Antimicrobial Susceptibility Testing – Aerobic Bacteria

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 antimicrobial susceptibility testing information for aerobic bacteria at Public Health Ontario’s (PHO) Laboratory. For information regarding other testing options, refer to the following PHO webpages:

Antimicrobial Susceptibility Testing – Anaerobes

Updates

New instructions added when requesting additional drug(s) for susceptibility testing on an isolate.
Update of the submission and collection notes

*Uncontrolled print copy. Valid only on day of print: 01 May 2024.

Testing Indications

Acceptance/Rejection Criteria

Primary specimens are not acceptable. Only pure cultures are accepted for testing.
If source of isolation of the culture is not indicated in the requisition, it will be rejected.
Mixed or non-viable cultures will not be tested. The submitter will be contacted by telephone and 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

Antimicrobial susceptibility testing- Aerobic bacteria

Reference Bacteriology Requisition

Pure, viable subculture of a 24 – 48 hour growth of organism on appropriate medium (e.g. Columbia sheep blood agar or broth media)

A swab of a pure culture in Amies charcoal transport media for susceptibility testing.

N/A

Submission and Collection Notes

Complete all fields on the Reference Bacteriology Requisition including:

Test(s) requested
Date of primary specimen collection
Primary source of isolation (mandatory)
Culture information (presumptive identification, gram morphology, catalase, oxidase)
Clinical/epidemiology information
The number of consecutive blood cultures positive for the submitted isolate when isolates are from blood cultures
Contact information

Where full bacterial identification and susceptibility testing is required, e.g. for an immunocompromised patient, please clearly indicate this on the requisition.

If a swab of pure culture in Amies charcoal transport medium or pure culture in broth medium are received, the TAT will be delayed by at least 24 hours.

Primary specimens are unacceptable; they should be processed in the originating lab and the isolate sent for testing. A report will be issued to indicate that the test has been rejected if primary specimen is sent.

Do not submit multiple isolates from the same specimen/site of infection - for polymicrobic infections, only submit most clinically important pathogens for testing.

For clinical specimens, 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. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

Limitations

When the identification of bacteria from clinical specimens is unknown, it can be difficult to determine upfront if it is clinically relevant and if susceptibility testing is required for patient care.

In such cases PHO’s laboratory will perform identification. If the microorganism identified represents a common contaminant or commensal flora of the source, an antimicrobial susceptibility testing is not necessary or appropriate.

Storage and Transport

Place the sealed culture in a biohazard bag and properly seal. Store at 2-8°C while awaiting for shipping. Transport a fresh subculture to ensure viability on receipt. It should be shipped to PHO’s laboratory within 48 hours of isolation. All cultures must be shipped in accordance to the Transportation of Dangerous Good Act.

Special Instructions

To request for certain additional drug for antimicrobial susceptibility testing (AST) when isolate submitted had been tested and susceptibility testing report had been received:

Look for the final report of the particular isolate received from PHO’s laboratory. Determine the date when the final report of the isolate previously sent for susceptibility testing was finalized.
Note: Additional drug request for susceptibility testing on a previously tested isolate for susceptibility testing is only accepted by PHO’s laboratory if request is received within 5 days after receiving the final report of it.
Fill out a new requisition form and indicate on submitter lab no. the PHO’s lab number of the isolate that you would want to request additional drug(s) for its susceptibility testing.
On the Test(s) Requested part of the requisition, select antimicrobial susceptibility then write legibly- Additional AST for: [name of drug], [name of drug].
Call PHO’s Laboratory Customer Service Center at 1-877-604-4567 to add any additional antimicrobial drug susceptibility testing and ask the appropriate number to fax the completed requisition

To request specific antimicrobial drug for an isolate to be submitted, indicate the name of the drug on the requisition.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Antimicrobial Susceptibility testing is performed from Monday – Saturday at Public Health Ontario’s laboratory, Toronto site.

TAT is up to 8 days from date of receipt at PHO’s laboratory.

Test Methods

Antimicrobial Susceptibility testing on a bacterial isolate is performed as per current CLSI guidelines.

Testing methods in use are:

Agar dilution
Micro-broth dilution (Sensititre™)
E test (Epsilometer or Exponential Gradient Method)

Algorithm

Consistent with principles of appropriate test utilization, the following will take place for the testing of the bacteria listed in Table A cultured from urine, lower respiratory tract specimens (e.g. sputa), wound/drainage:
- For bacteria not normally considered to be pathogenic, susceptibility testing will not routinely be performed
- For mid-stream urine, urine from in and out catheter or urine from indwelling catheters sources of submitted culture of isolate, only limited identification and no susceptibility testing will be performed on the organisms listed in Table A
For cultures of isolates from blood, the following algorithm will be implemented:
- If the culture is only isolated from one set of blood culture bottles that turns positive within 24 hour time frame, and is identified as any of the organisms listed in Table A, limited identification is performed and no susceptibility testing is set up
- NOTE: The list of organisms in Table A does not apply to neonatal blood cultures or those from individuals identified on the requisition as being immunocompromised
For isolates from critical specimens (e.g. CSFs), surgically collected specimens (e.g. tissues) and other sterile sites specimens (e.g. synovial fluids) full identification and susceptibility testing will continue to be performed.
Other than the scenarios described in Table A, full identification and/or susceptibility testing will continue to be performed on sterile site isolates; for all others, identification and susceptibility testing will be performed only on special request by the healthcare provider based on clinical relevance
Susceptibility testing will be performed on organisms commonly considered to be contaminants/commensal flora only on special request by the healthcare provider based on clinical relevance. Contact PHO’s Laboratory Customer Service Center for these requests.

