Enterobacterales – Isolate Identification, Susceptibility, and Typing

Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.

Background
This page provides identification, susceptibility, and typing information from Enterobacterales cultured isolates at Public Health Ontario (PHO) submitted by clinical microbiology laboratories. The order Enterobacterales includes a wide range of organisms which are traditionally glucose fermenting, facultatively anaerobic Gram-negative bacilli (e.g. Enterobacteriaceae, Erwiniaceae, Pectobacteriaceae, Yersiniaceae, Hafniaceae, Morganellaceae, Budviciaceae, Thorselliaceae, Gallaecimonadaceae, and Bruguierivoracaceae).

For primary testing of Enterobacterales from fecal specimens, refer to the following PHO webpage: Enteric Bacteria (including Salmonella, Shigella, STEC, Plesiomonas, Yersinia, Aeromonas, Vibrio, or Campylobacter) – Culture and PCR

For confirmation of ESBL or CPE only, refer to the following PHO webpages:

Updates

As of March 26, 2025, the previous page called “Enteric – Bacteria – Confirmatory Testing” has been separated into Enterobacterales (this page), Aeromonadales, Vibrionales, and Campylobacterales. Serotyping methodology for Salmonella and STEC has also been modified and is now predicted from whole genome sequencing (WGS) as opposed to traditional typing. This page was also updated to include specific information on organisms tested, routine submission of isolates for surveillance, requisition requirements, requests for additional susceptibility testing, turnaround time for subtyping, methods used, and interpretations including inducible resistance.

*Uncontrolled print copy. Valid only on day of print: 01 May 2025.

Testing Indications

Medical microbiology laboratories unable to confirm identification of Enterobacterales isolates locally may submit their isolate to PHO for further identification. Laboratories may also submit isolates for susceptibility testing if clinically needed and may submit isolates for cluster subtyping if needed for outbreak investigations (e.g., Serratia neonatal unit outbreaks).

Virulence confirmation for Shiga toxin-producing E. coli (STEC) and hypervirulent Klebsiella pneumoniae (hvKp) are available upon request. Virulence confirmation for other non-STEC E. coli intestinal pathotypes (e.g. EPEC, EIEC, ETEC, EAEC) is only available for outbreak investigations after PHO preapproval. Similarly, virulence confirmation for K1 extraintestinal pathogenic E. coli is only available after PHO preapproval.

In addition, all laboratories in Ontario should routinely send the following culture isolates to PHO for surveillance and outbreak investigations: Salmonella, STEC, Shigella, Yersinia, and Cronobacter. Accurate identification and typing of these organisms are necessary to ensure appropriate clinical and public health management.

Acceptance/Rejection Criteria

Primary specimens, mixed cultures, non-viable cultures, cultures in inappropriate medium, mislabelled cultures, unlabelled cultures, and requisitions with missing information (e.g. primary source of isolation) will be rejected if received.
Requests for EPEC, EIEC, ETEC, and EAEC confirmation without PHO preapproval will also be rejected if received.

Specimen Collection and Handling

Specimen Requirements

Test Requested

Required Requisition(s)

Specimen Type

Minimum Volume

Collection Kit

Identification, confirmation, susceptibility, and/or typing (specify organism)

Reference Bacteriology Requisition

Pure viable cultured isolate of organism

N/A

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

Semi-solid Amies charcoal transport medium swab

Submission and Collection Notes

Only clinical microbiology laboratories can submit a cultured isolate for testing.

Complete all fields of the requisition form.

Clearly identify on the requisition the organism submitted, including the level of identification so far obtained and biochemical characteristics assessed.

Clearly indicate if Shiga toxin production is suspected for submitted E. coli isolates (e.g. local stx toxin or O157 positive result).

Clearly indicate if hypervirulence is suspected for submitted K. pneumoniae isolates (e.g. compatible clinical picture).

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 source is sterile site, otherwise non-sterile site.

Turnaround time will be delayed if an isolate swab is received due to the need for plating.

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 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 on the same day that the fresh incubation is completed. Refrigerated (2-8°C) cultured isolates must be shipped to PHO on ice packs within 2 days. All cultured isolates must be shipped in accordance to the Transportation of Dangerous Good Act.

Special Instructions

If susceptibility testing is requested, clearly indicate which antibiotic(s) are of interest.

