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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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
Use direct inoculum (for all Staphs) - don’t grow up to turbid suspension
Microbiology

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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