.PDF MRSA 1 PDF

NATIONAL STANDARD METHOD
INVESTIGATION OF
SPECIMENS FOR
SCREENING FOR MRSA
BSOP 29
Issued by Standards Unit, Department for Evaluations, Standards and Training

Centre for Infections
INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

Issue no. 5.1 Issue date: 13.10.08 Issued by: Standards Unit, Department for Evaluations, Standards and Training, Page 1 of 24
BSOP 29i5.1
This NSM should be used in conjunction with the series of NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
STATUS OF NATIONAL STANDARD METHODS
National Standard Methods, which include standard operating procedures (SOPs), algorithms and
guidance notes, promote high quality practices and help to assure the comparability of diagnostic
information obtained in different laboratories. This in turn facilitates standardisation of surveillance
underpinned by research, development and audit and promotes public health and patient confidence
in their healthcare services. The methods are well referenced and represent a good minimum
standard for clinical and public health microbiology. However, in using National Standard Methods,
laboratories should take account of local requirements and may need to undertake additional
investigations. The methods also provide a reference point for method development.
National Standard Methods are developed, reviewed and updated through an open and wide
consultation process where the views of all participants are considered and the resulting documents
reflect the majority agreement of contributors.
Representatives of several professional organisations, including those whose logos appear on the
front cover, are members of the working groups which develop National Standard Methods. Inclusion
of an organisation’s logo on the front cover implies support for the objectives and process of preparing
standard methods. The representatives participate in the development of the National Standard
Methods but their views are not necessarily those of the entire organisation of which they are a
member. The current list of participating organisations can be obtained by emailing
standards@hpa.org.uk.
The performance of standard methods depends on the quality of reagents, equipment, commercial
and in-house test procedures. Laboratories should ensure that these have been validated and shown
to be fit for purpose. Internal and external quality assurance procedures should also be in place.
Whereas every care has been taken in the preparation of this publication, the Health Protection
Agency or any supporting organisation cannot be responsible for the accuracy of any statement or
representation made or the consequences arising from the use of or alteration to any information
contained in it. These procedures are intended solely as a general resource for practising
professionals in the field, operating in the UK, and specialist advice should be obtained where
necessary. If you make any changes to this publication, it must be made clear where changes have
been made to the original document. The Health Protection Agency (HPA) should at all times be
acknowledged.
The HPA is an independent organisation dedicated to protecting people’s health. It brings together
the expertise formerly in a number of official organisations. More information about the HPA can be
found at www.hpa.org.uk.
The HPA aims to be a fully Caldicott compliant organisation. It seeks to take every possible
precaution to prevent unauthorised disclosure of patient details and to ensure that patient-related
records are kept under secure conditions1.
More details can be found on the website at www.evaluations-standards.org.uk. Contributions to the

development of the documents can be made by contacting standards@hpa.org.uk.
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Suggested citation for this document:

Health Protection Agency (2008). Investigation of specimens for screening for MRSA. National
Standard Method BSOP 29 Issue 5.1. http://www.hpa-standardmethods.org.uk/pdf_sops.asp.

BSOP 29i5.1
INDEX
STATUS OF NATIONAL STANDARD METHODS ................................................................................ 2
INDEX...................................................................................................................................................... 3
AMENDMENT PROCEDURE ................................................................................................................. 4
SCOPE OF DOCUMENT ........................................................................................................................ 5
INTRODUCTION ..................................................................................................................................... 5
TECHNICAL INFORMATION/LIMITATIONS ......................................................................................... 8
1 SAFETY CONSIDERATIONS ......................................................................................................... 9

1.1 SPECIMEN COLLECTION ............................................................................................................... 9
1.2 SPECIMEN TRANSPORT AND STORAGE .......................................................................................... 9
1.3 SPECIMEN PROCESSING............................................................................................................... 9
2 SPECIMEN COLLECTION .............................................................................................................. 9
2.1 OPTIMAL TIME FOR SPECIMEN COLLECTION ................................................................................... 9
2.2 CORRECT SPECIMEN TYPE AND METHOD OF COLLECTION ............................................................... 9
2.3 ADEQUATE QUANTITY AND APPROPRIATE NUMBER OF SPECIMENS .................................................. 9
3 SPECIMEN TRANSPORT AND STORAGE ................................................................................... 9
3.1 TIME BETWEEN SPECIMEN COLLECTION AND PROCESSING .............................................................. 9
3.2 SPECIAL CONSIDERATIONS TO MINIMISE DETERIORATION ............................................................... 9
4 SPECIMEN PROCESSING ............................................................................................................. 9
4.1 TEST SELECTION ......................................................................................................................... 9
4.2 APPEARANCE .............................................................................................................................. 9
4.3 MICROSCOPY .............................................................................................................................. 9
4.4 CULTURE AND INVESTIGATION .................................................................................................... 10
4.5 IDENTIFICATION ......................................................................................................................... 10
4.6 ANTIMICROBIAL SUSCEPTIBILITY TESTING .................................................................................... 11
5 REPORTING PROCEDURE.......................................................................................................... 11
5.1 MICROSCOPY ............................................................................................................................ 11
5.2 CULTURE .................................................................................................................................. 11
6 REPORTING TO THE HPA (LOCAL AND REGIONAL SERVICES AND CDSC CENTRE FOR
INFECTIONS) ................................................................................................................................ 11
7 ACKNOWLEDGEMENTS AND CONTACTS................................................................................ 12
APPENDIX 1: SUBMISSION OF STAPHYLOCOCCUS AUREUS ISOLATES TO THE

