NATIONAL STANDARD METHOD

IDENTIFICATION OF CLOSTRIDIUM SPECIES

BSOP ID 8 Issued by Standards Unit, Evaluations and Standards Laboratory Centre for Infections

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 1 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

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 [email protected]. 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 [email protected].

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Suggested citation for this document: Health Protection Agency (2008). Identification of Clostridium species. National Standard Method BSOP ID 8 Issue 3. http://www.hpa-standardmethods.org.uk/pdf_sops.asp.

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 2 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

INDEX
STATUS OF NATIONAL STANDARD METHODS ................................................................................ 2 INDEX...................................................................................................................................................... 3 AMENDMENT PROCEDURE ................................................................................................................. 4 SCOPE OF DOCUMENT ........................................................................................................................ 5 INTRODUCTION ..................................................................................................................................... 5 TECHNICAL INFORMATION/LIMITATIONS ......................................................................................... 5 1 2 3 SAFETY CONSIDERATIONS ......................................................................................................... 6 TARGET ORGANISMS ................................................................................................................... 6 IDENTIFICATION............................................................................................................................. 7 3.1 3.2 3.3 3.4 3.5 3.6 4 5 MICROSCOPIC APPEARANCE ........................................................................................................ 7 PRIMARY ISOLATION MEDIA .......................................................................................................... 7 COLONIAL APPEARANCE............................................................................................................... 7 TEST PROCEDURES ..................................................................................................................... 8 FURTHER TESTS .......................................................................................................................... 8 STORAGE AND REFERRAL............................................................................................................. 8

IDENTIFICATION OF CLOSTRIDIUM SPECIES - FLOW CHART ............................................... 9 REPORTING .................................................................................................................................. 10 5.1 5.2 5.3 5.3 5.5 5.6 PRESUMPTIVE IDENTIFICATION ................................................................................................... 10 CONFIRMATION OF IDENTIFICATION ............................................................................................. 10 MEDICAL MICROBIOLOGIST ......................................................................................................... 10 CCDC...................................................................................................................................... 10 CENTRE FOR INFECTIONS ........................................................................................................... 10 INFECTION CONTROL STAFF ....................................................................................................... 10

6 7

REFERRALS ................................................................................................................................. 11 ACKNOWLEDGEMENTS AND CONTACTS................................................................................ 12

REFERENCES ...................................................................................................................................... 13

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 3 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

AMENDMENT PROCEDURE
Controlled document reference Controlled document title BSOP ID 8 Identification of Clostridium species

Each National Standard Method has an individual record of amendments. The current amendments are 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 in the laboratory. Amendment Number/ Date 3/ 14.07.08 Issue no. Discarded 2.1 Insert Issue no. 3 Page Section(s) involved Amendment

1 All

Front Page All

NIMAG logo added PDF links amended to read reference document title References reviewed and updated

13

References

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 4 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

IDENTIFICATION OF CLOSTRIDIUM SPECIES

SCOPE OF DOCUMENT
This National Standard Method (NSM) describes the identification of Clostridium species. There are many species of clostridia, which may be found naturally in animal faeces and the environment. Only species associated with humans will be discussed in this NSM.

INTRODUCTION
Taxonomy
The genus Clostridium currently contains approximately 100 species. In 1994 the heterogeneity of this species was confirmed by 16S rRNA gene sequencing. As a result five new genera and eleven new species were proposed2, none of which appear to be relevant to human infections3.

