Consensus Date: 29 March 2010
1 PHLN summary laboratory definition
1.1.1 Definitive Criteria
- Isolation of Listeria species from normally sterile sites OR
- Isolation of Listeria species from faeces
1.1.2 Suggestive Criteria
- Isolation of Gram positive bacilli from normally sterile site, demonstrating tumbling motility at room temperature
1.1.3 Special Considerations
- Isolation of Listeria species from a non-sterile body surface are not necessarily indicative of invasive infection, but may be used to prompt further definitive investigations such as blood culture, if clinically indicated
1.1.4 Links to related documents
- CDNA (clinical) case definition for listeriosis: http://www.health.gov.au/internet/main/publishing.nsf/Content/cda-surveil-nndss-casedefs-cd_listera.htm
- Fact Sheet on Listeria: http://www.health.gov.au/internet/main/publishing.nsf/Content/ageing-clinicians-type.htm/$File/Listeria.pdf
2 IntroductionThe genus Listeria contains a number of distinct species; however, only two members of the genus are regarded as pathogenic to humans, namely Listeria monocytogenes and Listeria ivanovii. Listeria monocytogenes has risen to prominence in recent years with frequent isolation from a wide range of foods. There is direct causal evidence of infection following consumption of food heavily contaminated by this species. L.ivanovii causes rare infections. Disease attributed to L.innocua has been reported.
L. monocytogenes is a risk to pregnant mothers, the very young and the aged, and patients on immunosuppressive therapy. Given the widespread distribution of this organism in food, education targeted at these risk groups has been used as the most effective means to decrease the risk of infection.
L. monocytogenes causes a transient flu-like illness. Invasion of the blood stream can lead to bacteraemia and/or meningitis, and in the case of pregnant women, the organism can cross the placental barrier and infect the unborn child, resulting in premature abortion and/or stillbirth. In rare cases, it can cause symptoms typical of food poisoning (vomiting and diarrhoea) some 19 hours after consumption of contaminated food.
Though the genus Listeria has not changed since the first edition of this Laboratory Case Definition, developments in molecular biology have led to improvements in molecular laboratory methods including hybridisation assays, PCR assays and molecular typing. The availability of a complete, annotated genome for both the pathogenic and some of the non-pathogenic Listeria species is likely to result in a range of new molecular methods for Listeria detection and characterisation.
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3.1 Culture for Listeria speciesListeria species are small, facultative anaerobic, non-spore forming, Gram positive bacilli, that grow readily on blood agar. In addition, a number of selective agars have been developed for use in food microbiology. On blood agar L monocytogenes produces incomplete β-haemolysis.
3.1.1 MediaFor optimal isolation of Listeria species, direct plating onto blood agar is recommended. This may be
direct from clinical specimens or from subcultures of blood culture medium. In addition, when foodborne cases are being investigated, culture from faeces is often useful. In such cases, selective media such as Oxford agar or PALCAM are recommended, as well as enrichment in a Listeria selective broth.
3.1.2 Suitable specimens:
- Blood – Blood samples should be inoculated immediately into blood culture bottles. These may be stored at room temperature during transport to the laboratory and subsequently incubated at 37oC.
- Brain/Abscess/foetal material swabs – Swabs should be placed in an appropriate transport medium and transported to the laboratory quickly.
- Faeces/rectal swab – Faeces collected in an appropriate sterile container, transported to the laboratory ASAP (if longer than 2h keep at 4°C), may be stored at 4° C for 24 hours before culture. Rectal swabs may be put into a tube of transport medium containing modified Stuart's medium, transported to the laboratory ASAP. May be stored at RT for 24 hours before culture.
3.1.3 Test sensitivityNo mathematical data available.
Depends on the quality and type of the specimen, the type of media chosen, and whether enrichment is used prior to direct plating. Antibiotic treatment may inhibit the growth of Listeria species.
3.1.4 Test specificityNo mathematical data available.
The isolation of a Listeria-like organism from a normally sterile site, confirmed by specific biochemical and phenotypic techniques. The isolation of Listeria monocytogenes is notifiable in most States of Australia.
3.1.5 Predictive valuesA negative blood or other culture does not exclude the diagnosis of listeriosis.
3.1.6 Suitable acceptance criteriaOn Blood agar, small β-haemolytic colonies which demonstrate characteristic tumbling motility at room temperature (optimal at 25oC) in broth cultures and which biochemically fits an identification of L. monocytogenes. L. ivanovii can be distinguished by its distinctive CAMP test reaction and different biochemical reactions.
3.1.7 Suitable internal controlsProperly documented, relevant, quality control program for each type and batch of medium used.
3.1.8 Suitable test validation criteriaIsolation of L. monocytogenes, confirmed by biochemical and other phenotypic parameters, is the reference standard.
3.1.9 Suitable external QC programmeRoyal College of Pathologists of Australia Quality Assurance Programs Pty. Ltd. (RCPA)
3.1.10 Special considerationsL. monocytogenes can be differentiated from L. ivanovii by the CAMP test. It is important that laboratories correctly identify the species present in a specimen.
3.2 Identification of Listeria speciesThere are two levels of action in the identification of Listeria sp.:
- A. Diagnostic laboratories – Most diagnostic laboratories should be capable of biochemically confirming the identity of Listeria monocytogenes.
B. Reference laboratories – Isolates, particularly where a cluster is involved, should be forwarded to a State reference laboratory for serotyping, molecular characterisation and molecular comparison with food isolates.
3.2.1 Conventional biochemical tests
188.8.131.52 Suitable specimenA pure culture on solid medium.
184.108.40.206 MediaVarious specific biochemical substrates
220.127.116.11 Test sensitivityThis depends on the biochemical tests performed. The demonstration of tumbling motility at 25oC is usually a reliable characteristic for presumptive recognition of a Listeria species. Many laboratories also use Aesculin hydrolysis as an additional screening test. Sugar fermentation tests may exhibit variable reactions. A number of commercial strip tests are available.