STAPHYLOCOCCUS REFERENCE LABORATORY OF THE LABORATORY OF HEALTHCARE
ASSOCIATED INFECTION (LHCAI, CFI, COLINDALE) (2005) ......................................................... 13
APPENDIX 2: CHARACTERISTICS OF UK EPIDEMIC MRSA.......................................................... 15
APPENDIX 3 ......................................................................................................................................... 17
REFERENCES ...................................................................................................................................... 18

BSOP 29i5.1
AMENDMENT PROCEDURE
Controlled document BSOP 29

reference
Controlled document title Investigation of specimens for screening for MRSA
Each National Standard Method has an individual record of amendments. The current amendments
are listed on this page. The amendment history is available from standards@hpa.org.uk.
On issue of revised or new pages each controlled document should be updated by the copyholder in
the laboratory.
Amendment Issue no. Insert Page Section(s) involved Amendment

Number/ Discarded Issue
Date no.
6/ 5 5.1 All All ESL replaced with
13.10.08 DEST
17 Appendix Flow chart inserted

BSOP 29i5.1
INVESTIGATION OF SPECIMENS FOR
SCREENING FOR MRSA
Types of specimens: MRSA screening specimens
SCOPE OF DOCUMENT
This National Standard Method describes the processing of screening specimens to detect meticillin
resistant Staphylococcus aureus (MRSA). Certain specimens may require additional routine culture.
NB In this document “meticillin” has been used in place of the established “methicillin” in accordance
with the current International Pharmacopoeia guidelines.
INTRODUCTION
Meticillin was the first penicillinase-resistant penicillin and has been widely used in testing
susceptibility of S. aureus to penicillinase-resistant β-lactam agents. Hence, despite the fact that
meticillin is no longer available and oxacillin and cefoxitin have replaced it for susceptibility testing,
resistant strains are commonly known as MRSA. However, MRSA may also be referred to as oxacillin
resistant S. aureus (ORSA).
MRSA strains are a continuing and increasing problem in many hospitals. Colonised and infected
patients represent the most important reservoir of MRSA strains in hospitals. MRSA are mainly
transmitted by direct person-to-person contact, usually via the hands of health care workers2-6, and
through environmental contamination7. Screening for MRSA provides a means of identifying patients
and staff who may be involved in transmission of the organism.
Emergence of meticillin resistant strains of S. aureus
MRSA were first described in the 1960s8 and their occurrence varied within and between countries.
During the late 1970s and early 1980s, strains of S. aureus resistant to multiple antibiotics including
meticillin and gentamicin were increasingly responsible for outbreaks of hospital infection worldwide9,10
and several clonal types have shown extensive international spread11. In the UK, the Laboratory of
Healthcare Associated Infection has defined and monitored the emergence of epidemic strains of
MRSA (EMRSA). The EMRSA strains are defined as strains affecting at least two patients in at least
two hospitals. Some EMRSA strains have affected some hospitals and current strains (notably
EMRSA -15 and -16) are particularly widespread12. EMRSA are distinguishable by phage typing and
other techniques such as molecular typing13,14. Antimicrobial susceptibility patterns may also provide
a means of tentatively differentiating types of EMRSA in diagnostic microbiology laboratories.
In England and Wales the spread of MRSA was well controlled until the 1990s. Between 1989 and
1991 only 1.6% of S. aureus bacteraemia isolates were meticillin resistant12. However, meticillin
resistance rates increased steadily throughout the 1990s to 13.2% in 199515 and are now in excess of
40% in several regions16. In the 1990s, there were also significant increases in the percentages of
isolates resistant to erythromycin, clindamycin, ciprofloxacin, gentamicin, trimethoprim and
rifampicin15. MRSA are often resistant to many of the therapeutic agents available for the treatment of
severe staphylococcal disease.
Most MRSA infections are healthcare-associated, but an increasing number of infections are
community-acquired, with patients having no established risk factors for acquisition of MRSA. While
infections with community-acquired MRSA (CA-MRSA) are usually mild they can be severe.
Presence of the Panton-Valentine leucocidin (PVL) is common among CA-MRSA and isolates are
often resistant only to β-lactam antibiotics17 but overall 2% of isolates carry PVL genes18.