Characteristics of Clostridium species

Clostridium species are Gram-positive rods (some are Gram-variable), often arranged in pairs or short chains, with rounded or sometimes pointed or square end. They are often pleomorphic. Clostridium species vary considerably in their oxygen tolerance. Some species such as Clostridium novyi type A and Clostridium haemolyticum are among the strictest of obligate anaerobes and may require extended incubation on pre-reduced or freshly prepared plates and total handling in an anaerobic chamber. Conversely, Clostridium tertium, Clostridium histolyticum and Clostridium carnis are aerotolerant and will form colonies on blood agar plates incubated in an atmosphere of air with 5-10% added CO23. Virtually all of the members of the genus, except Clostridium perfringens, are motile with peritrichous flagellae and form oval or spherical endospores that may distend the cell. They may be saccharolytic or proteolytic and are usually catalase-negative. Many species produce potent exotoxins4. Toxins of Clostridium species Clinically significant Clostridium species produce a variety of toxins. It is the production of these toxins which leads to the distinctive clinical features of the diseases they cause, eg tetanus and botulism result from the production of neurotoxins that are amongst the most lethal substances known to man5. Clostridial toxins are biologically active proteins that are antigenic in nature and can therefore be neutralised with specific antisera. Detection of a particular toxin in a patient sample may be diagnostic and therefore render isolation of the organism unnecessary (eg Clostridium difficile). Clostridium perfringens is the most commonly isolated Clostridium species. Five types (A-E) may be distinguished by the combinations of major lethal toxins they produce3.

Principles of Identification
Clues to the identity of certain pathogenic species may be obtained by observing characteristics such as colonial appearance, Gram stain appearances and the presence or absence of -haemolysis. Other phenotypic tests may also be applied to obtain a presumptive identification in conjunction with the use of a good laboratory manual such as the Wadsworth-KTL Anaerobe Laboratory Manual6. It is important to ensure the culture is pure, as the fine spreading growth of some Clostridium species may mask contaminating organisms. If confirmation of identity is required, isolates should be referred to the Anaerobe Reference Laboratory, Cardiff. If Clostridium botulinum is suspected, samples of patients serum, faeces and implicated foodstuff should be referred directly to the Food Safety Microbiology Laboratory, Colindale.

TECHNICAL INFORMATION/LIMITATIONS
N/A
IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 5 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

SAFETY CONSIDERATIONS7-18
Hazard Group 2 organisms Refer to current guidance on the safe handling of all Hazard Group 2 organisms documented in this NSM. 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
Clostridium species reported to have caused human disease4 Commonly isolated C. perfringens C. septicum C. tertium C. difficile Rarely isolated C. novyii type A C. sordellii Very rarely isolated C. tetani C. histolyticum C. botulinum Commonly isolated non-pathogenic clostridia C. sporogenes C. ramosum C. innocuum C. paraputrificum C. cadaveris C. bifermentans C. fallax C. clostridioforme

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 6 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

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3.1

IDENTIFICATION
MICROSCOPIC APPEARANCE
(See BSOPTP 39 - Staining Procedures) Gram stain Gram-positive rods, which may possess a single endospore. Some species may be Gramvariable. Spore stain Used to determine the shape and position of the spore (phase contrast microscopy is an alternative option). C. perfringens C. botulinum C. difficile C. novyi C. sordellii C. septicum C. tetani (Does not sporulate on ordinary media) Oval, subterminal Oval, subterminal Oval, subterminal Oval, subterminal Oval, subterminal Round, terminal

3.2 3.3

PRIMARY ISOLATION MEDIA

Agar containing blood incubated anaerobically at 35C - 37C for 40 48 h.

COLONIAL APPEARANCE
Colonial appearance varies with species and brief descriptions of the most common species are given here Organism Characteristics of growth on agar containing blood after anaerobic incubation at 35C 37C for 40 48 h Large, smooth, regular convex colonies, but may be rough and flat with an irregular edge. Usually has a double zone of -haemolysis; produces lecithinase Large (3 mm), irregularly circular, smooth, greyish, translucent with a fibrillar edge that may spread. Most strains are -haemolytic; produces lipase Glossy, grey, circular colonies with a rough edge; fluoresce greenyellow under UV light. They are usually non-haemolytic, with a characteristic farmyard smell. Raised, circular colonies, which become flattened and irregular in old cultures. Colonies tend to fuse forming a spreading growth with a double zone of -haemolysis. Type A produces lecithinase and lipase Grey-white, convex, circular colonies with crenated edges, which may spread. They may be -haemolytic; produce lecithinase; indole positive Usually produce a thick swarming growth with a narrow zone of -haemolysis Fine swarming growth (may be difficult to see) which may appear -haemolytic

