ESBL - Extended Spectrum Beta-Lactamase Confirmation

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Background
This page provides information for Extended Spectrum Beta-Lactamase (ESBL) production confirmation testing on appropriate isolates using conventional methods at Public Health Ontario’s (PHO) laboratory. For information regarding other testing options, refer to the following PHO webpages:

Antimicrobial Susceptibility Testing – Aerobic Bacteria

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Testing Indications

ESBL confirmation testing is performed only on Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis. Resistance to the third generation of cephalosporins may indicate ESBL production.

Acceptance/Rejection Criteria

Primary specimens are not acceptable. Only pure cultures are accepted for testing.
Mixed or non-viable cultures will not be tested. A written report will be issued to indicate that the test has been rejected.
Mislabelled or unlabelled cultures will not be tested. A report will be sent to the submitting laboratory stating “Culture received un-labelled” or “Requisition identification does not match the identification information on the culture received. Please resubmit if required.”
The submitting laboratory will be contacted when isolates derived from critical sites” (e.g. brain abscess) are submitted inappropriately (e.g. improper transport conditions or mislabeled/unlabeled).

Specimen Collection and Handling

Specimen Requirements

Test Requested

Required Requisition(s)

Specimen Type

Minimum Volume

Collection Kit

Extended Spectrum beta-lactamase confirmation

Reference Bacteriology Requisition

Pure viable subculture of 24 – 48 hours growth of organism on an appropriate medium (Columbia sheep blood agar plate)

A swab of a pure subculture of the organism in Amies transport medium

N/A

Submission and Collection Notes

Complete all fields on the Reference Bacteriology Requisition, including:

Test(s) requested
Culture information
Clinical/epidemiology
Date of primary specimen collection
Primary source of isolation (Mandatory)
Submitter’s Contact information

Primary specimens are unacceptable and will be rejected. The specimens should be processed in the originating lab and only the suspect isolate sent.

Label the culture container(s) with the patient’s first and last name, date of collection of the specimen source, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

If a swab or broth is received, the turnaround time will be delayed by at least 24 hours.

Limitations

When the identification of bacteria from clinical specimens is unknown, it can be difficult to determine if ESBL testing is required since testing is only limited to four species. In such cases PHO’s laboratory will first perform identification and then decide whether ESBL testing is indicated.
Organisms that have hyper production of non-ESBL Beta-Lactamases combined with altered permeability may produce false-positive results on phenotypic ESBL tests.

Storage and Transport

Specimens should be stored at 2-8°C following collection and shipped to PHO’s laboratory within 48 hours of isolation.

Place culture in a biohazard bag and seal. Store at 2-8°C while awaiting shipping.

All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

ESBL confirmation testing is performed daily at PHO’s laboratory, Toronto site.

Turnaround time is up to 3 business days from receipt at the PHO’s laboratory.

Test Methods

Isolates are confirmed for ESBL by using a phenotypic method (disk diffusion clavulanate inhibition test) recommended by Clinical and Laboratory Standards Institute (CLSI).¹

Interpretation

A ≥ 5 mm increase in a zone diameter for ceftazidime and/or cefoxitime tested in combination with clavulanic acid compared to the zone diameter when tested alone = ESBL

A < 5 mm increase in a zone diameter for ceftazidime and/or cefoxitime tested in combination with clavulanic acid compared to the zone diameter when tested alone = Not ESBL

Scenario	Zone size of	Minus zone size of	≥ 5 mm?	Result Interpretation
1	ceftazidime- clavulanic acid cefoxitime-clavulanic acid	ceftazidime cefoxitime	Yes No	ESBL
2	ceftazidime- clavulanic acid cefoxitime-clavulanic acid	ceftazidime cefoxitime	Yes Yes	ESBL
3	ceftazidime- clavulanic acid cefoxitime-clavulanic acid	ceftazidime cefoxitime	No Yes	ESBL
4	ceftazidime- clavulanic acid cefoxitime-clavulanic acid	ceftazidime cefoxitime	No No	Not ESBL

Reporting

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

References

CLSI Performance Standards for Antimicrobial Susceptibility Testing 34th Edition. M100.