BSOP 29i5.1
Prevalence
EMRSA-3, EMRSA-15 and EMRSA-16 are the epidemic strains most prevalent in the United Kingdom
at the present time12. Their prevalence is higher in certain parts of the country, with London currently
having the highest MRSA bacteraemia rates19. EMRSA-17 has been described more recently in
several hospitals in the South and Midlands20.
Healthcare-associated infections with MRSA are now posing a major threat to patients admitted to
many hospitals in the UK. The cause of the dramatic rise in MRSA infections in the UK is probably
multi-factorial. The prevalent strains have a particular ability to spread. This may also be related to
changed hospital practice with more inter-ward transfers21 and low staffing levels on some wards. In
addition, there is now a significant reservoir of patients with MRSA in the community and in some
nursing homes throughout the country. MRSA have been reported to replace meticillin susceptible S.
aureus (MSSA)22, but most studies indicate that infections with MRSA tend to occur in addition to the
background rate which might be expected due to MSSA23-28.
Guidelines for the control of MRSA
Guidelines for the control of MRSA in healthcare facilities29 have been produced by a working party of
the Hospital Infection Society, the British Society for Antimicrobial Chemotherapy and the Infection
Control Nurses Association. These guidelines recommend a risk assessment approach and advise
Infection Control Committees to adapt them locally when designing infection control policies. Other
recent recommendations have been published by the Scottish Infection Standards and Strategy
Group30, and the Department of Health31.
Virulence
Numerous studies32 have shown that the majority of patients from whom MRSA strains are isolated
are colonised rather than infected with the organism, although the proportion of colonised patients
who become infected varies between 5 and 60% depending on the population studied21,33,34. Factors
predisposing to superficial colonisation include procedures involving “hands-on” care such as occur on
acute surgical, renal dialysis and critical care units. The risk of colonisation proceeding to infection is
increased in the presence of any breach in the skin, such as surgical wounds and devices penetrating
the skin, eg prostheses and catheters, which provide a portal of entry for bacteria35-38. Several case
control studies have demonstrated that MRSA are similar in virulence to MSSA strains39,40. More
severe infection with CA-MRSA is mainly related to production of PVL41-43. When treated
appropriately with vancomycin, the mortality rate from healthcare-associated MRSA infections is
comparable with that from infections due to meticillin susceptible strains44.
Multiple drug resistance
The most prevalent EMRSA strains in the UK remain susceptible to several antibiotics including the
glycopeptides vancomycin and teicoplanin (see Appendix 2). However, MRSA strains showing
reduced susceptibility to vancomycin have been described in several countries including Japan45, the
USA46, France47, and the UK48, and clinical isolates with high-level resistance have been described in
the USA49,50. This eventuality should be considered when patients from other countries are admitted
to hospital, especially to intensive care, burns and other specialist units51 and also in any patient with
MRSA in whom there is an apparent treatment failure with a glycopeptide antibiotic48,52. Some strains
now demonstrate resistance to as many as 20 antimicrobial compounds, including antiseptics and
disinfectants53 and this trend in acquisition of extra resistances appears to be increasing19,54,55.
Despite this there are several agents that are appropriate for the treatment of MRSA infections and
new agents are being developed and introduced56.
Mechanisms of resistance
Intrinsic resistance to β-lactams in clinical strains of S. aureus is often heterogeneous. High-level
resistance is expressed by a minority of cells on ordinary media at 37°C but more uniformly in
hypertonic media or at 30°C57,58. Although most MRSA produce a β-lactamase, this is not responsible
for their resistance to meticillin. All MRSA contain the mecA gene and this is the essential
determinant of meticillin resistance. MecA is a 2,130-bp segment of DNA coding for a penicillin-
binding protein (PBP2’ or PBP2a) characterised by a low affinity for most β-lactams, and which is
thought to take over the functions of all other PBPs when they are saturated by meticillin or other β-
lactam antibiotics59. MSSA do not produce this protein and their DNA will not hybridise with a probe
BSOP 29i5.1
specific for the mecA gene. The genetic determinant of PBP 2a is transcribed in all MRSA cells and
all phenotypic classes of MRSA, but additional factors affect the expression of meticillin-resistance60.
The mecA gene is part of a mobile genetic element, the staphylococcal chromosomal cassette mec
(SCCmec), which is incorporated in the chromosome61. Five distinct types of SCCmec, designated I,
II III, IV and V have been described to date62,63. Most hospital-acquired MRSA are types I, II or III
whereas most CA-MRSA are types IV or V17, although EMRSA-15 is type IV.
The presence of the mecA gene and either an oxacillin MIC of >2mg/L64,65 a meticillin MIC of
>4mg/L65,66,or a cefoxitin MIC of >4mg/L65, are accepted criteria for meticillin resistance.
