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MULTI
LOCUS SEQUENCE TYPING
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.
See:-
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
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