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

Microbiology

Antimicrobial Susceptibility Testing – Part I

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

CLS 418 Clinical Microbiology

Student Laboratory Session

Microbiology

• Bacteriostatic: inhibits new organism growth• Bactericidal: kills organism• Antibiogram: record of resistance patterns

within an institution or area

Definitions

Antimicrobial Susceptibility Testing - Antibiotics

Microbiology

• Inhibit cell wall synthesis– All beta-lactam antibiotics– Vancomycin

• Inhibit or disrupt membrane function – toxic– Polymyxin (topical)

• Inhibit protein synthesis– Aminoglycosides– Macrolide (erythromycin)

Mechanisms of Action


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• Inhibit DNA & RNA synthesis– Fluoroquinolones/quinolones:

ciprofloxacin• Inhibition of folic acid synthesis

– Sulfa drugs

Mechanisms of Action


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Mechanisms of Resistance – Enzymatic Inactivation

• Beta-lactamases– Hydrolyze the beta-lactam

ring

– Very specific/broad spectrum


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• Receptor for antibiotic• Antibiotic able to alter

cell wall synthesis

• Altered receptor for antibiotic

• Antibiotic is ineffective

Mechanisms of Resistance – Altered binding site


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• Streptococcus pneumoniae• MRSA• Enterococcus sp.

Mechanisms of Resistance – Altered binding site


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Altered Permeability

• Influx: restricts entry of antibiotic into cell

• Efflux: pumps antibiotic out of cell


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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

Antimicrobial Susceptibility Testing

Microbiology

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


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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


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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


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• Bacterial inoculum purity & size– Adjust to 0.5 McFarland density standard

(1.5 X 108 CFU/ml)– 150,000,000 CFU/ml


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• 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


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• Incubation– 35°C, ambient air 16-18 hours

– If organism requires CO2, specific interpretative criteria

• Antimicrobial concentrations used for testing• Interpretation of results


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Testing Methods – Diffusion Tests• Kirby-Bauer• Qualitative Result

– S, I, R


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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

Antimicrobial Susceptibility Testing - Diffusion

Microbiology

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

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

E-Test (Epsilometer Test)Antimicrobial Susceptibility Testing - Diffusion

• Enterobacter cloacae vs. Imipenem

• MIC = Lowest concentration of antibiotic that visibly inhibits growth of organism

• MIC = 2.0 ug/ml• Interpretation = Susceptible

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

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• 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)

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• 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.


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• 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”



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• 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


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


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• Haemophilus influenzae• Moraxella catarrhalis

(ID)• Pathogenic Neisseria • Anaerobes (ID)• Staphylococcus sp. (if

test “S” to penicillin, ampicillin)

Beta-lactamase Test


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• 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


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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

Documents

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