Borderline resistance
Some strains of Staphylococcus aureus may be encountered which are not mecA positive but which
exhibit a borderline resistance. This may be due to hyper-production of β-lactamase (particularly
obvious when testing oxacillin susceptibility) or alteration of PBPs67. There is some evidence from
animal models that hyper-production of β-lactamase is not clinically significant68, but further data on
virulence and effectiveness of therapy of patients infected with borderline-resistant strains are needed
to determine whether control measures are warranted69,70.
Techniques for detecting resistance
Molecular techniques for the detection of mecA are viewed as the “gold standard” for determining
resistance. Polymerase chain reaction (PCR) for amplification of the staphylococcal mecA gene can
be performed within a few hours. PCR is not currently available in many diagnostic laboratories and
the additional costs of using PCR must be justified. However, PCR is valuable for confirmation of
equivocal resistance and commercial kits are available71. Conventional oxacillin susceptibility tests
are markedly affected by test conditions and the use of cefoxitin in disc diffusion tests has been shown
to be less affected by test conditions and to be more reliable than tests with oxacillin72,73. Both disc
diffusion and breakpoint methods are widely used. However, other methods giving more rapid results
may be considered, such as the latex agglutination-based method that detects the PBP2a protein74
which is commercially available from several suppliers.
Screening for MRSA
In order to achieve the most effective use of finite hospital resources and to minimise morbidity due to
these organisms it is usual to have a policy of planned screening to guide control measures to protect
patients from MRSA colonisation and infection. Precisely what patient and staff screening is
performed will depend on the endemicity of the problem and the case-mix of the unit. If MRSA is
highly endemic, with constant challenges to the provider units, then a risk assessment process is
recommended. One approach is to concentrate on patients at greatest risk. Screening may also be
appropriate in areas with low patient risk, particularly so where there is extensive interaction and
transfer of patients with MRSA among wards or to acute care wards. Recommendations have been
published by the Working Party of the Hospital Infection Society, the British Society for Antimicrobial
Chemotherapy and the Infection Control Nurses Association29, the Scottish Infection Standards and
Strategy Group30, and the Department of Health31. Local Infection Control Committees may adapt
these guidelines to their local situation.
Methods of screening for MRSA
Conventional methods used for screening should detect strains of MRSA by inhibiting contaminants
and selecting S. aureus strains which are meticillin resistant. Direct plating on selective medium has
the advantage that results may be available within 24h, but most studies indicate that direct plating is
less sensitive than broth enrichment followed by plating on solid media71. Whether this is the case
with more recently developed chromogenic media remains to be determined. Sodium chloride,
antibiotics and other selective agents may be added to the media to reduce contamination, and
oxacillin or cefoxitin added to select meticillin resistant strains.
Enrichment broth (nutrient broth or cooked meat medium) containing 7% sodium chloride (NaCl) was
recommended by a HIS/BSAC/ICNA working party66, although several other different media have
been used71,75-78 and no specific recommendation was made in the recent HIS/BSAC/ICNA
guidelines71. Enrichment broth containing 7% NaCl may inhibit the growth of some isolates of MRSA
if present in small numbers76. Antibiotics suggested as an alternative to NaCl include aztreonam79,
BSOP 29i5.1
nalidixic acid plus colistin80, or cefoxitin; these have been evaluated in solid media but not in
enrichment broth.
For direct plating and plating from enrichment broth the recent HIS/BSAC/ICNA working party71
compared published performance data for several media and suggested using Baird Parker agar with
ciprofloxacin (BPC) or mannitol salt agar (MSA) 81-83 with 7% NaCl. MSA and variations of MSA84-86
have been widely used, but have the disadvantage that direct agglutination tests for identification of
S. aureus on MSA are unreliable75,83 or growth of MRSA is slow87. BPC has been used where the
majority of MRSA are known to be ciprofloxacin resistant75,88 and, although ciprofloxacin susceptible
MRSA will be missed when screening with this medium, the isolation rate with BPC has been reported
to be higher than with MSA75,87,88. The HIS/BSAC/ICNA working party71 suggested that recently
developed chromogenic MRSA screening media appeared promising and more recent reports
consistently show chromogenic media to perform well89-93.
Ideally, a screening method should allow the growth of all MRSA, inhibit or differentiate other
organisms, and allow direct identification tests to be performed on colonies. Unfortunately some of
these requirements conflict and compromise is necessary. There are few comparative studies of the
effectiveness of different approaches in the clinical situation.
A significant limitation of all culture-based screening methods is the dependency on growth of

