Enteric Bacteria (including Salmonella, Shigella, STEC, Plesiomonas, Yersinia, Aeromonas, Vibrio, or Campylobacter) – Culture and PCR

Testing Indications

At PHO, primary testing of clinical specimens for enteric bacteria is restricted to local health unit requests for case management and cluster/outbreak investigations ONLY.

Acceptance/Rejection Criteria

• Specimens received from submitters other than local health units will be rejected if received.
• Non-stool specimens (e.g. rectal swabs, intestinal washing content) will also be rejected if received.
• Limit specimen collection from ten symptomatic persons at the onset of the outbreak, when the causative agent is still unknown.

Storage and Transport

Place the sealed specimen container in a biohazard bag and properly seal the bag. Specimens should be stored at 2-8°C and shipped to PHO on ice packs within 48 hours of collection. If specimens might not be able to arrive at PHO within 48 hours of collection, they should be stored frozen (- 20°C or lower) and shipped on ice pack as soon as possible. All specimens 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:

1. 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.
2. 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)]”.
3. 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.

Test Frequency and Turnaround Time (TAT)

Fecal specimen testing for Salmonella, Shigella, STEC, Plesiomonas, Yersinia, Aeromonas, Vibrio, or Campylobacter is set up daily on weekdays (Monday to Friday) at PHO’s laboratory, Toronto location. Turnaround time for detection is up to 6 days from receipt date, and if requested, turnaround time for susceptibility results is up to 8 days from receipt date. Where applicable, serotype identification results are up to 21 days from receipt date.

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, Yersinia, Vibrio, and Campylobacter is performed as needed at the National Microbiology Laboratory (NML) in Winnipeg. Turnaround time for cluster subtyping is up to 15 days for outbreak isolates and 21 days for routine isolates from receipt at PHO. 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 at 416-235-6556/1-877-604-4567 prior to sample submission.

Fecal specimen testing for Salmonella, Shigella, Plesiomonas, Yersinia, Aeromonas, Vibrio, and Campylobacter is performed by culture. Fecal specimen testing for STEC is performed by laboratory-developed stx1 and stx2 gene PCR testing from the primary specimen as well as culture.¹

If growth is observed, isolate identification is confirmed using 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.

Susceptibility testing is performed by minimal inhibitory concentration (MIC) measurement following the Clinical and Laboratory Standards Institute (CLSI) M100 or M45.^2,3

Salmonella serovar typing is mainly performed by whole genome sequencing (WGS) and Salmonella in Silico Typing Resource (SISTR) prediction.⁴ STEC serotyping is mainly performed by WGS E. coli Typer (ECTyper) prediction.⁵ Shigella, Yersinia, and Vibrio cholerae serotyping is performed by traditional antisera agglutination.

Salmonella, Shigella, STEC, and V. cholerae 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.⁶

Algorithm

If no suspected organism is requested: Routine testing will include Salmonella, Shigella, STEC, Yersinia, and Campylobacter. Additional testing for Plesiomonas, Aeromonas, and Vibrio are only offered upon request or for enteric outbreaks of unknown etiology.

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

If Salmonella typhi, S. paratyphi, or Shigella is isolated: Unless specific agents are requested, antimicrobial agents routinely tested include ampicillin, fluoroquinolones, trimethoprim-sulfamethoxazole, and azithromycin by agar plate dilution, and if resistant, ceftriaxone and carbapenems are further tested. Resistance to three or more 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).

If Vibrio cholerae isisolated: Unless specific agents are requested, antimicrobial agents routinely tested include ampicillin and trimethoprim-sulfamethoxazole by broth microdilution.  Additional susceptibility testing for these organisms may be offered for select agents if requested.

If other organisms are isolated: Additional susceptibility testing for these organisms may be offered for select agents if the patient is under 1 year old, has impaired immunity, is hospitalized, or if requested. Additional cluster subtyping for outbreak investigations may also be offered if requested.

Interpretation

If no organism is isolated: The list of organisms tested will be resulted as not isolated.

If an organism is isolated: 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, Yersinia, and Vibrio cholerae. For isolated STEC cases, stx1 and/or stx2 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.

For organisms theoretically known to harbour an inducible class C ESBL mechanism (e.g. Yersinia enterocolitica, Yersinia mollaretii, Yersinia ruckeri, Edwardsiella tarda, Aeromonas), 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.”⁷

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, Shigella, STEC,pathogenic Yersinia, Vibrio cholerae, and Campylobacter are reported to the local Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

Currently, routine results of Salmonella, Shigella, STEC, and Vibrio cholerae 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

1. 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.
2. Clinical & Laboratory Standards Institute. M100 Performance Standards for Antimicrobial Susceptibility Testing. Wayne, PA.
3. Clinical & Laboratory Standards Institute. M45 Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria. Wayne, PA.
4. 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.
5. 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.
6. Public Health Agency of Canada. PulseNet Canada. 2022 Jun. Available online at: https://www.canada.ca/en/public-health/programs/pulsenet-canada.html
7. Jacoby GA. AmpC β-lactamases. Clin Microbiol Rev. 2009;22(1):161–82.