NATIONAL STANDARD METHOD

IDENTIFICATION OF ESCHERICHIA COLI O157

BSOP ID 22 Issued by Standards Unit, Evaluations and Standards Laboratory Centre for Infections

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page 1 of 13 BSOP ID 22i3 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 organisations 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 peoples 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. The reader is informed that all taxonomy in this document was correct at time of issue.
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Suggested citation for this document: Health Protection Agency (2010). Identification of Escherichia coli O157. National Standard Method BSOP ID 22 Issue 3. http://www.hpa-standardmethods.org.uk/pdf_sops.asp.

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page 2 of 13 BSOP ID 22i3 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

INDEX
STATUS OF NATIONAL STANDARD METHODS ................................................................................ 2 INDEX...................................................................................................................................................... 3 AMENDMENT PROCEDURE ................................................................................................................. 4 IDENTIFICATION OF ESCHERICHIA COLI O157 ................................................................................ 5 SCOPE OF DOCUMENT ........................................................................................................................ 5 INTRODUCTION ..................................................................................................................................... 5 TECHNICAL INFORMATION ................................................................................................................. 6 1 2 3 SAFETY CONSIDERATIONS ......................................................................................................... 7 TARGET ORGANISMS ................................................................................................................... 7 IDENTIFICATION............................................................................................................................. 7 3.1 3.2 3.3 3.4 3.5 4 5 MICROSCOPIC APPEARANCE ........................................................................................................ 7 ISOLATION MEDIA ......................................................................................................................... 7 COLONIAL APPEARANCE............................................................................................................... 7 TEST PROCEDURES ..................................................................................................................... 8 STORAGE AND REFERRAL ............................................................................................................ 8

IDENTIFICATION OF VERO CYTOTOXIN-PRODUCING E. COLI O 157 FLOW CHART ....... 9 REPORTING .................................................................................................................................. 10 5.1 5.2 5.3 5.4 5.5 5.6 PRESUMPTIVE IDENTIFICATION ................................................................................................... 10 CONFIRMATION OF IDENTIFICATION ............................................................................................. 10 MEDICAL MICROBIOLOGIST ......................................................................................................... 10 NOTIFICATION TO THE HPA........................................................................................................ 10 CENTRE FOR INFECTIONS .......................................................................................................... 11 INFECTION CONTROL STAFF........................................................................................................ 11

REFERRALS ................................................................................................................................. 11 6.1 REFERENCE LABORATORY ......................................................................................................... 11

ACKNOWLEDGEMENTS AND CONTACTS................................................................................ 11

REFERENCES ...................................................................................................................................... 12

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 3 of 13 BSOP ID 22i3 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

AMENDMENT PROCEDURE
Controlled document reference Controlled document title BSOP ID 22 Identification of Escherichia coli O 157

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 Number/ Date 3/ 28.09.10 Issue no. Discarded 2 Insert Issue no. 3 Page Section(s) involved Amendment

Principles of Identification

Section updated to link to BSOP 30 and to mention isolates of presumptive (locally confirmed) E. coli O157. Text amended to VTEC O157 may cause severe illness that is sometimes fatal. Note added. Only CT-SMAC recommended explanation. Section updated. agar with

Safety considerations

7 7

Target organisms Isolation media

Colonial appearance Test procedures

Option for chromogenic identification plate added. Section expanded.

Storage and referral Flowchart Reporting

10 11

Updated Updated to include the new Health Protection Regulations 2010. Updated

12

References

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 4 of 13 BSOP ID 22i3 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

IDENTIFICATION OF ESCHERICHIA COLI O157

SCOPE OF DOCUMENT
This National Standard Methods (NSMs) describes the identification of presumptive Vero cytotoxinproducing Escherichia coli O157 (VTEC O157) isolated from faeces. These strains are associated with a wide spectrum of disease including haemolytic uraemic syndrome (HUS).

INTRODUCTION
Taxonomy
Vero cytotoxin-producing E. coli O157 (VTEC O157) is a member of the genus Escherichia and the family Enterobacteriaceae.

