Microbiology

Antimicrobial Susceptibility Testing – Part II

Karen Honeycutt, M.Ed., MT(ASCP)SM

CLS 418 Clinical Microbiology

Student Laboratory Session

Microbiology

• Commercial• Antibiotic at

various concentrations

• Inoculate with standardized inoculum

• MIC• Interpret (NCCLS)

MIC: MicrodilutionAntimicrobial Susceptibility Testing - MIC

Microbiology

MIC: MicrodilutionAntimicrobial Susceptibility Testing - MIC

Growth control

Sterility control

MIC

ug/ml

Ampicillin

8.0 16.0 32.0>32.0

Piperacillin

16.0 32.0 64.0 128.0128.0

Aztreonam

8.0 16.0 32.0<8.0

Microbiology

MIC: Agar DilutionAntimicrobial Susceptibility Testing - MIC

R = resistant controlS = susceptible controlA, B, C, D = isolates of

S. aureus

24 hr inc.

R = resistant controlS = susceptible controlA = resistant (MRSA)

B, C, D = susceptibleisolates of S. aureus

Microbiology

Detection of MRSAAntimicrobial 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

Microbiology

Detection of MRSAAntimicrobial 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

Microbiology

• 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) - don’t grow up to turbid suspension

Detection of MRSA

Antimicrobial Susceptibility Testing - MRSA

Microbiology

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 manufacturer’s protocol)

Microbiology

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

• Synergistic– break down cell wall– aminoglycoside to

ribosome

Susceptibility Testing: Enterococcus sp.

Antimicrobial Susceptibility Testing

Microbiology

• 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”

Susceptibility Testing: Enterococcus sp.

Antimicrobial Susceptibility Testing

Microbiology

• 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

Susceptibility Testing: Enterococcus sp.

Antimicrobial Susceptibility Testing - VRE

Microbiology

• Detect resistance to penicillin

• Screen: K-B with oxacillin on MHA w/5% sheep blood

• < 20 mm zone = presumptive evidence of resistance to penicillin

Streptococcus pneumoniae – screen for penicillin susceptibility

Antimicrobial Susceptibility Testing – S. pneumoniae

Microbiology

• Nitrocefin (yellow) changes color (red) when beta-lactam ring hydrolyzed

• Nitrocefin disc most sensitive method

• Reaction time varies

Beta-lactamase Test

Antimicrobial Susceptibility Testing

Microbiology

• Haemophilus influenzae• Anaerobes (ID)• Staphylococcus sp. (if

test “S” to penicillin, ampicillin)

Beta-lactamase Test

Antimicrobial Susceptibility Testing

Microbiology

• 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

Detection of specific resistance mechanisms

Antimicrobial Susceptibility Testing

Microbiology

Predictable Patterns

• If Gram-positive bacteria, then “S” to vancomycin

• If beta-hemolytic Streptococci, then “S” to penicillin

• Pg 7 of notes

Antimicrobial Susceptibility Testing - Patterns