colonies. The value of screening would be greater if results were available more rapidly, and there is
a clear need to develop rapid screening strategies. Molecular methods for the detection of S. aureus
and the mecA gene are available71 and have been applied to screening for MRSA, usually in
conjunction with a broth enrichment step94,95. The possibility of false positives due to mixtures of
meticillin susceptible S. aureus and meticillin resistant coagulase-negative staphylococci carrying the
mecA gene can be limited by inclusion of oxacillin in the enrichment broth, but this may reduce
isolation of MRSA and the time for enrichment culture reduces the benefit of rapid PCR detection.
Direct identification of MRSA on screening swabs by a molecular method that links identification of
MRSA with the presence of mecA has been described96 and is commercially available. Evaluations
indicate good performance and results in 2-3 h97-101.
Recommended methods
Routine screening by direct plating:
A chromogenic selective MRSA agar.
Screening by enrichment:
In particular circumstances (eg checking patients for clearance of MRSA) screening by an enrichment
method may be used. Several swabs from the same patient can be combined in the same 7% NaCl
nutrient broth. This is a cost-effective method where the aim is to determine the presence, rather than
the site, of MRSA carriage.
Both direct plating and enrichment methods may be used. Enrichment delays reporting of results by
24 h but negative results with a more sensitive technique (enrichment) may be required before MRSA
control measures are discontinued for that patient102. The advantage of enrichment over direct plating
has yet to be confirmed with chromogenic media.
Detection of a presumptive MRSA strain should be followed by its full identification as S. aureus,
confirmation of meticillin resistance and testing susceptibility to other antimicrobial agents. This last
may be helpful in identifying EMRSA strains or local types (see Appendix 2).
Screening by molecular methods:
Use of a commercial method applied directly to screening swabs may be considered if very rapid
results are required.
TECHNICAL INFORMATION/LIMITATIONS
Chromogenic media are affected by light and plates should be stored in the dark and not left in the
light before or after inoculation. Incubation times for chromogenic media should be as recommended
by the manufacturers.
BSOP 29i5.1
1 SAFETY CONSIDERATIONS103-108
1.1 SPECIMEN COLLECTION
N/A
1.2 SPECIMEN TRANSPORT AND STORAGE
Sealed plastic bag.
1.3 SPECIMEN PROCESSING
Containment Level 2.
The above guidance should be supplemented with local COSHH and risk assessments.
2 SPECIMEN COLLECTION
2.1 OPTIMAL TIME FOR SPECIMEN COLLECTION
N/A
2.2 CORRECT SPECIMEN TYPE AND METHOD OF COLLECTION
Screening swabs, catheter urine, etc as appropriate.
Swabs may be received dry109,110, in Amies transport medium with charcoal111 or in enrichment
broth.
Specimens for molecular methods should follow the recommendations for the method.
2.3 ADEQUATE QUANTITY AND APPROPRIATE NUMBER OF SPECIMENS
N/A
3 SPECIMEN TRANSPORT AND STORAGE