Characteristics
VTEC O157 is a Gram-negative rod. On sorbitol MacConkey agar (SMAC) or SMAC containing cefixime and tellurite (CT-SMAC), the colonies are colourless and 2-3mm in diameter. VTEC O157 differs from other members of the genus Escherichia in that it does not usually ferment sorbitol2 (a characteristic that is exploited by the selective medium3) and is -glucuronidase-negative. Most VTEC O157 are motile and possess the flagellar antigen H7, but at least 20% in England and Wales are phenotypically non-motile. They are facultatively anaerobic. Strains are oxidase-negative and usually produce gas from glucose. Some strains show atypical biochemistry eg they are anaerogenic, non lactose-fermenting, indole-negative or urea-positive. Some VTEC O157 strains have been found to ferment sorbitol and to be -glucuronidase-positive4,5. VTEC O157 is highly infective, and the infective dose is less than 50 organisms6. Laboratory acquired infections have been documented7,8.

Principles of Identification
BSOP 30 Investigation of faecal specimens for bacterial pathogens recommends that all diarrhoeal stools are screened for the presence of E.coli O157. Presumptive VTEC O157 isolates from primary culture are identified by colonial appearance on CT-SMAC, serology (agglutination with O157-specific antisera) and biochemical tests. Some commercial biochemical tests may give a doubtful or a low percentage profile for E. coli O157 because the fermentation of sorbitol is heavily weighted for other E. coli strains9, and care must be taken with the interpretation of the profile. All isolates of presumptive (locally confirmed) E. coli O157 should be referred to the Reference Laboratory (Laboratory of Gastrointestinal Pathogens (LGP), Centre for Infection (CfI), HPA) for confirmation of identification, testing for the presence of vero cytotoxin genes, serotyping, and phagetyping. All identification tests should ideally be performed from non-selective agar to take into account the variations that may occur with biochemical tests such as sorbitol fermentation. Where the clinical evidence is suggestive of VTEC infection (particularly in children under 15 years and adults over 65 years) and no presumptive sorbitol non-fermenting E. coli O157 colonies are observed on CT-SMAC agar, we recommend that clinical laboratories should: Test sorbitol fermenting colonies for agglutination with E. coli O157 antiserum Confirm the identification of agglutination-positive O157 colonies as E. coli Send presumptive isolate(s) to the Reference Laboratory (LEP, CfI, HPA Colindale) for confirmation, detection of VT genes and phage typing

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 5 of 13 BSOP ID 22i3 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

Faecal samples from cases with appropriate clinical symptoms from whom VTEC O157 have not been isolated should be submitted to the Laboratory of Gastrointestinal Pathogens for detection of VTEC strains belonging to serogroups other than O157. It is essential that all commercial and in-house test procedures have evidence of adequate validation demonstrating they are fit for purpose. It is also essential that appropriate ongoing Quality Assurance procedures should be in place.

TECHNICAL INFORMATION
N/A

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 6 of 13 BSOP ID 22i3 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

SAFETY CONSIDERATIONS6,7,10-21
VTEC O157 is in Hazard Group 3 All work with suspected isolates of VTEC O157 must be performed under Containment level 3 conditions. VTEC O157 may cause severe illness that is sometimes fatal. Laboratory acquired infections have been reported. Low numbers are required for an infective dose6,7. Laboratory procedures that give rise to infectious aerosols must be conducted in a microbiological safety cabinet. The above guidance should be supplemented with local COSHH and risk assessments. Compliance with postal and transport regulations is essential.

TARGET ORGANISMS
Vero cytotoxin-producing E. coli O157 NB: this procedure may result in the identification of isolates of presumptive E. coli O157 that do not produce vero cytotoxin and some organisms that give equivocal results in section 3.4.

3
3.1

IDENTIFICATION
MICROSCOPIC APPEARANCE
Grams stain Gram-negative rods

3.2

ISOLATION MEDIA
CT-SMAC agar incubated in air at 35 - 37C for 16 - 24 h. Enrichment culture in modified tryptone soya broth (MTSB) may be required in outbreaks (BSOP 30 - Investigation of faeces specimens for bacterial pathogens). CT-SMAC agar is used since classical sorbitol nonfermenting VTEC O157 are relatively resistant to potassium tellurite compared with other E. coli. There is a risk of isolation failure on SMAC agar lacking cefixime and tellurite.

