Services Available

The Unit provides a range of accredited specialist microbiology tests on Staphylococcus aureus (including MRSA) and other Staphylococcus spp.

The ability to link suspected related cases of infection and exclude unrelated cases is important for the epidemiological study of Staphylococcus spp in healthcare and community settings. Various methods can be used to track possible transmission events. Currently, the front-line tool used for typing S. aureus is spa typing [external link]. Fine strain discrimination (second-line typing) of isolates which give indistinguishable or closely related spa types is afforded by Pulsed-Field Gel Electrophoresis (PFGE). 

Updates on the number of PVL cases (including PVL-MRSA, so-called community-associated MRSA) occurring in England are published in the Health Protection Report (HPR); Vol 5 No 7; 18 February 2011 PVL–Staphylococcus aureus infections: an update [external link]
Download PDF version of report [external PDF]

Urgent requests

Specialist reference services are available for urgent public health investigations, outbreaks and incident management, either healthcare- or community-based.  Specimen submissions regarded by the sending laboratory as especially important or urgent should be notified to SRU by telephone (020 8327 7227) to ensure the appropriate level of priority is accorded to these specimens immediately upon receipt.

Depending on the nature of the enquiry and the complexity of the investigation, specimens can be "fast-tracked" to provide spa typing or PVL test results within 24 hours of receipt of a pure culture. Telephone SRU in advance of submission to discuss (020 8327 7227).

Staphylococcus aureus

(Please see referral criteria)

• spa typing - culture on agar slope
• Serodiagnosis - serum (not less than 200 µl)
• Toxin gene detection - culture on agar slope
• Molecular comparison - culture on agar slope

Phage typing of S. aureus

From Monday October 4th 2010, a 'phage typing service for S. aureus will no longer be available from HPA Centre for Infections.

The International Set of S. aureus 'phages, together with their propagating strains, are available from the  National Collection of Type Cultures (NCTC).

Staphylococci (coagulase negative)

• Speciation and molecular comparison - culture on agar slope

Streptococcus  pyogenes

• Serodiagnosis  - serum (not less than 200 µl)

spa typing of S. aureus

Isolates of S. aureus are characterised by spa typing which involves DNA sequencing of short sequence repeats in the polymorphic X region of the protein A gene (spa) of S. aureus (for further information, see http://www.spaserver.ridom.de). The region consists of a variable number of these repeat units which are usually 24bp in length. Each new base composition of a repeat is assigned an alpha-numerical code (r01, r02 etc) and the repeat succession determines the spa type (e.g. t001, t002 etc) as illustrated:

DNA sequencing of polymorphic X region of protein A gene

Spa typing is used throughout Europe and world-wide for reliable, accurate and discriminatory typing of S. aureus (both MSSA & MRSA). Over 7,000 spa types have been described to date; spa non-typable strains are rare (less than 0.1%).

There is a standard international nomenclature which is web-enabled (http://www.spaserver.ridom.de) and the data are directly comparable between centres and countries. For some S. aureus lineages, the technique has a discriminatory index approaching that of PFGE.

In commonly occurring lineages, DNA sequencing of the spa gene allows presumptive identification of Healthcare- and Community-Associated MRSA strains identified in the UK and MLST clonal complex designation (see Table).

If you have any queries, please contact Dr Angela Kearns, Staphylococcus Reference Unit, LHCAI, CfI (angela.kearns@hpa.org.uk; tel: 0208 3277227).

Molecular typing (PFGE) for epidemiological investigations, monitoring and surveillance of S. aureus (see services list):

Currently, inter-strain comparison of epidemiologically related S. aureus isolates which give indistinguishable or closely related spa types is by pulsed-field gel electrophoresis (PFGE). For MSSA and MRSA, this method has a high discriminatory index and remains the gold standard for inter-strain comparative purposes to assist with epidemiological investigations.

PFGE-based analyses can also be used to help identify the 17 epidemic types of healthcare-associated MRSA which have been described in the UK (EMRSA-1 to -17).

Currently, EMRSA-15 and -16 predominate in the UK healthcare settings. For epidemiological purposes, these can be sub-typed by PFGE and are assigned a "type" based on the nomenclature defined in the original papers: 

• EMRSA-15 variant B1 to B27 [external link] 
• EMRSA-16 variant A1 to A33 [external link]

Where newer PFGE sub-types are identified, these are assigned an arbitrary nomenclature of EMRSA-15 new variant A, B, C etc or EMRSA-16 new variant A, B, C etc.

N.B. These arbitrary codings are applicable to a particular investigation, but not between incidents which are temporally and geographically distinct. Where comparison of isolates between different investigations is required, please contact us to discuss this.

PFGE methodology

In this method, un-sheared bacterial DNA prepared in agarose blocks is cut using rare cutting restriction endonucleases to produce, optimally, 20 - 40 fragments. These fragments are then separated through 1.2% agarose in an electric field of 6V/cm alternating in two directions at an incident angle of 120° with the temperature maintained at 12°C and with pulse times increasing from short to long as appropriate for the organisms being compared (see table). Following electrophoresis, the gel is stained with ethidium bromide and visualised by ultra violet transillumination. Banding patterns may be interpreted by eye or with the aid of BioNumerics software (Applied Maths, Kortrijk, Belgium).

Organism	Restriction endonuclease	PFGE parameters
Staphylococcus aureus	SmaI	1 - 80 s pulses for 30 h
Coag. neg. staphylococci	SmaI	1 - 45 s pulses for 30 h

PCR-based detection of staphylococcal toxin genes in isolates from clinical and food-related disease:

The following 14 virulence genes are screened for using multiplex PCR [external link]:

• Toxic shock syndrome toxin (tst)
• Enterotoxins (sea, seb, sec, sed, see, seg, seh, sei and sej)

For the above, our studies have shown a 99% correlation with gene detection and expression.

• Panton-Valentine leukocidin (lukS-PV/lukF-PV)
  - particularly associated with primary pyogenic skin infections [external link] (abscesses, boils etc) especially where these are recurrent; occasionally PVL-positive strains of S. aureus are associated with more invasive disease such as bone and joint infections [external link], purpura fulminans [external link]or necrotising pneumonia [external link]. Updates on the number of PVL cases (including PVL-MRSA, so-called community-associated MRSA) occurring in England are published in the Health Protection Report (HPR); Vol 5 No 7; 18 February 2011 PVL–Staphylococcus aureus infections: an update [external link]
  Download PDF version of report [external PDF]
• Exfoliatins (eta, etb and etd)
  - particularly associated with scalded skin syndrome and impetigo

Phenotypic and genotypic identification of Coagulase-negative staphylococci.

This service uses the API ID32STAPH kit to provide preliminary data and isolates are subsequently tested using PCRs specific for S. epidermidis, S. haemolyticus and S. hominis as appropriate. For epidemiological purposes, molecular (PFGE-based) fingerprinting of multiple CNS isolates is available on request.