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IDENTIFICATION OF ESCHERICHIA COLI O157Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page 1 of 13

BSOP ID 22i3This NSM should be used in conjunction with the series of NSMs from the Health Protection Agency

www.evaluations-standards.org.uk Email: [email protected]

NATIONAL STANDARD METHOD

IDENTIFICATION OFESCHERICHIA COLI O157

BSOP ID 22

Issued by Standards Unit, Evaluations and Standards LaboratoryCentre for Infections

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IDENTIFICATION OF ESCHERICHIA COLI O157Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page 2 of 13



STATUS OF NATIONAL STANDARD METHODSNational Standard Methods, which include standard operating procedures (SOPs), algorithms andguidance notes, promote high quality practices and help to assure the comparability of diagnosticinformation obtained in different laboratories. This in turn facilitates standardisation of surveillance

underpinned by research, development and audit and promotes public health and patient confidencein their healthcare services. The methods are well referenced and represent a good minimumstandard for clinical and public health microbiology. However, in using National Standard Methods,laboratories should take account of local requirements and may need to undertake additionalinvestigations. The methods also provide a reference point for method development.

National Standard Methods are developed, reviewed and updated through an open and wideconsultation process where the views of all participants are considered and the resulting documentsreflect the majority agreement of contributors.

Representatives of several professional organisations, including those whose logos appear on thefront cover, are members of the working groups which develop National Standard Methods. Inclusionof 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 StandardMethods but their views are not necessarily those of the entire organisation of which they are amember. The current list of participating organisations can be obtained by [email protected] .

The performance of standard methods depends on the quality of reagents, equipment, commercialand in-house test procedures. Laboratories should ensure that these have been validated and shownto 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 ProtectionAgency or any supporting organisation cannot be responsible for the accuracy of any statement orrepresentation made or the consequences arising from the use of or alteration to any informationcontained 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 wherenecessary. If you make any changes to this publication, it must be made clear where changes havebeen made to the original document. The Health Protection Agency (HPA) should at all times beacknowledged.

The HPA is an independent organisation dedicated to protecting peoples health. It brings togetherthe expertise formerly in a number of official organisations. More information about the HPA can befound at www.hpa.org.uk .

The HPA aims to be a fully Caldicott compliant organisation. It seeks to take every possibleprecaution to prevent unauthorised disclosure of patient details and to ensure that patient-relatedrecords are kept under secure conditions 1.

More details can be found on the website at www.evaluations-standards.org.uk . Contributions to thedevelopment of the documents can be made by contacting [email protected] .

The reader is informed that all taxonomy in this document was correct at time of issue.

Suggested citation for this document:Health Protection Agency (2010). Identification of Escherichia coli O157 . National Standard MethodBSOP ID 22 Issue 3. http://www.hpa-standardmethods.org.uk/pdf_sops.asp .

Please note the references are now formatted using Reference Manager software. If you alter or delete text without Reference Manager installed on your computer, the references will not be updated automatically.

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IDENTIFICATION OF ESCHERICHIA COLI O157Issue no: 3 Issue date: 28.09.10 Issued by: Standards Unit, Evaluations, Department for Evaluations, Standards and Training Page 3 of 13



INDEXSTATUS 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 SAFETY CONSIDERATIONS ......................................................................................................... 7

2 TARGET ORGANISMS ................................................................................................................... 7 3 IDENTIFICATION............................................................................................................................. 7

3.1 MICROSCOPIC APPEARANCE ........................................................................................................ 7 3.2 ISOLATION MEDIA ......................................................................................................................... 7 3.3 COLONIAL APPEARANCE ............................................................................................................... 7 3.4 TEST PROCEDURES ..................................................................................................................... 8 3.5 S TORAGE AND REFERRAL ............................................................................................................ 8

