Antimicrobial Susceptibility Testing – Part II Karen Honeycutt, M.Ed., MT(ASCP)SM CLS 418 Clinical Microbiology Student Laboratory Session Microbiology

MIC: Microdilution Commercial Antibiotic at various concentrations Antimicrobial Susceptibility Testing - MIC MIC: Microdilution Commercial Antibiotic at various concentrations Inoculate with standardized inoculum MIC Interpret (NCCLS) Microbiology

MIC: Microdilution Antimicrobial Susceptibility Testing - MIC MIC Growth control Sterility control MIC ug/ml Ampicillin 8.0 16.0 32.0 >32.0 Piperacillin 64.0 128.0 Aztreonam <8.0 Microbiology

Antimicrobial Susceptibility Testing - MIC MIC: Agar Dilution 24 hr inc. R = resistant control S = susceptible control A, B, C, D = isolates of S. aureus R = resistant control S = susceptible control A = resistant (MRSA) B, C, D = susceptible isolates of S. aureus Microbiology

Detection of MRSA Methicillin “R” S. aureus or multi-drug “R” Antimicrobial Susceptibility Testing - MRSA Detection of 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 MRSA If resistant to oxacillin, then MRSA Antimicrobial Susceptibility Testing - MRSA Detection of 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

Detection of MRSA Heteroresistant colonies “R” strains grow best at: Antimicrobial Susceptibility Testing - MRSA Detection of 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) - don’t grow up to turbid suspension Microbiology

Detection of Vancomycin “R” Enterococci Antimicrobial Susceptibility Testing - VRE Detection of Vancomycin “R” Enterococci 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

Susceptibility Testing: Enterococcus sp. Antimicrobial Susceptibility Testing Susceptibility Testing: Enterococcus sp. Systemic infections (blood, body fluids, wounds, etc.) treat with cell wall active antibiotic and aminoglycoside Synergistic break down cell wall aminoglycoside to ribosome Microbiology

Susceptibility Testing: Enterococcus sp. Antimicrobial Susceptibility Testing Susceptibility Testing: Enterococcus sp. 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” Microbiology

Susceptibility Testing: Enterococcus sp. Antimicrobial Susceptibility Testing - VRE Susceptibility Testing: Enterococcus sp. 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 Microbiology

Streptococcus pneumoniae – screen for penicillin susceptibility Antimicrobial Susceptibility Testing – S. pneumoniae Streptococcus pneumoniae – screen for penicillin susceptibility Detect resistance to penicillin Screen: K-B with oxacillin on MHA w/5% sheep blood < 20 mm zone = presumptive evidence of resistance to penicillin Microbiology

Antimicrobial Susceptibility Testing Beta-lactamase Test Nitrocefin (yellow) changes color (red) when beta-lactam ring hydrolyzed Nitrocefin disc most sensitive method Reaction time varies Microbiology

Beta-lactamase Test Haemophilus influenzae Anaerobes (ID) Antimicrobial Susceptibility Testing Beta-lactamase Test Haemophilus influenzae Anaerobes (ID) Staphylococcus sp. (if test “S” to penicillin, ampicillin) Microbiology

Detection of specific resistance mechanisms Antimicrobial Susceptibility Testing Detection of specific resistance mechanisms 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 Microbiology

Predictable Patterns If Gram-positive bacteria, then “S” to vancomycin Antimicrobial Susceptibility Testing - Patterns Predictable Patterns If Gram-positive bacteria, then “S” to vancomycin If beta-hemolytic Streptococci, then “S” to penicillin Pg 7 of notes Microbiology