3.3

COLONIAL APPEARANCE
Typical VTEC O157 colonies on CT-SMAC agar are colourless or slightly greyish in appearance and 1-2 mm in diameter. Some rare variant strains of VTEC O157 ferment sorbitol and may grow poorly on CTSMAC/SMAC, although this property is variable. Although CT-SMAC offers a degree of selection for presumptive VTEC O157, growth of other organisms may be observed. Mixed growth from faecal specimens may contain other sorbitol non-fermenters.

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 7 of 13 BSOP ID 22i3 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

Organism

Colour and size of colonies on CT-SMAC

Shigella flexneri Salmonella Typhimurium

Pink colonies. 0.5 - 1 mm diameter Pale pink pinpoint colonies

E. coli (non-O157)

Generally sorbitol fermenters. Pink colonies. Pinpoint to 0.25 mm diameter

3.4

TEST PROCEDURES
Agglutination Use VTEC O157 antiserum (latex or other commercial reagent). It is important to perform the appropriate control for autoagglutination. There have been reports of false positive agglutination test results for VTEC O157 due to other sorbitol-negative species such as E. hermannii 22. Biochemical tests Commercial identification kit In-house identification kit Some commercial kits will identify the isolate as a low discrimination E. coli. The lack of sorbitol fermentation will be listed as a test weighting against the isolate being an E. coli. It is important that when reading the commercial kit this factor is considered when the result is interpreted. Chromogenic identification plates are available and may be valuable as an alternative for confirmation of identification of E.coli 23. Subculture to lactose containing media This may be the purity plate from the commercial identification kit. VTEC O157 are almost always lactose-positive but rare isolates have been found to be lactose non-fermenters. Isolation failure Where the clinical evidence is suggestive of VTEC infection (particularly in children under 15 years and adults over 65 years) and no presumptive sorbitol non-fermenting E. coli O157 colonies are observed on CT-SMAC, we recommend that clinical laboratories should: Test sorbitol fermenting colonies for agglutination with E. coli O157 antiserum Confirm the identification of agglutination-positive O157 colonies as E. coli

Faecal samples from appropriate cases from whom VTEC O157 have not been isolated should be submitted to the Reference Laboratory for detection of VTEC belonging to serogroups other than O157 by culture and DNA-based methods.

3.5

STORAGE AND REFERRAL

All purified isolates of presumptive (locally confirmed) E. coli O157 (sorbitol non-fermenters or sorbitol fermenting) should be saved on nutrient agar slopes. Cultures should be referred promptly to the Reference Laboratory for biochemical confirmation, detection of VT genes, serotyping and phage typing.

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 8 of 13 BSOP ID 22i3 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

IDENTIFICATION OF VERO CYTOTOXINPRODUCING E. COLI O157 FLOW CHART

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 9 of 13 BSOP ID 22i3 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

Note: Refer to clinical details: in cases and particularly clusters of cases where isolation or identification fails, but the symptoms are consistent with VTEC O157 infection, the following actions are recommended: Send a faecal sample to the Reference Laboratory Send a serum sample to the Reference Laboratory for the testing for the presence of antibodies to E. coli O157 lipopolysaccharide

5
5.1

REPORTING24,25
PRESUMPTIVE IDENTIFICATION
Presumptive identification of E. coli O157 is based on appropriate growth characteristics, biochemical tests, colonial appearance and agglutination with O157 antiserum or commercial antigen kits.

5.2 5.3

CONFIRMATION OF IDENTIFICATION
Refer to Laboratory of Gastrointestinal Pathogens, Centre for Infections, HPA, Colindale.

MEDICAL MICROBIOLOGIST
Inform the medical microbiologist of presumptive or confirmed E. coli O157 strains. According to local protocol, the medical microbiologist should also be informed if the request card bears relevant information which suggests infection with E. coli O157 eg Enterocolitis (especially if complicated by haemolytic-uraemic syndrome, neurological dysfunction and/or confusional states) Recent travel, farming (or visits to farms) Veterinary or laboratory work Food poisoning Investigation of outbreak situations