Table A

Organisms commonly considered to be contaminants/commensal flora for which limited susceptibility testing will be performed (this is not to be considered a complete list)

Source	Organism
Single Positive Blood Culture^{1, 3-6}	Coagulase- negative Staphylococcus andrelated organisms (e.g. Micrococcus spp, Rothia spp), not including S. lugdunensis “Viridans group Streptococci” Corynebacterium spp and related organisms (e.g. Arthrobacter spp), not including C. diphtheriae Bacillus spp and related organisms (e.g. Paenibacillus spp, Lysinibacillus spp etc), not including B. anthracis Neisseria spp other than N. meningitidis or N. gonorrhoeae Propionibacterium sppand Cutibacterium spp
Urine⁸	Lactobacillus spp Viridans group Streptococcus Coagulase- negative Staphylococcus andrelated organisms (e.g. Micrococcus spp, Rothia spp) Corynebacterium spp and related organisms (e.g. Arthrobacter spp), not including C. urealyticum, C. glucoronolyticum and C. pseudogenitalium Bacillus spp and related organisms (e.g. Paenibacillus spp, Lysinibacillus spp etc.), not including B. anthracis Neisseria spp other than N. meningitidis, N. gonorrhoeae
Lower Respiratory Tract Specimens^2,7,9	Viridans group Streptococcus Neisseria sppincluding N. meningitidis (identification only) Coagulase- negative Staphylococcus andrelated organisms (e.g. Micrococcus spp, Rothia spp), not including S. lugdunensis Corynebacterium spp and related organisms (e.g. Arthrobacter spp), not including C. pseudodiphtheriticum and C. diphtheriae Moraxella sppother than M. catarrhalis Enterococcus spp
Wound swab and Drainage³	Coagulase -negative Staphylococcus, unless isolated from chest/sternum Coagulase -negative Staphylococcus, not including S. lugdunensis “Viridans groupStreptococci” Corynebacterium spp and related organisms (e.g. Arthrobacter spp), not including C. diphtheriae Bacillus spp and related organisms (e.g. Paenibacillus spp, Lysinibacillus spp etc.), not including B. anthracis Micrococcus spp Neisseria spp other than N. meningitidis, N. gonorrhoeae Propionibacterium sppand Cutibacterium spp

Extended Antibiotic Panel: The following extended panel of antibiotics are available on special request: ceftazidime/avibactam, ceftolozane/tazobactam, meropenem/varbobactam, imipenem/ relebactam, and plazomicin.

Antimicrobial Susceptibility Testing of the extended panel is performed on isolates that meets any of the criteria below:

Carbapenemase -Producing Enterobacterales (CPE) from sterile site samples, in addition to Colistin, Tigecycline and Aztreonam
On special requests as follows:

Isolates that are multidrug resistant to first line and second line antibiotics
Sterile site isolates (on request)
Non-sterile site isolates after discussion with PHO Laboratory microbiologist

The extended panel is performed by micro-broth dilution method.

Interpretation

Antimicrobial susceptibility testing results are interpreted as per current CLSI guidelines.

For the organism/antimicrobial where there are no CLSI guidelines, MICs will be reported when applicable with the following comment: “No clinical data available to assist in interpretation of MIC results.”

Reporting

Organism/antimicrobial susceptibility results are reported to the ordering physician or health care provider as indicated on the requisition.

With certain organisms, both MICs and Interpretations will be reported, whereas with others, only interpretations will be reported.

References

Institute for Quality Management in Healthcare (IQMH): Consensus Practice Recommendations – BACT-Blood Cultures. Revision date: 2017-09-28.
Institute for Quality Management in Healthcare (IQMH): Consensus Practice Recommendations – BACT Sputum specimens. 2013-10-07.
Institute for Quality Management in Healthcare (IQMH): Consensus Practice Recommendations – BACT Superficial wound swabs. 2013-12-09.
Miller JM, Binnicker MJ, Campbell S, Carroll KC, et al. A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology. Clinical Infectious Diseases, Volume 67, Issue 6, 31 August 2018, Pages e1–e94, doi.org/10.1093/cid/ciy381
Clinical and Laboratory Standards Institute (CLSI). Principles and procedures for blood culture; approved guideline. CLSI document M47-A, Vol. 27, No. 17. Wayne, Pennsylvania, USA. 2007.
Baron EJ, Weinstein MP, Dunne Jr WM, Yagupsky P, Welch DF, Wilson DM. 2005. Cumitech 1C, Blood Cultures IV. Coordinating ed., EJ Baron. ASM Press, Washington DC.
Sharp SE, Robinson A, Saubolle MA, Santa Cruz M, Carroll K, Baselski V. 2004. Cumitech 7B, Lower respiratory tract infections. Coordinating ed., SE Sharp. ASM Press Washington, DC.
McCarter YS, Burd EM, Hall, GS, Zervos M. 2009. Cumitech 2C, Laboratory Diagnosis of Urinary Tract Infections. Coordinating ed. SE Sharp. ASM Press, Washington, DC.
Waites KB, Saubolle MA, Talkinton DF, Moser SA, Baselski V. 2006, Cumiteech 10A, Laboratory Diagnosis of Upper Respiratory Tract Infections. Coordinating ed. SE Sharp. ASM Press, Wahington, DC.