4 IDENTIFICATION OF VERO CYTOTOXIN-PRODUCING E. COLI O157 FLOW CHART....... 9

5 REPORTING.................................................................................................................................. 10 5.1 P RESUMPTIVE IDENTIFICATION ................................................................................................... 10 5.2 CONFIRMATION OF IDENTIFICATION ............................................................................................. 10 5.3 MEDICAL MICROBIOLOGIST ......................................................................................................... 10 5.4 NOTIFICATION TO THE HPA........................................................................................................ 10 5.5 CENTRE FOR INFECTIONS .......................................................................................................... 11 5.6 INFECTION CONTROL STAFF ........................................................................................................ 11

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

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

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

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AMENDMENT PROCEDURE

Controlled documentreference

BSOP ID 22

Controlled document title Identification of Escherichia coli O157

Each National Standard Method has an individual record of amendments. The current amendmentsare listed on this page. The amendment history is available from [email protected] .

On issue of revised or new pages each controlled document should be updated by the copyholder inthe laboratory.

AmendmentNumber/ Date

Issue no.Discarded

InsertIssueno.

Page Section(s) involved Amendment

3/ 28.09.10

2 3 5 Principles ofIdentification

Section updated to linkto BSOP 30 and tomention isolates ofpresumptive (locallyconfirmed) E. coli O157.

7 Safetyconsiderations

Text amended to VTECO157 may cause severeillness that is sometimesfatal.

7 Target organisms Note added.

7 Isolation media Only CT-SMAC agarrecommended withexplanation.

7 Colonialappearance

Section updated.

8 Test procedures Option for chromogenicidentification plateadded.

8 Storage andreferral

Section expanded.

10 Flowchart Updated

11 Reporting Updated to include thenew Health ProtectionRegulations 2010.

12 References Updated

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IDENTIFICATION OF ESCHERICHIA COLI O157

SCOPE OF DOCUMENTThis National Standard Methods (NSMs) describes the identification of presumptive Vero cytotoxin-producing Escherichia coli O157 (VTEC O157) isolated from faeces. These strains are associatedwith a wide spectrum of disease including haemolytic uraemic syndrome (HUS).

INTRODUCTION

TaxonomyVero cytotoxin-producing E. coli O157 (VTEC O157) is a member of the genus Escherichia and thefamily Enterobacteriaceae.

CharacteristicsVTEC O157 is a Gram-negative rod. On sorbitol MacConkey agar (SMAC) or SMAC containingcefixime and tellurite (CT-SMAC), the colonies are colourless and 2-3mm in diameter. VTEC O157differs from other members of the genus Escherichia in that it does not usually ferment sorbitol 2 (acharacteristic that is exploited by the selective medium 3) and is -glucuronidase-negative. Most VTECO157 are motile and possess the flagellar antigen H7, but at least 20% in England and Wales arephenotypically non-motile. They are facultatively anaerobic. Strains are oxidase-negative and usuallyproduce gas from glucose. Some strains show atypical biochemistry eg they are anaerogenic, nonlactose-fermenting, indole-negative or urea-positive. Some VTEC O157 strains have been found toferment sorbitol and to be -glucuronidase-positive 4,5 .

VTEC O157 is highly infective, and the infective dose is less than 50 organisms 6. Laboratory acquiredinfections have been documented 7,8 .

Principles of IdentificationBSOP 30 Investigation of faecal specimens for bacterial pathogens recommends that all diarrhoealstools are screened for the presence of E.coli O157. Presumptive VTEC O157 isolates from primaryculture are identified by colonial appearance on CT-SMAC, serology (agglutination with O157-specificantisera) and biochemical tests. Some commercial biochemical tests may give a doubtful or a lowpercentage profile for E. coli O157 because the fermentation of sorbitol is heavily weighted for other E.coli strains 9, 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 ofGastrointestinal Pathogens (LGP), Centre for Infection (CfI), HPA) for confirmation of identification,testing for the presence of vero cytotoxin genes, serotyping, and phagetyping. All identification testsshould ideally be performed from non-selective agar to take into account the variations that may occurwith biochemical tests such as sorbitol fermentation.

