Download beta-lactam antibiotics

Antimicrobial Susceptibility
Testing – Part I
Karen Honeycutt, M.Ed., MT(ASCP)SM
CLS 418 Clinical Microbiology
Student Laboratory Session
Microbiology
Antimicrobial Susceptibility Testing - Antibiotics
Definitions
• Bacteriostatic: inhibits new organism growth
• Bactericidal: kills organism
• Antibiogram: record of resistance patterns
within an institution or area
Microbiology
Antimicrobial Susceptibility Testing - Antibiotics
Mechanisms of Action
• Inhibit cell wall synthesis
– All beta-lactam antibiotics
– Vancomycin
• Inhibit or disrupt membrane function – toxic
– Polymyxin (topical)
• Inhibit protein synthesis
– Aminoglycosides
– Macrolide (erythromycin)
Microbiology
Antimicrobial Susceptibility Testing - Antibiotics
Mechanisms of Action
• Inhibit DNA & RNA synthesis
– Fluoroquinolones/quinolones:
ciprofloxacin
• Inhibition of folic acid synthesis
– Sulfa drugs
Microbiology
Antimicrobial Susceptibility Testing - Antibiotics
Mechanisms of Resistance –
Enzymatic Inactivation
• Beta-lactamases
– Hydrolyze the beta-lactam
ring
– Very specific/broad
spectrum
Microbiology
Antimicrobial Susceptibility Testing - Antibiotics
Mechanisms of Resistance – Altered
binding site
• Receptor for antibiotic
• Antibiotic able to alter
cell wall synthesis
• Altered receptor for
antibiotic
• Antibiotic is ineffective
Microbiology
Antimicrobial Susceptibility Testing - Antibiotics
Mechanisms of Resistance – Altered
binding site
• Streptococcus pneumoniae
• MRSA
• Enterococcus sp.
Microbiology
Antimicrobial Susceptibility Testing - Antibiotics
Altered Permeability
• Influx: restricts entry
of antibiotic into cell
• Efflux: pumps
antibiotic out of cell
Microbiology
Antimicrobial Susceptibility Testing
Intrinsic vs. Acquired Resistance
• Intrinsic – consistently inherited
characteristic (predictable)
– K. pneumoniae: Ampicillin (R)
– Gram-negative rods: Vancomycin (R)
• Acquired – mutations & acquisition of
genes, variability in susceptibility
patterns within a species
Microbiology
Antimicrobial Susceptibility Testing
General Considerations
• In vitro methods – determine an
organism’s acquired resistance
• Acquired resistance – mutations &
acquisition of genes
– variability in susceptibility patterns within a
species
– Example:
• Escherichia coli susceptible to Ampicillin
• Escherichia coli resistant to Ampicillin
Microbiology
Antimicrobial Susceptibility Testing
Standardization!!!!!!!!!
• Optimal growth conditions – any growth
inhibition due to antimicrobial agent
• Maintain antimicrobial integrity (store
properly) – any inhibition of growth
attributed to resistance mechanisms
• Reproducible and consistent results
• CLSI (NCCLS) standards – reference
Microbiology
Antimicrobial Susceptibility Testing
Standardization of Testing
Components
• Bacterial inoculum purity &
size
– Direct method: 4-5 colonies
same morphology, 16-24 hrs
old, non-selective agar,
emulsify in saline or supplied
media
Microbiology
Antimicrobial Susceptibility Testing
Standardization of Testing
Components
• Bacterial inoculum purity & size
– Adjust to 0.5 McFarland density standard
(1.5 X 108 CFU/ml)
– 150,000,000 CFU/ml
Microbiology
Antimicrobial Susceptibility Testing
Standardization of Testing
Components
• Growth medium: Mueller-Hinton agar base
–
–
–
–
–
Clear, nutrient agar
pH 7.2 to 7.4
Cation concentration
Agar depth = 4mm
If necessary for organism growth, add blood or
serum supplements to M-H base
Microbiology
Antimicrobial Susceptibility Testing
Standardization of Testing Components
• Incubation
– 35°C, ambient air 16-18 hours
– If organism requires CO2, specific interpretative
criteria
• Antimicrobial concentrations used for testing
• Interpretation of results
Microbiology
Antimicrobial Susceptibility Testing
Testing Methods – Diffusion Tests
• Kirby-Bauer
• Qualitative Result
– S, I, R
Microbiology
Antimicrobial Susceptibility Testing - Diffusion
Kirby-Bauer or Disk Diffusion
• Lawn of growth w/standardized inoculum
• Disk placement within 1-3 min, but not longer
than 15 min. of agar inoculation
• Incubation within 15 min of disk placement
Microbiology
Antimicrobial Susceptibility Testing - Diffusion
Kirby-Bauer or Disk Diffusion
• Interpretation
– Growth: confluent & pure
– Measure in mm diameter of zone across disk
(transmitted light)
– CLSI criteria: convert zone size to “S” “I” or
“R”
Microbiology
Antimicrobial Susceptibility Testing - Diffusion
E-Test (Epsilometer Test)
• Concentration
gradient-coated plastic
strip
• Set up like K-B
• Provides quantitative
results
• S. pneumoniae,
anaerobes,
H. influenzae,
Nocardia
• Expensive
Microbiology
Antimicrobial Susceptibility Testing - Diffusion
E-Test (Epsilometer Test)
• Enterobacter cloacae vs.
Imipenem
• MIC = Lowest concentration of
antibiotic that visibly inhibits
growth of organism
• MIC = 2.0 ug/ml
• Interpretation = Susceptible
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
• Moraxella catarrhalis
(ID)
• Pathogenic Neisseria
• 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