To request additional antimicrobial agents for susceptibility testing after susceptibility testing was already finalized by PHO:

Determine if and when the susceptibility testing report was finalized at PHO (“Final” written on the report). Additional susceptibility requests on previously tested isolates are only accepted if the request is received within 5 days of the final report.
Fill out a new requisition form and indicate in the “Submitter lab no.” field the lab number provided by PHO instead. Under Test(s) Requested section, select “antimicrobial susceptibility” and in “other – (specify)”, write “Additional AST for [name of drug(s)]”.
Call PHO Laboratory Customer Service at 416-235-6556/1-877-604-4567 and ask for the appropriate number to fax the completed requisition with the indicated additional susceptibility agents requested.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Enterobacterales cultured isolates identification and/or susceptibility testing is set up daily on weekdays (Monday to Friday) at PHO’s laboratory, Toronto location. If requested, turnaround time for genus or species identification results is up to 4 days from receipt date, and turnaround time for susceptibility results is up to 8 days from receipt date. Where applicable, serotype identification results is up to 21 days from receipt date.

STEC confirmation is performed daily on weekdays upon request at PHO’s laboratory, Toronto location. Turnaround time is up to 6 days from receipt at PHO. Virulence confirmation for hvKp and other non-STEC E. coli pathotypes is performed upon request at the National Microbiology Laboratory (NML) in Winnipeg. Turnaround time for results is up to 15 days from receipt at PHO.

Preliminary cluster subtyping for Salmonella and STEC outbreak detection is performed daily on weekdays (Monday to Friday) at PHO’s laboratory, Toronto location. Cluster subtyping for Shigella is performed weekly at the NML. Cluster subtyping for Serratia and other Enterobacterales is performed as needed at the NML. Turnaround time for cluster subtyping is up to 15 days for outbreak isolates and 21 days for routine isolates from receipt at PHO’s laboratory. Note: unless requested, cluster 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 Laboratory Customer Service as 416-235-6556/1-877-604-4567 prior to sample submission.

Test Methods

Enterobacterales identification of cultured isolates is based on colony morphology, Gram stain morphology, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, Bruker RUO systems), and where applicable, a wide range of biochemical and antisera agglutination reactions. Results may be further investigated by 16S targeted Sanger sequencing where applicable.

If STECis mentioned on the requisition, cultured isolates confirmed to be E. coli will be further tested by laboratory-developed stx1 and stx2 gene PCR testing on the isolate at PHO.¹ If hvKp is mentioned on the requisition, cultured isolates confirmed to be K. pneumoniae will be further tested by laboratory-developed K1, K2, K5, rmpA1/rmpA2, iucA, iroB, ybtA, and clbA PCR testing on the isolate at NML.² If other non-STEC E. coli pathotypes are mentioned on the requisition and testing was pre-approved by PHO, cultured isolates confirmed to be E. coli will be further tested by laboratory-developed PCR testing on the isolate at NML.^3,4

Susceptibility testing is performed by minimal inhibitory concentration (MIC) measurement following the Clinical and Laboratory Standards Institute (CLSI) M100.⁵

Salmonella serovar typing is mainly performed by whole genome sequencing (WGS) and Salmonella In Silico Typing Resource (SISTR) prediction.⁶ STEC serotyping is mainly performed by WGS E. coli Typer (ECTyper) prediction.⁷ Shigella and Yersinia serotyping is performed by traditional antisera agglutination.

Salmonella, STEC, and Shigella cluster subtyping for outbreak detection is performed by WGS following PulseNet Canada protocols, comparing 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.⁸

Algorithm

If identification or confirmation is requested: Cultured isolates will be identified at PHO’s laboratory using the identification methods described above.

If Salmonella, Shigella or STEC is isolated: Routine cluster subtyping for surveillance is automatically performed for all first positive isolates per patient (per body site).

If susceptibility testing is requested: When the identification provided on the requisition is sufficient to conduct testing, cultured isolates are tested for susceptibility without confirmation of identification. If the identification provided is insufficient, the submitted isolate is first identified at PHO using the identification methods described above. Unless specific agents are requested, antimicrobial agents routinely tested for Enterobacterales (with the exception of Salmonella and Shigella) include ampicillin, cefazolin, ceftriaxone, amoxicillin-clavulanate, piperacillin-tazobactam, ciprofloxacin, levofloxacin, gentamicin, and trimethoprim-sulfamethoxazole by agar plate dilution. For Salmonella and Shigella, routine agents include ampicillin, fluoroquinolones, trimethoprim-sulfamethoxazole, and azithromycin by agar plate dilution, and if resistant, ceftriaxone and carbapenems are further tested. Resistance to three or four of these antimicrobial classes will be labelled as multidrug resistant (MDR), and resistance to five or more of these antimicrobial classes will be labelled as extensively drug resistant (XDR). Additional susceptibility testing for these organisms may be offered for select agents if requested.

