Staphylococcus aureus
Multilocus sequence typing of Staphylococcus aureus
MLST has been developed for Staphylococcus aureus by Mark Enright (now situated at the University of Bath) in the laboratory of Professor Brian Spratt, Imperial College , London, in collaboration with the laboratories of Drs Nick Day and Sharon Peacock at the John Radcliffe Hospital, Oxford.
The S. aureus MLST database contains the alleles profiles of 155 isolates from invasive disease from the Oxfordshire area –
M.C. Enright, N.P.J. Day, C.E. Davies, S.J. Peacock and B.G. Spratt, J. Clin. Microbiol. vol. 38,1008-1015 (2000).
These isolates represent both methicillin-sensitive (MSSA) and resistant (MRSA) lineages and include the allelic profiles of examples of the major epidemic MRSA clones circulating in the United Kingdom (eMRSA 3, 15 and 16).
Work is ongoing to characterise the major global MRSA clones including examples of MRSA with intermediate sensitivity to vancomycin (vancomycin intermediately-susceptible S. aureus [VISA]), and allelic profiles of these isolates will be available later this year.
Obtaining an allelic profile and comparing your strains with those in our database
The allelic profile of a S. aureus 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 S. aureus can be obtained as follows –
arcc | aroe | glpf | gmk_ | pta_ | tpi_ | yqil
and can be used to ensure that your sequences have been trimmed correctly.
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 Staphylococcus aureus 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 S. aureus 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 3/4, 4/7, 5/7 or 6/7 matches to the submitted allelic profile. The similarity of your query strain to other strains can be displayed as a dendrogram.
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) or for advanced querying.
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The seven loci and the primers and conditions used for PCR
The S. aureus MLST scheme uses internal fragments of the following seven house-keeping genes:-
arc (Carbamate kinase)
aro (Shikimate dehydrogenase)
glp (Glycerol kinase)
gmk (Guanylate kinase)
pta (Phosphate acetyltransferase)
tpi (Triosephosphate isomerase)
yqi (Acetyle coenzyme A acetyltransferase)
Staphylococcus aureus DNA extraction protocol
1. Resuspend 1/8 of a plate of overnight confluent growth from a blood agar plate in 400 ul lysis solution (see below)
2. Incubate at 37ºC 30 min
3. Heat to 95ºC for 10 min
4. Place on ice and add 1ml phenol/chloroform isoamyl alcohol (25:24:1) and mix thoroughly by inversion
5. Centrifuge 10 000 g 20 min.
6. Recover aqueous layer to fresh tube and precipitate DNA with the addition of 1ml absolute. ethanol
7. Place on ice for 15 min then pellet DNA by centrifugation at 10 000g for 20 min.
8. Resuspend DNA in 50 ul water.
Lysis solution
Lysozyme 5000 units ml-1 0.5 ml
Lysostaphin 500 units ml-1 0.5 ml
EDTA 0.5 M 0.2 ml
Tris 1M 0.1 ml
De-ionised water 8.7 ml
PCR conditions
PCR amplification is carried out on chromosomal DNA using an extension time of 30 seconds, and an annealing temperature of 55C, 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 by adding:
PCR/Sequencing primers
arc up – 5′ TTG ATT CAC CAG CGC GTA TTG TC -3′
arc dn – 5′ AGG TAT CTG CTT CAA TCA GCG -3′
aro up – 5′ ATC GGA AAT CCT ATT TCA CAT TC -3′
aro dn – 5′ GGT GTT GTA TTA ATA ACG ATA TC -3′
glp up – 5′ CTA GGA ACT GCA ATC TTA ATC C -3′
glp dn – 5′ TGG TAA AAT CGC ATG TCC AAT TC -3′
gmk up – 5′ ATC GTT TTA TCG GGA CCA TC -3′
gmk dn – 5′ TCA TTA ACT ACA ACG TAA TCG TA -3′
pta up – 5′ GTT AAA ATC GTA TTA CCT GAA GG -3′
pta dn – 5′ GAC CCT TTT GTT GAA AAG CTT AA -3′
tpi up – 5′ TCG TTC ATT CTG AAC GTC GTG AA -3′
tpi dn – 5′ TTT GCA CCT TCT AAC AAT TGT AC -3′
yqi up- 5′ CAG CAT ACA GGA CAC CTA TTG GC -3′
yqi dn- 5′ CGT TGA GGA ATC GAT ACT GGA AC -3′