Listeria – Identification and Subtyping

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Background
This page provides identification and subtyping information for Listeria cultured isolates at Public Health Ontario (PHO) submitted from clinical microbiology laboratories.

For susceptibility testing of cultured isolates, refer to the following PHO webpage:

Antimicrobial Susceptibility Testing – Aerobic Bacteria

For primary testing of Listeria from food and environmental products, refer to the following PHO webpage:

Food Testing

Important: PHO does NOT provide primary testing for Listeria on clinical specimens, such as blood or cerebrospinal fluid (CSF). Patients suspected of listeriosis should have primary testing performed by their community or hospital laboratory. Additional guidance is available on our Listeriosis Clinical Testing Information webpage.

Update
As of July 12, 2024, this webpage was updated to include additional background information previously found in the 2019 Labstract titled “Listeria monocytogenes – Clinical and Food Testing Guidelines for Foodborne Illness Outbreaks”.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 27 juill. 2024.

Testing Indications

All positive isolates of Listeria (including Listeria monocytogenes) should be forwarded from clinical microbiology laboratories to PHO for further identification and subtyping. Accurate identification of Listeria species and subtyping of Listeria monocytogenes isolates are necessary to ensure appropriate clinical and public health management.

Acceptance/Rejection Criteria

Specimens listed below will be rejected

Primary specimens (e.g. blood, CSF), or
Mixed or non-viable cultured isolate, or
Mislabelled or un-labelled cultured isolate.

Specimen Collection and Handling

Specimen Requirements

Test Requested

Required Requisition(s)

Specimen Type

Minimum Volume

Collection Kit

Listeria identification and/or subtyping and/or surveillance

Reference Bacteriology Requisition

Pure viable cultured isolate of Listeria

N/A

Solid blood agar medium (or any non-selective solid agar that supports organism growth)

Semi-solid Amies charcoal transport medium swab

Submission and Collection Notes

Only clinical microbiology laboratories may submit a cultured isolate following primary isolation. PHO’s laboratory does not provide collection kits for primary isolation nor performs primary testing for Listeria from clinical specimens. For clinical testing recommendations, follow the information page here: Listeriosis Clinical Testing Information.

Submit a fresh, pure, and viable subculture of the Listeria bacterial colony on an appropriate non-selective medium that supports the growth of the organism (e.g. blood agar). A swab of the pure subculture in bacterial transport medium is an acceptable alternative, however turnaround time will be delayed if a swab is received due to the need for plating.

Submit only one cultured isolate from the same primary specimen source of isolation per patient. If the organism was isolated from multiple specimen sources, the order of preference of specimen submission is CSF first, otherwise blood, otherwise any other sterile site.

Complete all fields of the Reference Bacteriology Requisition.

Label the culture container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. Failure to provide this information may result in rejection or testing delay.

If the patient is part of a cluster/outbreak investigation, contact PHO’s laboratory Customer Service at 416-235-6556/1-877-604-4567 prior to sample submission.

Storage and Transport

Place the sealed culture in a biohazard bag and properly seal bag. Cultured isolates must be stored at 2-8°C if they cannot be shipped to PHO’s laboratory on the same day that the incubation is completed. Refrigerated (2-8°C) cultured isolates must be shipped to PHO’s laboratory on ice packs within 2 days. All cultured isolates must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Listeria cultured isolates are identified daily on weekdays (Monday to Friday) at PHO’s Toronto laboratory site. Turnaround time for identification results is up to 4 days from receipt at PHO’s laboratory.

Preliminary Listeria monocytogenes subtyping is performed daily on weekdays (Monday to Friday) at PHO’s Toronto laboratory site, and confirmatory subtyping is performed weekly at the National Microbiology Laboratory (NML) in Winnipeg. Turnaround time for subtyping is up to 15 days following organism identification at PHO’s laboratory. Note: subtyping results are not provided to submitters and only used for internal surveillance purposes.

STAT and Critical Specimens Testing

Priority testing for cluster/outbreak investigations is available upon request. If needed, contact PHO’s laboratory Customer Service at 416-235-6556/1-877-604-4567 prior to sample submission.

Test Methods

Listeria identification of cultured isolates is performed by colony morphology, Gram stain morphology, catalase test, oxidase test, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), umbrella motility at 30°C, beta-hemolysis on blood agar, CAMP test, esculin hydrolysis, methyl red and Voges-Proskauer test, triple sugar iron test, dextrose fermentation, rhamnose fermentation, and xylose fermentation.^1,2

Listeria monocytogenes subtyping is performed by whole genome sequencing (WGS). Sequence analysis is performed using standardized PulseNet Canada pipelines and compared to both provincial databases (preliminary or ‘fast match’ result) and national databases (confirmatory result) to identify closest isolate matches by whole genome multilocus sequence typing (wgMLST). Following confirmatory results by NML, a cluster number may be assigned to the isolate if it is found to be genetically related to other isolates within the same cluster.³

Algorithm

Cultured isolates submitted by microbiology laboratories are first identified at PHO’s laboratory using the identification methods described above. Following identification, any first positive patient isolate of Listeria monocytogenes per body site (e.g. blood or CSF) is automatically reflexed for subtyping at PHO and NML in parallel. Repeat positive isolates from the same patient and body site does not routinely undergo subtyping, but may be considered if positive isolates are recovered from different sterile body sites.

Interpretation

Results Interpretation:

Aerobic Culture Test Result

Interpretation

Isolate identified as:
Listeria monocytogenes

Identification of the isolate is confirmed to be Listeria monocytogenes. If this is the first positive isolate from the patient (per body site), it will be automatically reflexed to subtyping for surveillance purposes.

Isolate identified as:

Listeria innocua, Listeria ivanovii, or other Listeria species

Listeria species other than Listeria monocytogenes are mostly considered non-pathogenic in humans. Clinical correlation is required. Subtyping will not be performed.

Cultured isolates received unviable or mixed will be reported as such without further identification or subtyping results.

Reporting

Results of cultured isolate identification are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18), and/or submitter as indicated on the requisition.

Isolates confirmed as Listeria monocytogenes are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

At this time, results of Listeria monocytogenes subtyping are not reported to the submitter and are only used internally by PHO and Public Health Agency of Canada (PHAC) epidemiologists through PulseNet Canada for surveillance purposes. When isolates are identified as potentially related by subtyping, PHO collaborates with local health units and, where applicable, the Outbreak Investigation Coordination Committee (OICC) partners to corroborate epidemiological evidence to the subtyping results.

References

CLSI. Abbreviated Identification of Bacteria and Yeast; Approved Guideline—Second Edition. CLSI document M35-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
Kenneth P. Smith, and Rebecca M. Harris, 2023. Listeria and Erysipelothrix, In: Carroll KC, Pfaller MA Manual of Clinical Microbiology, 13th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.MCM.ch27
Public Health Agency of Canada. PulseNet Canada. 2022 Jun. Available online at: https://www.canada.ca/en/public-health/programs/pulsenet-canada.html