3.1 TIME BETWEEN SPECIMEN COLLECTION AND PROCESSING
Specimens should be transported and processed as soon as possible.
3.2 SPECIAL CONSIDERATIONS TO MINIMISE DETERIORATION
If processing of swabs is delayed, refrigeration is preferable to storage at ambient temperature.
Delays of over 48 h are undesirable.
Swabs may be placed directly in enrichment broth on the ward. Swabs in enrichment broths
should not be refrigerated. If ward staff are involved they should be adequately trained.
4 SPECIMEN PROCESSING
4.1 TEST SELECTION
N/A
4.2 APPEARANCE
N/A
4.3 MICROSCOPY
N/A

BSOP 29i5.1
4.4 CULTURE AND INVESTIGATION
4.4.1 PRE-TREATMENT
N/A
4.4.2 SPECIMEN PROCESSING
Direct culture
Inoculate each agar plate with swab or other sample (BSOP 54 – Inoculation of culture media).
Enrichment culture
Remove the cap aseptically from the container and place the swab(s) in the broth, break off (or
cut) the swab-stick(s) and replace the cap.
4.4.3 CULTURE MEDIA, CONDITIONS AND ORGANISMS
For all specimens*:

Clinical details/ Standard media Incubation Cultures Target
conditions read organism
Temp ° C Atmos Time
Direct culture Chromogenic selective

MRSA medium
37 Aerobic 18 h - 48 h** daily MRSA
AND/OR
Enrichment culture Nutrient broth 30 Aerobic 18 h - 24 h N/A
containing 7% NaCl
*** then subculture to
Chromogenic selective
37 Aerobic 18 h - 48 h** daily MRSA
MRSA medium
* Consider a molecular method if rapid results are required
**For chromogenic media refer to manufacturer’s instructions for recommended incubation times
***The bottle should contain a volume of broth sufficient to cover the swabs. The NaCl concentration should be reduced if
locally prevalent strains are known to be inhibited by 7% NaCl
4.5 IDENTIFICATION
4.5.1 MINIMUM LEVEL
S. aureus species level, meticillin resistant
4.5.2 REFERRAL TO REFERENCE LABORATORIES
See Appendix 1
Staphylococcus Reference Laboratory

Laboratory for Healthcare Associated Infection
Centre for Infections, Health Protection Agency
61 Colindale Avenue
London
NW9 5EQ
Contact Health Protection Agency main switchboard:

Tel + (44) 0820 200 4400

Issue no. 5.1 Issue date: 13.10.08 Issued by: Standards Unit, Department for Evaluations, Standards and Training, Page 10 of
24
BSOP 29i5.1
4.6 ANTIMICROBIAL SUSCEPTIBILITY TESTING
Refer to BSOP 45 - Susceptibility testing.
5 REPORTING PROCEDURE
5.1 MICROSCOPY
N/A
5.2 CULTURE
Negatives
“MRSA not isolated”
Positives
“MRSA isolated”
5.2.1 CULTURE REPORTING TIME

Clinically urgent culture results to be telephoned or sent electronically when available.
Written report, 72 h stating, if appropriate, that a further report will be issued.
5.3 SUSCEPTIBILITY TESTING

Report susceptibilities as clinically indicated.
MRSA should not be reported as susceptible to any currently available β-lactams8.
6 REPORTING TO THE HPA112 (LOCAL AND

REGIONAL SERVICES AND CDSC CENTRE FOR
INFECTIONS)
Refer to the following:
Health Protection Agency publications:
"Laboratory reporting to the HPA. A guide for diagnostic laboratories"
“Hospital infection control : Guidance on the control of infection in hospitals"
Local guidelines including Infection Control Policy and Memorandum of Understanding

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BSOP 29i5.1
7 ACKNOWLEDGEMENTS AND CONTACTS
This National Standard Method has been developed, reviewed and revised by the National
Standard Methods Working Group for Bacteriology
(http://www.hpa-standardmethods.org.uk/wg_bacteriology.asp). The contributions of many
individuals in clinical bacteriology laboratories and specialist organisations who have provided
information and comment during the development of this document, and final editing by the
Medical Editor are acknowledged.
The National Standard Methods are issued by Standards Unit, Department for Evaluations,
Standards and Training, Centre for Infections, Health Protection Agency, London.
For further information please contact us at:
Standards Unit
Department for Evaluations, Standards and Training
Centre for Infections
Health Protection Agency
Colindale
London
NW9 5EQ
E-mail: standards@hpa.org.uk

