Antimicrobial Susceptibility Testing for Bacteriology Laboratories

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Antimicrobial Susceptibility Testing for
Bacteriology Laboratories
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ObjectivesAt the conclusion of this
presentation participants will be able to

• Explain how to implement current NCCLS
  antimicrobial susceptibility testing (AST) and
  reporting recommendations.
• Describe reliable methods for detecting
  resistance among
• Enterobacteriaceae
• Pseudomonas aeruginosa
• Staphylococcus aureus
• Enterococcus spp.
• Streptococcus pneumoniae and Streptococcus spp.

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At the conclusion of this presentation
Participants will be able to(cont)

• Describe effective reporting of AST results.
• List steps that can be taken to verify AST
  results obtained on bacteria isolated from
  patients.
• Discuss NCCLS-recommended QC procedures and how
  to trouble-shoot out-of-control QC results

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What Does the Laboratory Need to Knowabout
Antimicrobial Susceptibility Testing (AST) ?

• Which organisms to test?
• What methods to use?
• What antibiotics to test?
• How to report results?

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What Does a Laboratory Need to Know about AST?
(cont)

• How to determine the clinical significance of
  results?
• How to ensure accuracy of results?
• Quality control / quality assurance
• When to call the MD, infection control, public
  health?

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What Does a Laboratory Need to Know about AST?
(cont)

• When to ask for help?
• Where to go for help?

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Brief Review of Routine AST Methods
AST
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Routine Susceptibility Tests

• Disk diffusion (Kirby Bauer)
• Broth micro-dilution MIC
• NCCLS reference method
• Etest

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• Disk Diffusion
• Test

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Prepare inoculum suspension
Select colonies
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Mix well
Standardize inoculum suspension
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Swab plate
Remove sample
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Incubate overnight
Add disks
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Measure Zones
Transmitted Light
Reflected Light
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Measure Zones Transmitted Light

• Staphylococci oxacillin, vancomycin
• Enterococci vancomycin

Measure all other drug/bug combinations with
reflected light
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Zone Interpretive Criteria (mm)
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Disk Diffusion Test

• Qualitative results
• Susceptible
• Intermediate may respond if infection is at
  body site where drug concentrates (e.g. urine) or
  if higher than normal dose can be safely given
• Resistant

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Modify methods for fastidious bacteria
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Clinical Conditions when MICs are Useful

• Endocarditis
• Meningitis
• Septicemia
• Osteomyelitis
• Immunosuppressed patients (HIV, cancer, etc.)
• Prosthetic devices
• Patients not responding despite S

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MIC

• Minimal inhibitory concentration
• The lowest concentration of antimicrobial agent
  that inhibits the growth of a bacterium
• Interpret
• Susceptible
• Intermediate
• Resistant

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Inoculum Preparation MIC Testing (NCCLS
Reference Method)

• Standardize inoculum suspension
• Final inoculum concentration
• 3 5 x 105 CFU/ml
• (3 5 x 104 CFU/well)

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Prepare inoculum suspension
Microdilution MIC tray
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Dilute mix inoculum suspension
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Pour inoculum into reservoir and inoculate MIC
tray
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Incubate overnight
Inoculate purity plate
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Reflected light
Transmitted light
Examining purity plate
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Optimal Use of Purity Plates

• Sub final test suspension to non-selective medium
  (after inoculating MIC test)
• Streak for isolation (avoid several specimens per
  plate - may not reveal contaminants if no
  isolated colonies)
• Examine before reading MIC (usually at 16-20 h)
• Re-incubate if antibiogram questionable

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Read MICs
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MICs
-

0.5
1
2
4
8
16
32
64
gt64
gt64
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Reporting Time Disk Diffusion or MIC

• Staphylococci oxacillin, vancomycin, cefoxitin
• Enterococci vancomycin
• Resistance may be reported any time growth is
  observed after a minimum of 16 h incubation
• After minimum of 16 h, read test
• if R, report
• If S, re-incubate and read again at 24 h

NCCLS M2-A8, M7-A6
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MIC on a strip abbiodisk.com
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(No Transcript)
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S. pneumoniae Penicillin MIC 3 ?g/ml
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MIC Interpretive Criteria (?g/ml)
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MICs Recommended (NCCLS M100-S14)

• Viridans Streptococcus penicillin
• S. pneumoniae penicillin, cefotaxime/ceftriaxone
  (sterile sites)
• Enterococcus vancomycin Int results
• Staphylococcus vancomycin zone ?14 mm

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Credits Janet Fick Hindler, MCLS MT(ASCP) UCLA
Medical Center Los Angeles, CA