18.104.22.168 Test specificityNo mathematical data available. The CAMP test and acid formation from specific sugars are used to differentiate between isolates of L.monocytogenes and L. ivanovii.
22.214.171.124 Predictive valuesA negative result in one test does not preclude L. monocytogenes. Further tests may be necessary to confirm the identification.
126.96.36.199 Suitable test criteriaAn isolate that exhibits biochemical characteristics consistent with documented reactions for a Listeria species. The major distinguishing feature of L.monocytogenes is fermentation of Rhamnose, lack of fermentation of Xylose and hydrolysis of aesculin.
188.8.131.52 Suitable internal controlsEach batch of biochemical substrate tested with positive and negative control strains. Results of all testing recorded and the records maintained.
184.108.40.206 Suitable validation criteriaCorrect biochemical reactions exhibited by a standard Listeria strain.
220.127.116.11 Suitable external QC programRCPA Quality Assurance Programs Pty. Ltd.
3.2.2 Kits/automated systems for biochemical identificationVarious kits and automated machines exist for the identification of Listeria species. Of the numerous kits available in Australia, the API Listeria and the MICRO-ID Listeria are most commonly used. MicroScan (Baxter, Baxter Healthcare Corporation, West Sacramento, USA), Vitek (bioMerieux, Marcy- l'Etoile, France), MicroStation (Biolog, Heyward, USA) and Cobas (Becton Dickinson Instrument Systems, Sparks, USA) are the major systems in use.
The information stated in sections 18.104.22.168 - 22.214.171.124 applies to both kits and automated identification systems.
126.96.36.199 Special considerationsFor accurate results, follow the manufacturer’s procedure.
3.2.3 Molecular identificationMolecular probes have been developed for the detection and identification of L.monocytogenes, particularly for detection in food samples. However, to date molecular probes are only in use in some reference laboratories and have not been made commercially available for use by clinical laboratories.
3.3 Serotyping of L. monocytogenesAlthough there are at least 13 serotypes of L.monocytogenes, almost all disease is caused by serotypes 4b, 1/2a and 1/2b. Serotyping of Listeria isolates therefore has limited epidemiological value.
3.3.1 Suitable specimenPure bacterial culture.
3.3.2 MaterialsL.monocytogenes is subdivided into 13 serovars based on cellular O and flagellar H antigens; 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e and 7. Serotyping is usually confirmed in reference laboratories with stocks of type-specific antisera.
3.3.3 Test sensitivityUnless an isolate is a rough colony variant/phenotype, most L.monocytogenes isolates can be serotyped.
3.3.4 Test specificityNo data available.
3.3.5 Predictive valuesFailure to serotype a strain does not mean it does not have a serotype.
3.3.6 Suitable test acceptance criteriaAgglutination against a type-specific antisera.
3.3.7 Suitable internal controlsRegular serotyping of a range of different, previously characterised L.monocytogenes serotypes.
3.3.8 Suitable validation criteriaUnequivocal demonstration of a recognised serotype.
3.3.9 Suitable External QC programNone
3.4 Further subtypingFurther typing for epidemiological purposes is available in a small number of reference laboratories. Their methods include molecular serotyping, binary molecular typing, MLVA, MLST, Ribotyping and DNA macrorestriction (often known as “PFGE”). The methods chosen for use in specific jurisdictional laboratories differ according to public health operational priorities but can be regarded as complementary. In the event of multi-jurisdictional laboratory-based investigations, isolates or DNA extracts can be exchanged between reference laboratories within the PHLN. L.monocytogenes isolates should be forwarded to the nearest reference laboratory for confirmation, further analysis and storage.
3.5 SerodiagnosisSerology is unreliable and is not recommended.
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4 Agreed typing & subtyping methodsA standard nomenclature has yet to be agreed. Other than for binary genotyping systems such as MLST, where the naming is strictly arbitrary, genotyping methods are prone to multiple sources of variation. Even when binary genotyping systems are used, there may still be an implicit bias in genotype data interpretation by the specific mathematic algorithm adopted by interpretive software. The performance of specific Listeria genotyping methods should be discussed with the public health laboratory performing the analysis. The scientific interpretation of genotyping results is best done by the scientists who did the genotyping.
4.1 Laboratory Nomenclature for National Database Dictionary
4.1.1 Organism Name(s) List
- Listeria monocytogenes
- Listeria ivanovii
- Listeria species
4.1.2 Typing/Subtyping Nomenclature List(s)Varies by jurisdiction
5 References1Murray, P.R., Baron, E.J., Jorgensen, J.H., Landry, M.L. and Pfaller, M.A. 2007. Manual of Clinical Microbiology, ASM Press, Washington, D.C. Vol 1. pp 474-484.
2Hocking, A.D, Arnold, G., Jenson, I., Newton, K. and Sutherland, P. (eds.) Foodborne Microorganisms of Public Health Significance. 1997. 5th ed. AIFST (NSW Branch) Food Microbiology Group.
3Glaser, P, Franguel, L., Buchrieser, C. et al. Comparative genomics of Listeria species. Science 2001; 294:849-52.
4McLauchlin J, Jones D. Erysipelothrix and Listeria. in Topley & Wilson's Microbiology and Microbial Infections. eds Collier C, Balows A, Sussman M. vol 2. Systematic Bacteriology. vol. eds Balows A, Duerden B. Arnold, London, 1998. pp 683-708.
5MacFaddin, J.F. Biochemical tests for the identification of medical bacteria. Lippincott, Williams & Wilkins, Philadelphia, 2000.