Interpretation

If identification, confirmation, or typing is requested: Identification will be confirmed to the species level if feasible, otherwise the taxonomic classification most closely aligned to the organism will be reported. Additional serotype result will be provided for Salmonella, STEC, Shigella, and Yersinia. For isolated STEC cases, stx1 and/or stx2 gene status will also be reported. For other E. coli pathotypes or hvKp cases, virulence gene status will also be reported.

If susceptibility testing is performed: Results will be provided for each antimicrobial agent tested as either “susceptible”, “susceptible dose-dependent”, “intermediate”, or “resistant” according to the applicable CLSI clinical breakpoint for systemic therapy use. MIC values may be provided upon request or if there are no applicable CLSI clinical breakpoints. If the identification provided on the requisition was sufficient, the following will be stated: “Antimicrobial susceptibility testing results and interpretations are based on the submitter’s identification. Identification was not requested and was not performed at PHO.”

For organisms known to harbour an inducible class A or class C ESBL mechanism (more established with Enterobacter species, Citrobacter freundii complex, Klebsiella aerogenes, Hafnia species, and to a lesser extent Morganella, Providencia, Serratia, Cronobacter, Buttiauxella, Cedecea, Edwardsiella tarda, Erwinia rhapontici, Ewingella americana, Yersinia enterocolitica, Yersinia mollaretii, Yersinia ruckeri, Proteus penneri, Proteus vulgaris), the following statement will be added: “Resistance to extended-spectrum penicillins, beta-lactam/beta-lactamase inhibitor combinations, cephalosporins, and aztreonam may develop during therapy with these agents.”^9,10

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.

Isolates confirmed as Salmonella, STEC, Shigella, pathogenic Yersinia, or CPE are reported to the local Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

At this time, routine results of Salmonella, STEC, and Shigella cluster 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

Chui L, Couturier MR, Chiu T, Wang G, Olson AB, McDonald RR, Antonishyn NA, Horsman G, Gilmour MW. Comparison of Shiga toxin-producing Escherichia coli detection methods using clinical stool samples. J Mol Diagn. 2010 Jul;12(4):469-75. doi: 10.2353/jmoldx.2010.090221. Epub 2010 May 13. PMID: 20466837; PMCID: PMC2893631.
National Microbiology Laboratory. Klebsiella pneumoniae Hypervirulence. Guide to Services. Available online at: https://cnphi.canada.ca/gts/reference-diagnostic-test/11316
National Microbiology Laboratory. E. coli Toxin Detection. Guide to Services. Available online at: https://cnphi.canada.ca/gts/reference-diagnostic-test/4566
National Microbiology Laboratory. E. coli K1 Virulence Gene Detection. Guide to Services. Available online at: https://cnphi.canada.ca/gts/reference-diagnostic-test/14520
Clinical & Laboratory Standards Institute. M100 Performance Standards for Antimicrobial Susceptibility Testing. Wayne, PA.
Yoshida CE, Kruczkiewicz P, Laing CR, Lingohr EJ, Gannon VP, Nash JH, Taboada EN. The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies. PLoS One. 2016 Jan 22;11(1):e0147101. doi: 10.1371/journal.pone.0147101. PMID: 26800248; PMCID: PMC4723315.
Bessonov K, Laing C, Robertson J, Yong I, Ziebell K, Gannon VPJ, Nichani A, Arya G, Nash JHE, Christianson S. ECTyper: in silico Escherichia coli serotype and species prediction from raw and assembled whole-genome sequence data. Microb Genom. 2021 Dec;7(12):000728. doi: 10.1099/mgen.0.000728. PMID: 34860150; PMCID: PMC8767331.
Public Health Agency of Canada. PulseNet Canada. 2022 Jun. Available online at: https://www.canada.ca/en/public-health/programs/pulsenet-canada.html
Tamma PD, Heil EL, Justo JA, Mathers AJ, Satlin MJ, Bonomo RA. Infectious Diseases Society of America 2024 Guidance on the Treatment of Antimicrobial-Resistant Gram-Negative Infections. Clin Infect Dis. 2024 Aug 7:ciae403. doi: 10.1093/cid/ciae403. Epub ahead of print. PMID: 39108079.
Jacoby GA. AmpC β-lactamases. Clin Microbiol Rev. 2009;22(1):161–82.