24
BSOP 29i5.1
APPENDIX 1: SUBMISSION OF STAPHYLOCOCCUS
AUREUS ISOLATES TO THE STAPHYLOCOCCUS
REFERENCE LABORATORY OF THE LABORATORY OF
HEALTHCARE ASSOCIATED INFECTION (LHCAI, CFI,
COLINDALE) (2005)
To enable LHCAI to maintain current low turn round times and improve the quality of the service,
please:
1) Request typing only if you intend to act upon the results
2) Ensure that the Consultant Microbiologist and/or Infection Control Team have confirmed there are
good reasons for submission
3) For all requests, state hypothesis to be tested, ie how typing will make a difference
In outbreaks (a temporal and spatial cluster above the normal baseline):
Send the minimum number of isolates needed to inform local practice (this should rarely be more than
half), and store temporally related isolates.
Give priority to isolates that cause invasive or serious infection during the course of an outbreak, but
avoid sending multiple isolates from single patients or environmental isolates without discussion with
LHCAI.
Wherever possible, use surrogate markers such as urease and antimicrobial resistances and include
representative isolates with significantly different phenotypes eg in antibiotic susceptibilities,
pigmentation and/or haemolysis.
In endemic situations:
If surrogate markers are being used to identify any locally endemic strains (particularly, for example to
identify your EMRSAs as EMRSA-15 and EMRSA-16) LHCAI are willing to check a few representative
isolates for you from time to time, eg 5 every 6 months.
Suspected toxin-mediated disease:
LHCAI would like to receive an isolate from cases of suspected toxin-mediated disease eg
staphylococcal toxic shock syndrome, impetigo and scalded skin syndrome for toxin gene profiling. In
addition, LHCAI would like to receive isolates from suspected PVL-related disease eg serious skin and
soft tissue infection and necrotising pneumonia for analysis. Further information is available at:
http://www.hpa.org.uk/cdr/archives/2003/cdr1503.pdf and
http://www.hpa.org.uk/cdr/archives/2005/cdr1105.pdf.
A brief questionnaire will be included with the report, which LHCAI would appreciate you completing
and returning to us.
Community-associated MRSA:
There are many different lineages of community MRSA, all of which are distinct from
healthcare-associated MRSA. Typically, CA-MRSA are heterogeneously resistant to oxacillin and
unusually susceptible to antimicrobials other than β-lactams, particularly ciprofloxacin. LHCAI would
like to receive such isolates for further characterisation. Further information is available at:
http://www.hpa.org.uk/cdr/archives/2005/cdr1105.pdf.
24
BSOP 29i5.1
Anomalous isolates:
State the anomaly/resistance to be investigated eg slide coagulase negative MRSA, and please check
for mixed culture, coagulase, catalase and Gram’s stain before sending.
Antibiotic resistance:
Request antibiotic susceptibility tests only when necessary to assist your local studies eg anomalous
or doubtful test results, unusual or clinically significant results, necessary quantitation (eg MIC of first
encountered mupirocin-resistant isolates), unexpected resistance (eg to vancomycin).
Food-related isolates
All isolates relating to incidents of food poisoning or food contamination should be sent to the Food
Safety Microbiology Laboratory (020 8200 4400 extn 7116).
Non-food-related isolates
Please send all non-food-related S. aureus to the Staphylococcus Reference Laboratory

accompanied by the request form. Further copies and information can be obtained by phoning 020
8327 7228. Queries about antibiotic resistance tests should be made to 020 8327 7237 (MICs) or 020
8327 7255 (mecA/mupA).
If in any doubt contact LHCAI and ask (020 8327 7227)

24
BSOP 29i5.1
APPENDIX 2: CHARACTERISTICS OF UK EPIDEMIC
MRSA
Data from the Laboratory of Healthcare-Associated Infection, CFI
Epidemic MRSA Phage patternb Antibiotic resistance Toxin

patternc genesd
EMRSA-1a (84)/85/88A/(90)/932 P T E (K) (G) S A
EMRSA-2 80/85/90/932 PE A
EMRSA-3a 75/83A/(83C)/932 P E (K) (G) (N) (Cip) (S) -
EMRSA-4 85/(90)/932 PTES A
EMRSA-5 29/6/42E/47/54/75/77/84/85/81 P T K G Rif A,B,C
EMRSA-6 90/932wk P T E K N Ba S A
EMRSA-7 85/932 PTES A,C
EMRSA-8 83A/83C/932 PTS -
EMRSA-9 77/84/932 PTEKGS -
EMRSA-10 29/75/77/83A/85 PTEKG A,B
EMRSA-11 83A/84/85 P T E K G N Ba S A
EMRSA-12 75/83A/83C/932 PTEKNFS -
EMRSA-13 29/6/42E/47/54 P T K G N Ba F S -
EMRSA-14 29/6/47/54 PTKNFS -
EMRSA-15a 75wk P (E) Cip C
EMRSA-16a 29/52/75/77/83A/83C P E (K) (G) (N) Cip A

TSST-1
a
EMRSA-17 29/77/932 P T (E) Rif F K G (N) S A
Tp Cip (Mup)
KEY:
a
Currently circulating UK Epidemic MRSA strains
b
Phage patterns at 100xRTD (routine test dilution) wk = weak reaction ( ) = variable
reaction
c
Antibiotic resistance pattern:
Ba, bacitracin; Cip, ciprofloxacin; E, erythromycin; F, fusidic acid; G, gentamicin; K,
kanamycin (or tobramycin); Mup, mupirocin; N, neomycin; P, penicillin; Rif, rifampicin; S,
streptomycin; T, tetracycline; Tp, teicoplanin borderline MIC 4 - 8 mg/L; ( ), Variable
susceptibility among isolates.
Neomycin resistance is difficult to detect by disc diffusion testing with some isolates. With
gentamicin susceptible isolates tobramycin or kanamycin are more reliable indicators of the
aadD gene responsible for neomycin resistance.