C. perfringens

C. botulinum/ sporogenes

C. difficile

C. novyi

C. sordellii /bifermentans C. septicum

C. tetani

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 7 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

Other Clostridium species

Colonial appearances vary, but may produce a spreading growth which may or may not be -haemolytic

3.4

TEST PROCEDURES
Nagler (see BSOPTP 22 - Nagler test) with C. perfringens antitoxin C. perfringens lecithinase is inhibited by the antitoxin as is that produced by C. bifermentans and C. sordellii. Species other than C. perfringens may produce lecithinase. Also examine for the production of lipase (pearly layer) on egg yolk agar. Reverse CAMP test can be used for differentiation of C. perfringens from other Clostridium species19. Commercial identification kits Results should be interpreted with caution in conjunction with other test results. If clinically indicated refer to the Anaerobe Reference Laboratory for further identification.

3.5 3.6

FURTHER TESTS
N/A

STORAGE AND REFERRAL

If required save the pure isolate in fastidious anaerobe broth or Robinsons cooked meat broth for referral to the Anaerobe Reference Laboratory.

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 8 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

IDENTIFICATION OF CLOSTRIDIUM SPECIES -

FLOW CHART

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 9 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

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5.1

REPORTING
PRESUMPTIVE IDENTIFICATION
If appropriate growth characteristics, colonial appearances and Gram stain of the culture are demonstrated and the isolate is metronidazole susceptible.

5.2

CONFIRMATION OF IDENTIFICATION
Following Nagler plate, or Reverse CAMP test for C.perfringens, commercial identification kit results and/or Reference Laboratory report.

5.3

MEDICAL MICROBIOLOGIST
Inform the medical microbiologist of all positive cultures from normally sterile sites. According to local protocols, the medical microbiologist should also be informed of a presumptive and confirmed Clostridium species. when the request card bears relevant information eg: Cases of trauma, penetrating injury, compound fracture or retained foreign body, or known injecting drug abuse (especially heroin) Septic abortion Suspicion of clostridial myonecrosis, (necrotising) myofasciitis, surgical wound infection (especially in cases with occlusive peripheral vascular disease and/or diabetes mellitus) Other serious medical conditions eg alcohol or substance abuse, immunodeficiency, cancer, or persons receiving treatment for cancer (including neutropenia and/or mucositis) Food poisoning (especially involving descending paralysis with cranial nerve involvement) and/or consumption of unusual or imported foods (suspicion of botulism) Investigation of outbreaks Pseudomembranous colitis or antibiotic-related diarrhoea Suspicion of tetanus

Follow local protocols for reporting to clinician

5.3 5.5 5.6

CCDC
Refer to local Memorandum of Understanding.

CENTRE FOR INFECTIONS20

Refer to current guidelines on CDSC and COSURV reporting.

INFECTION CONTROL STAFF

Inform the infection control team of presumptive and confirmed isolates of C. botulinum and C. difficile.

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6.1

REFERRALS
REFERENCE LABORATORY
For identification and for information on the tests offered, turn around times, transport procedure and the other requirements of the reference laboratory refer to: Anaerobe Reference Laboratory NPHS Microbiology Cardiff University Hospital of Wales Heath Park Cardiff CF14 4XW Telephone +44 (0) 29 2074 2171 or 2378 http://www.hpa.org.uk/cfi/arl/default.htm For toxin detection and for information on the tests offered, turn around times, transport procedure and the other requirements of the reference laboratory refer to: Food Safety Microbiology Laboratory Centre for Infections Health Protection Agency 61 Colindale Avenue London NW9 5HT http://www.hpa.org.uk/cfi/fsml/default.htm Contact CfI main switchboard: Tel. +44 (0) 20 8200 4400

