S., Li, M., Hogg, G., Spalburg, E., Van Embden, J.D. and Willems, R.J. 2002. Multilocus sequence typing scheme for Enterococcus faecium. J. Clin. Microbiol. 40: 1963-1971.

Candida albicans

Bougnoux, M.E., Morand, S. and d'Enfert, C. 2002. Usefulness of multilocus sequence typing for characterization of clinical isolates of Candida albicans. J. Clin. Microbiol. 40: 1290-1297.

Listeria monocytogenes

Salcedo C., Arreaza L, Alcala B, de la Fuente L, Vazquez JA. UDevelopment of a multilocus sequence typing method for analysis of Listeria monocytogenes clones. J Clin Microbiol. 2003 Feb;41(2):757-62.

Streptococcus agalactiae

Jones N., Bohnsack JF, Takahashi S, Oliver KA, Chan MS, Kunst F, Glaser P, Rusniok C, Crook DW, Harding RM, Bisharat N, Spratt BG. Multilocus Sequence Typing System for Group B Streptococcus. J Clin Microbiol. 2003 Jun;41(6):2530-2536.

 Please address queries to David Aanensen