Where the clinical evidence is suggestive of VTEC infection (particularly in children under 15 yearsand adults over 65 years) and no presumptive sorbitol non-fermenting E. coli O157 colonies areobserved 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) forconfirmation, detection of VT genes and phage typing

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Faecal samples from cases with appropriate clinical symptoms from whom VTEC O157 have not beenisolated should be submitted to the Laboratory of Gastrointestinal Pathogens for detection of VTECstrains belonging to serogroups other than O157.

It is essential that all commercial and in-house test procedures have evidence of adequate validationdemonstrating they are fit for purpose. It is also essential that appropriate ongoing Quality Assuranceprocedures should be in place.

TECHNICAL INFORMATION

N/A

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1 SAFETY CONSIDERATIONS 6,7,10-21

VTEC O157 is in Hazard Group 3

All work with suspected isolates of VTEC O157 must be performed under Containment level 3conditions.

VTEC O157 may cause severe illness that is sometimes fatal.

Laboratory acquired infections have been reported.

Low numbers are required for an infective dose 6,7 .

Laboratory procedures that give rise to infectious aerosols must be conducted in amicrobiological safety cabinet.

The above guidance should be supplemented with local COSHH and risk assessments.

Compliance with postal and transport regulations is essential.

2 TARGET ORGANISMSVero cytotoxin-producing E. coli O157

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

3 IDENTIFICATION

3.1 M ICROSCOPIC APPEARANCE Grams stainGram-negative rods

3.2 I SOLATION MEDIA CT-SMAC agar incubated in air at 35 - 37C for 16 - 24 h. Enrichment culture in modifiedtryptone soya broth (MTSB) may be required in outbreaks ( BSOP 30 - Investigation of faecesspecimens for bacterial pathogens ). CT-SMAC agar is used since classical sorbitol non-fermenting VTEC O157 are relatively resistant to potassium tellurite compared with otherE. coli . There is a risk of isolation failure on SMAC agar lacking cefixime and tellurite.

3.3 C OLONIAL APPEARANCE Typical VTEC O157 colonies on CT-SMAC agar are colourless or slightly greyish inappearance and 1-2 mm in diameter.

Some rare variant strains of VTEC O157 ferment sorbitol and may grow poorly onCTSMAC/SMAC, although this property is variable.

Although CT-SMAC offers a degree of selection for presumptive VTEC O157, growth of otherorganisms may be observed. Mixed growth from faecal specimens may contain other sorbitolnon-fermenters.

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Organism Colour and size of colonies on CT-SMAC

Shigella flexneri Pink colonies. 0.5 - 1 mm diameter

Salmonella Typhimurium Pale pink pinpoint colonies

E. coli (non-O157) Generally sorbitol fermenters. Pink colonies. Pinpoint to 0.25mm diameter

3.4 T EST PROCEDURES AgglutinationUse VTEC O157 antiserum (latex or other commercial reagent). It is important to perform theappropriate control for autoagglutination. There have been reports of false positiveagglutination test results for VTEC O157 due to other sorbitol-negative species such asE. hermannii 22 .

Biochemical testsCommercial identification kitIn-house identification kit

Some commercial kits will identify the isolate as a low discrimination E. coli. The lack of sorbitolfermentation will be listed as a test weighting against the isolate being an E. coli. It is importantthat 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 forconfirmation of identification of E.coli 23.

Subculture to lactose containing mediaThis may be the purity plate from the commercial identification kit.

VTEC O157 are almost always lactose-positive but rare isolates have been found to belactose non-fermenters.

Isolation failureWhere the clinical evidence is suggestive of VTEC infection (particularly in children under 15years and adults over 65 years) and no presumptive sorbitol non-fermenting E. coli O157colonies 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 isolatedshould be submitted to the Reference Laboratory for detection of VTEC belonging toserogroups other than O157 by culture and DNA-based methods.

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

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4 IDENTIFICATION OF VERO CYTOTOXIN-PRODUCING E. COL I O157 FLOW CHART

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Note: Refer to clinical details: in cases and particularly clusters of cases where isolation oridentification fails, but the symptoms are consistent with VTEC O157 infection, the following actionsare 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 REPORTING 24,25

5.1 P RESUMPTIVE IDENTIFICATION Presumptive identification of E. coli O157 is based on appropriate growth characteristics,biochemical tests, colonial appearance and agglutination with O157 antiserum or commercialantigen kits.