24
BSOP 29i5.1
d
Toxin genes: A,B,C Enterotoxins TSST1; Toxic Shock Syndrome Toxin-1

24
BSOP 29i5.1
APPENDIX 3
Prepare all specimens*
and /or
Direct culture
Enrichment culture
Chromogenic selective Nutrient broth

MRSA medium containing 7% NaCl***
Incubate at 37 C Incubate at 30 C
Aerobic Aerobic
18-48 h** 18-24 h
Read daily
Subculture to
MRSA Chromogenic selective
MRSA medium
Incubate at 37 C
Aerobic
18-48 h**
Read daily
MRSA
* Consider a molecular method if rapid results are required

** For chromogenic media refer to manufacturer’s instructions for recommended incubation times
*** The bottle should contain a volume of broth sufficient to cover the swabs. The NaCl concentration should be reduced if locally
prevalent strains are known to be inhibited by 7% NaCl

24
BSOP 29i5.1
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NATIONAL STANDARD METHOD

Issued by Standards Unit, Department for Evaluations, Standards and Training

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

More details can be found on the website at www.evaluations-standards.org.uk. Contributions to the

Suggested citation for this document:

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

STATUS OF NATIONAL STANDARD METHODS ................................................................................ 2

AMENDMENT PROCEDURE ................................................................................................................. 4

SCOPE OF DOCUMENT ........................................................................................................................ 5

TECHNICAL INFORMATION/LIMITATIONS ......................................................................................... 8

1 SAFETY CONSIDERATIONS ......................................................................................................... 9

7 ACKNOWLEDGEMENTS AND CONTACTS................................................................................ 12

APPENDIX 1: SUBMISSION OF STAPHYLOCOCCUS AUREUS ISOLATES TO THE

APPENDIX 2: CHARACTERISTICS OF UK EPIDEMIC MRSA.......................................................... 15

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

Controlled document BSOP 29

Amendment Issue no. Insert Page Section(s) involved Amendment

17 Appendix Flow chart inserted

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

A significant limitation of all culture-based screening methods is the dependency on growth of

3 SPECIMEN TRANSPORT AND STORAGE

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

4.4.3 CULTURE MEDIA, CONDITIONS AND ORGANISMS

For all specimens*:

Direct culture Chromogenic selective

* Consider a molecular method if rapid results are required

Staphylococcus Reference Laboratory

Contact Health Protection Agency main switchboard:

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

5.2.1 CULTURE REPORTING TIME

Written report, 72 h stating, if appropriate, that a further report will be issued.

5.3 SUSCEPTIBILITY TESTING

MRSA should not be reported as susceptible to any currently available β-lactams8.

6 REPORTING TO THE HPA112 (LOCAL AND

Health Protection Agency publications:

"Laboratory reporting to the HPA. A guide for diagnostic laboratories"

“Hospital infection control : Guidance on the control of infection in hospitals"

Local guidelines including Infection Control Policy and Memorandum of Understanding

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

For further information please contact us at:

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

1) Request typing only if you intend to act upon the results

In outbreaks (a temporal and spatial cluster above the normal baseline):

Suspected toxin-mediated disease:

Please send all non-food-related S. aureus to the Staphylococcus Reference Laboratory

If in any doubt contact LHCAI and ask (020 8327 7227)

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

Epidemic MRSA Phage patternb Antibiotic resistance Toxin

EMRSA-1a (84)/85/88A/(90)/932 P T E (K) (G) S A

EMRSA-3a 75/83A/(83C)/932 P E (K) (G) (N) (Cip) (S) -

EMRSA-4 85/(90)/932 PTES A

EMRSA-5 29/6/42E/47/54/75/77/84/85/81 P T K G Rif A,B,C

EMRSA-7 85/932 PTES A,C

EMRSA-8 83A/83C/932 PTS -

EMRSA-9 77/84/932 PTEKGS -

EMRSA-10 29/75/77/83A/85 PTEKG A,B

EMRSA-12 75/83A/83C/932 PTEKNFS -

EMRSA-14 29/6/47/54 PTKNFS -

EMRSA-15a 75wk P (E) Cip C

EMRSA-16a 29/52/75/77/83A/83C P E (K) (G) (N) Cip A

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA

INVESTIGATION OF SPECIMENS FOR SCREENING FOR MRSA