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 11 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

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). 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, Evaluations and Standards Laboratory, Centre for Infections, Health Protection Agency, London. For further information please contact us at: Standards Unit Evaluations and Standards Laboratory Centre for Infections Health Protection Agency Colindale London NW9 5EQ E-mail: [email protected]

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 12 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]

REFERENCES
1. Department of Health NHS Executive: The Caldicott Committee. Report on the review of patientidentifiable information. London. December 1997. 2. Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, et al. The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 1994;44:812-26. 3. Koneman EW, Allen S D, Janda W M, Schreckenberger P C, Winn WC J, editors. Color Atlas and Textbok of Diagnostic Microbiology. 5th ed ed. Philadelphia: Lippincott Williams & Wilkins; 1997. p. 709-84 4. Holt JG, Krieg N R, Sneath P H A, Staley J T, Williams S T, editors. Bergey's Manual of Determinative Bacteriology. 9th ed ed. Baltimore: Williams and Wilkins; 1994. p. 560 5. Hatheway CL. Toxigenic clostridia. Clin Microbiol Rev 1990;3:66-98. 6. Jousimies-Somer H, Summanen P, Citron D, et al. Anaerobic Bacteriology Manuel. Anaerobic Bacteriology Manuel. Sixth ed. Star Publishing Company; 2002. p. 54. 7. Advisory Committee on Dangerous Pathogens 2004 Approved List of Biological Agents. http://www.hse.gov.uk/pubns/misc208.pdf. p. 1-17. 8. Health and Safety Executive, editor. Biological Agents: Managing the risks in laboratories and healthcare premises. 5 A.D.

9. Public Health Laboratory Service Standing Advisory Committee on Laboratory Safety. Safety Precautions: Notes for Guidance. 4th ed. London: Public Health Laboratory Service (PHLS); 1993. 10. Control of Substances Hazardous to Health Regulations 2002. General COSHH. Approved Code of Practice and Guidance, L5. Suffolk: HSE Books; 2002. 11. Health and Safety Executive. 5 steps to risk assessment: a step by step guide to a safer and healthier workplace, IND (G) 163 (REVL). Suffolk: HSE Books; 2002. 12. Health and Safety Executive. A guide to risk assessment requirements: common provisions in health and safety law, IND (G) 218 (L). Suffolk: HSE Books; 2002. 13. Health Services Advisory Committee. Safety in Health Service laboratories. Safe working and the prevention of infection in clinical laboratories and similar facilities. 2nd ed. Suffolk: HSE Books; 2003. 14. NHS Estates. Health Building Note 15. Accommodation for pathology services. 1st ed. London: Her Majesty's Stationary Office (HMSO); 1991. (Out of print - 2nd edition in press). 15. BS EN 12469: 2000. Biotechnology - performance criteria for microbiological safety cabinets. London: British Standards Institution (BSI); 2000. 16. BS 5726: 1992. Microbiological safety cabinets. Part 2. Recommendations for information to be exchanged between purchaser, vendor and installer and recommendations for installation. London: British Standards Institution (BSI); 1992. 17. BS 5726: 1992. Microbiological safety cabinets. Part 4. Recommendations for selection, use and maintenance. London: British Standards Institution (BSI); 1992.

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18. Advisory Committee on Dangerous Pathogens. The management, design and operation of microbiological containment laboratories. Suffolk: HSE Books; 2001. 19. Buchanan AG. Clinical laboratory evaluation of a reverse CAMP test for presumptive identification of Clostridium perfringens. J Clin Microbiol 1982;16:761-2. 20. Health Protection Agency. Laboratory Reporting to the Health Protection Agency. Guide for diagnostic laboratories. 2008.

IDENTIFICATION OF CLOSTRIDIUM SPECIES Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 14 of 14 BSOP ID 8i3 This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency www.evaluations-standards.org.uk Email: [email protected]