Follow local protocols for reporting to clinician

5.4

NOTIFICATION TO THE HPA26,27

From 1 October 2010 provisions relating to diagnostic laboratories come into force. The Notification Regulations require diagnostic laboratories to notify the Health Protection Agency (HPA) when they identify the causative agents that are listed in Schedule 2 of the Regulations. Notifications must be provided in writing, on paper or electronically, within seven days. Urgent cases should be notified orally and as soon as possible, recommended within 24 hours. These should be followed up by written notification within seven days. For the purposes of the Notification Regulations, the recipient of laboratory notifications is the local HPA office. If a case has already been notified by a registered medical practitioner, the diagnostic laboratory is still required to notify the case if they identify any evidence of an infection caused by a notifiable causative agent. Notification under the Health Protection (Notification) Regulations 2010 does not replace voluntary reporting to the HPA. The vast majority of NHS laboratories voluntarily report a wide range of laboratory diagnoses of causative agents to the HPA and many HPA offices have agreements with local laboratories for urgent reporting of some infections. This should continue.

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 10 of 13 BSOP ID 22i3 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

(Note: The Health Protection Legislation Guidance (2010) includes reporting of HIV & STIs, HCAIs and CJD under Notification Duties of Registered Medical Practitioners: it is not noted under Notification Duties of Diagnostic Laboratories). Other arrangements exist in Scotland28 and Wales29.

5.5

INFECTION CONTROL STAFF

Inform the infection control team of presumptive and confirmed isolates of E. coli O157 and other VTEC causing a clinical picture characteristic of infection with E. coli O157.

6
6.1

REFERRALS
REFERENCE LABORATORY
For information on the tests offered, turn around times, transport procedure and the other requirements of the reference laboratory refer to: http://www.hpa.org.uk/cfi/lep/default.htm Laboratory of Gastrointestinal Pathogens Centre for Infections Health Protection Agency 61 Colindale Avenue London NW9 5EQ Contact CFI main switchboard: Tel. +44 (0) 020 8327 6173

ACKNOWLEDGEMENTS AND CONTACTS

This National Standard Method has been developed, reviewed and revised by the National Standard Methods Working Group for Clinical Bacteriology (http://www.hpa-standardmethods.org.uk/wg_bacteriology.asp). We would like to thank Dr Geraldine Smith of the Centre for Infections, and Dr David Tompkins of HPA Regional Microbiology Network for their significant contributions.. 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

IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 11 of 13 BSOP ID 22i3 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

REFERENCES
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IDENTIFICATION OF ESCHERICHIA COLI O157 Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 12 of 13 BSOP ID 22i3 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

19. British Standards Institution (BSI). Microbiological safety cabinets. Part 2: Recommendations for information to be exchanged between purchaser, vendor and installer and recommendations for installation. BS 5726:2005. British Standards Institution (BSI). London. 24-3-0005. 20. British Standards Institution (BSI). Microbiological safety cabinets. Part 4: Recommendations for selection, use and maintenance. BS 5726. London. 1992. 21. Advisory Committee on Dangerous Pathogens. The management, design and operation of microbiological containment laboratories. HSE Books. 2001. 22. Lior H, Borczyk AA. False positive identifications of Escherichia coli O157. Lancet 1987;1:333. 23. Fallon D, Ackland G, Andrews N, Frodsham D, Howe S, Howells K, et al. A comparison of the performance of commercially available chromogenic agars for the isolation and presumptive identification of organisms from urine. J Clin Pathol 2003;56:608-12. 24. Health Protection Agency. Essential operational guidance for HPA staff dealing with incidents of VTEC infection. The VTEC Operational Manual 2010. 25. Health Protection Agency. Background evidence for the Public Health Management of Infection with Verotoxigenic Escherichia coli (VTEC). The VTEC Support Document 2010. 26. Health Protection Agency. Laboratory Reporting to the Health Protection Agency: Guide for Diagnostic Laboratories. 2008. 27. Department of Health. Health Protection Legislation (England) Guidance 2010. http://www.dh.gov.uk/en/Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/D H_114510. p. 1-112. 28. Scottish Government. Public Health (Scotland) Act 2008. http://www.scotland.gov.uk/Topics/Health/NHSScotland/publicact/Implementation/Timetable3333/Part2Guidance/Q/EditMode/on/ForceUpdate/on 29. The Welsh Assembly Government. Health Protection Legislation (Wales) Guidance 2010. http://wales.gov.uk/topics/health/protection/communicabledisease/legislation/regulations/jsessioni d=GysLMLLbvmnvTHVkVQTv8Hr6LQzLC8G7G4vpgFYpY99JhK2vJsKd!-1351106478?lang=en.

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