5.2 C ONFIRMATION OF IDENTIFICATION Refer to Laboratory of Gastrointestinal Pathogens, Centre for Infections, HPA, Colindale.

5.3 M EDICAL 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 requestcard bears relevant information which suggests infection with E. coli O157 eg

Enterocolit is (especially if complicated by haemolytic-uraemic syndrome, neurologicaldysfunction 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 N OTIFICATION TO THE HPA 26,27

From 1 October 2010 provisions relating to diagnostic laboratories come into force. TheNotification 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. Urgentcases 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 theNotification Regulations, the recipient of laboratory notifications is the local HPA office. If acase has already been notified by a registered medical practitioner, the diagnostic laboratoryis still required to notify the case if they identify any evidence of an infection caused by anotifiable causative agent.

Notification under the Health Protection (Notification) Regulations 2010 does not replacevoluntary reporting to the HPA. The vast majority of NHS laboratories voluntarily report a widerange of laboratory diagnoses of causative agents to the HPA and many HPA offices haveagreements with local laboratories for urgent reporting of some infections. This shouldcontinue.

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(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 notedunder Notification Duties of Diagnostic Laboratories).

Other arrangements exist in Scotland 28 and Wales 29 .

5.5 I NFECTION CONTROL STAFF Inform the infection control team of presumptive and confirmed isolates of E. coli O157 andother VTEC causing a clinical picture characteristic of infection with E. coli O157.

6 REFERRALS6.1 R EFERENCE LABORATORY

For information on the tests offered, turn around times, transport procedure and the otherrequirements of the reference laboratory refer to: http://www.hpa.org.uk/cfi/lep/default.htm

Laboratory of Gastrointestinal PathogensCentre for InfectionsHealth Protection Agency

61 Colindale AvenueLondonNW9 5EQ

Contact CFI main switchboard: Tel. +44 (0) 020 8327 6173

7 ACKNOWLEDGEMENTS AND CONTACTSThis National Standard Method has been developed, reviewed and revised by the NationalStandard Methods Working Group for Clinical Bacteriology(http://www.hpa-standardmethods.org.uk/wg_bacteriology.asp ). We would like to thank DrGeraldine Smith of the Centre for Infections, and Dr David Tompkins of HPA Regional

Microbiology Network for their significant contributions.. The contributions of many individualsin clinical bacteriology laboratories and specialist organisations who have provided informationand comment during the development of this document, and final editing by the Medical Editorare 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 UnitDepartment for Evaluations, Standards and TrainingCentre for Infections

Health Protection AgencyColindaleLondonNW9 5EQ

E-mail: [email protected]

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19. British Standards Institution (BSI). Microbiological safety cabinets. Part 2: Recommendations forinformation to be exchanged between purchaser, vendor and installer and recommendations forinstallation. BS 5726:2005. British Standards Institution (BSI). London. 24-3-0005.

20. British Standards Institution (BSI). Microbiological safety cabinets. Part 4: Recommendations forselection, use and maintenance. BS 5726. London. 1992.

21. Advisory Committee on Dangerous Pathogens. The management, design and operation ofmicrobiological 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 theperformance of commercially available chromogenic agars for the isolation and presumptiveidentification of organisms from urine. J Clin Pathol 2003;56:608-12.

24. Health Protection Agency. Essential operational guidance for HPA staff dealing with incidents ofVTEC 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 forDiagnostic Laboratories. 2008.

27. Department of Health. Health Protection Legislation (England) Guidance 2010.http://www.dh.gov.uk/en/Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/DH_114510 . p. 1-112.

28. Scottish Government. Public Health (Scotland) Act 2008.http://www.scotland.gov.uk/Topics/Health/NHS-Scotland/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/jsessionid=GysLMLLbvmnvTHVkVQTv8Hr6LQzLC8G7G4vpgFYpY99JhK2vJsKd!-1351106478?lang=en .

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