Antimicrobial Susceptibility Testing

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Antimicrobial Susceptibility Testing Part II

• Karen Honeycutt, M.Ed., MT(ASCP)SM
• CLS 418 Clinical Microbiology
• Student Laboratory Session

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MIC Microdilution
Antimicrobial Susceptibility Testing - MIC

• Commercial
• Antibiotic at various concentrations
• Inoculate with standardized inoculum
• MIC
• Interpret (NCCLS)

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MIC Microdilution
Antimicrobial Susceptibility Testing - MIC
Growth control Sterility control MIC ug/ml
Ampicillin 8.0 16.0 32.0 gt32.0
Piperacillin 16.0 32.0 64.0 128.0 128.0
Aztreonam 8.0 16.0 32.0 lt8.0
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MIC Agar Dilution
Antimicrobial Susceptibility Testing - MIC
R resistant controlS susceptible controlA,
B, C, D isolates of S. aureus
R resistant controlS susceptible controlA
resistant (MRSA) B, C, D susceptibleisolates
of S. aureus
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Detection of MRSA
Antimicrobial Susceptibility Testing - MRSA

• Methicillin R S. aureus or multi-drug R
• Beta-lactamase susceptible penicillins
• penicillin, ampicillin
• 95 of all S. aureus R to these penicillins
• Beta-lactamase resistant penicillins
• oxacillin, methicillin, nafcillin
• drug of choice to treat Staphylococcus infections
• Oxacillin is the representative drug tested for
  the penicillinase R penicillins

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Detection of MRSA
Antimicrobial Susceptibility Testing - MRSA

• If resistant to oxacillin, then MRSA
• MRSA is resistant to all penicillinase-resistant
  penicillins
• oxacillin, methicillin, nafcillin
• Also resistant to all beta-lactam antibiotics
• Drug of choice becomes vancomycin

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Detection of MRSA
Antimicrobial Susceptibility Testing - MRSA

• Heteroresistant colonies
• within one colony S R strains
• R strains grow best at
• 33 to 35 C
• 2-4 NaCl in media
• pH 7.2 - 7.4
• Use direct inoculum (for all Staphs) - dont grow
  up to turbid suspension

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Detection of Vancomycin R Enterococci
Antimicrobial Susceptibility Testing - VRE

• Vancomycin R Enterococcus sp. (E. faecium)
• Very difficult to treat
• No standard treatment protocol
• Usually confirmed by 2 methods (MIC, agar
  dilution, K-B)
• Incubate a full 24 hours before interpretation is
  S (refer to manufacturers protocol)

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Susceptibility Testing Enterococcus sp.
Antimicrobial Susceptibility Testing

• Systemic infections (blood, body fluids, wounds,
  etc.) treat with cell wall active antibiotic and
  aminoglycoside
• Synergistic
• break down cell wall
• aminoglycoside to ribosome

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Susceptibility Testing Enterococcus sp.
Antimicrobial Susceptibility Testing

• Synergy Screen
• Test synergy with high level aminoglycoside
• gentamicin 500 ug/ml
• streptomycin 1000 ug/ml
• If S to penicillin and high level
  aminoglycoside then should be synergistic
• Penicillin S
• Streptomycin Synergy S
• Gentamicin Synergy S

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Susceptibility Testing Enterococcus sp.
Antimicrobial Susceptibility Testing - VRE

• Penicillin S Strep Syn S Gent Syn R
• Penicillin synergistic w/ Streptomycin
• Penicillin R Vancomycin S Strep Syn S
  Gent Syn S
• Vancomycin synergistic w/Streptomycin Gentamcin
• Penicillin R Vancomycin S Strep Syn R
  Gent Syn R
• No synergistic response

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Streptococcus pneumoniae screen for penicillin
susceptibility
Antimicrobial Susceptibility Testing S.
pneumoniae

• Detect resistance to penicillin
• Screen K-B with oxacillin on MHA w/5 sheep
  blood
• lt 20 mm zone presumptive evidence of resistance
  to penicillin

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Beta-lactamase Test
Antimicrobial Susceptibility Testing

• Nitrocefin (yellow) changes color (red) when
  beta-lactam ring hydrolyzed
• Nitrocefin disc most sensitive method
• Reaction time varies

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Beta-lactamase Test
Antimicrobial Susceptibility Testing

• Haemophilus influenzae
• Anaerobes (ID)
• Staphylococcus sp. (if test S to penicillin,
  ampicillin)

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Detection of specific resistance mechanisms
Antimicrobial Susceptibility Testing

• MRSA Detection of PBP2a rapid latex
  agglutination test detects the altered protein
  encoded for by the mecA gene
• Genotypic methods detection of genes or
  plasmids encoding for resistance at the molecular
  level

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Predictable Patterns
Antimicrobial Susceptibility Testing - Patterns

• If Gram-positive bacteria, then S to vancomycin
• If beta-hemolytic Streptococci, then S to
  penicillin
• Pg 7 of notes