Multilocus sequence typing of Streptococcus pneumoniae
The pneumococcal MLST database currently
contains over 5000 isolates, obtained from serious invasive
disease, acute otitis media and nasopharyngeal carriage,
as well as penicillin-resistant and multiply antibiotic-resistant
The database will be expanded by the addition
of further invasive isolates and antibiotic-resistant isolates,
plus further isolates from other pneumococcal diseases and
from nasopharyngeal carriage.
It is envisaged that the allelic profiles
of reference isolates of all published clones of antibiotic-resistant
pneumococci will be maintained in our database, which will
allow the characterisation of penicillin-resistant pneumococci
via the Internet. Our studies have shown that members of the
major antibiotic-resistant clones usually have the same allelic
profile, or differ from that profile at only a single locus
(Zhou, J., Enright, M.C., and Spratt, B.G. Identification
of the major Spanish clones of penicillin-resistant pneumococci
via the Internet using multilocus sequence typing.J. Clin. Microbiol., 38, 977-986, 2000).
This database will be maximally
useful if you deposit the allelic profiles and epidemiological
information on your strains at this site. If you are not able
to carry out MLST in your own laboratory, it would also be
helpful if a typical isolate of any novel penicillin-resistant
or multi-resistant clone, or any other novel clone of
interest, could be sent to us, so that we can determine its
allelic profile and enter it in the database.
Acknowledging the use of the MLST database in your
Please acknowledge the use of this site
in your publications as follows: 'We acknowledge the use of
the pneumococcal MLST database which is located at Imperial
College London and is funded by the Wellcome Trust'.
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an allelic profile and comparing your strains with those in
The allelic profile of a pneumococcal 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. The sequences of the seven loci
from a typical pneumococcus can be obtained here
and can be used to ensure that your sequences have been trimmed
You then need to access
our databases, which involves a simple registration process,
that allows us to inform you of new developments by e-mail.
Select the Streptococcus
pneumoniae database, and the multiple locus and
allelic profile query, followed by submit. You
then 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 pneumococcal 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 pneumococcus?
If a query allele is more than one or two percent different
from any known allele, the strain may not be a pneumococcus.
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 pneumococcal alleles in the MLST database,
it is likely that the strain is an 'atypical' pneumococcus
or a closely-related species. Check if the strain is
serotypeable as such strains will not have a pneumococcal
Some non-typeable pneumococci are authentic non-capsulated
isolates and these have typical pneumococcal alleles at six
or all seven loci. Some of these alleles may not all
be in the database, but they should be similar in sequence
to those in the database. N.B. Authentic pneumococci do sometimes
have diverged alleles at one of the seven loci.
A new facility is the ability to
concatenate the DNA sequences at six of the seven MLST loci (ddl
is omitted as this gene from penicillin-resistant isolates
often contains highly diverged sequences) and to
compare this concatenated sequence with the concatenated sequences of a
reference set of pneumococci and pneumococcal-like organisms.
If your isolate clusters on the resulting tree within the
pneumococcal strains you can be confident that it is a
pneumococcus. If it is clearly separated from the
pneumococcal strains on the tree it is almost certainly not an
seven loci and the primers and conditions used for PCR
The pneumococcal MLST
scheme uses internal fragments of the following seven house-keeping
The primer pairs used
for the PCR amplification of internal fragments of these genes
5'-GCC TTT GAG GCG ACA GC and
5'-TGC AGT TCA (G/A)AA ACA T(A/T)T TCT AA
5'-ATG GAC AAA CCA GC(G/A/T/C) AG(C/T) TT and
5'-GCT TGA GGT CCC AT(G/A) CT(G/A/T/C) CC
5'-GGC ATT GGA ATG GGA TCA CC and
CCC GCA GCT GAC AC
5'-GCC AAC TCA GGT CAT CCA GG and
5'- TGC AAC CGT AGC ATT GTA AC
5'-TTA TTC CTC CTG ATT CTG TC and
5'-GTG ATT GGC CAG AAG CGG AA
5'-TTA TTA GAA GAG CGC ATC CT and
5'-AGA TCT GCC TCC TTA AAT AC.
5'-TGC (C/T)CA AGT TCC TTA TGT GG and
5'-CAC TGG GT(G/A) AAA CC(A/T) GGC AT
PCR amplification is carried
out on chromosomal DNA using an extension time of 30 seconds,
and an annealing temperature of 50oC, 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 using QIAquick (Qiagen) 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 with d-Rhodamine-labeled
terminators (PE Applied Biosystems).
L., McDougal L, Zhou J, Spratt BG, Tenover FC, George
R, Hakenbeck R, Hryniewicz W, Lefevre JC, Tomasz A,Klugman
KP. Nomenclature of major antimicrobial-resistant clones of
Streptococcus pneumoniae defined by the pneumococcal molecular
epidemiology network.J. Clin. Microbiol. 39, 2565-71,
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.
M. and Spratt, B.G. A multilocus sequence typing scheme
for Streptococcus pneumoniae: identification of clones
associated with serious invasive disease. Microbiology 144,
Z.-Y. , Enright, M.C., Wilkinson, P., Griffiths, D.,
and Spratt, B.G. Identification of three major clones of
multiply antibiotic-resistant Streptococcus pneumoniae
in Taiwanese hospitals using multilocus sequence typing.
J. Clin. Microbiol. 36, 3514-3519, 1998.
B.G. Multilocus sequence typing: Molecular typing of
bacterial pathogens in an era of rapid DNA sequencing and
the Internet. Current Opinion in Microbiology, 2,
M.C., Fenoll, A., Griffiths, D., and Spratt B.G.
The three major Spanish clones of penicillin-resistant Streptococcus
pneumoniae are the most common clones recovered from recent
cases of meningitis in Spain. J. Clin. Microbiol.
37, 3210-3216, 1999.
T.J., Daniels, M., Enright, M.C., and Spratt, B.G.
Serotype 14 variants of the Spanish penicillin-resistant
serotype 9V clone of Streptococcus pneumoniae arose by large
recombinational replacements of the cpsA-pbp1a region.
Microbiology 145, 2023 - 2031, 1999
J., Enright, M.C., and Spratt, B.G. Identification of
the major Spanish clones of penicillin-resistant pneumococci
via the Internet using multilocus sequence typing.
J. Clin. Microbiol., 38, 977-986, 2000.
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.