Download Enterobacteriaceae GNRs

Antimicrobial Susceptibility
Testing – Part II
Karen Honeycutt, M.Ed., MT(ASCP)SM
CLS 418 Clinical Microbiology
Student Laboratory Session
Microbiology
Antimicrobial Susceptibility Testing - MIC
MIC: Microdilution
• Commercial
• Antibiotic at
various
concentrations
• Inoculate with
standardized
inoculum
• MIC
• Interpret (NCCLS)
Microbiology
Antimicrobial Susceptibility Testing - MIC
MIC: Microdilution
Growth
control
MIC
ug/ml
Sterility
control
Ampicillin
8.0
16.0
>32.0
32.0
Piperacillin
16.0
32.0
64.0
Aztreonam
8.0
Microbiology
16.0
32.0
128.0
128.0
<8.0
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
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
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
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
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
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
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
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
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
Antimicrobial Susceptibility Testing
Beta-lactamase Test
• Haemophilus influenzae
• Anaerobes (ID)
• Staphylococcus sp. (if
test “S” to penicillin,
ampicillin)
Microbiology
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
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