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 Antimicrobial Susceptibility Testing - Antibiotics

Microbiology Inhibit DNA & RNA synthesis –Fluoroquinolones/quinolones: ciprofloxacin Inhibition of folic acid synthesis –Sulfa drugs Mechanisms of Action Antimicrobial Susceptibility Testing - Antibiotics

Microbiology Mechanisms of Resistance – Enzymatic Inactivation Beta-lactamases –Hydrolyze the beta-lactam ring –Very specific/broad spectrum Antimicrobial Susceptibility Testing - Antibiotics

Microbiology Receptor for antibiotic Antibiotic able to alter cell wall synthesis Altered receptor for antibiotic Antibiotic is ineffective Mechanisms of Resistance – Altered binding site Antimicrobial Susceptibility Testing - Antibiotics

Microbiology Streptococcus pneumoniae MRSA Enterococcus sp. Mechanisms of Resistance – Altered binding site Antimicrobial Susceptibility Testing - Antibiotics

Microbiology Altered Permeability Influx: restricts entry of antibiotic into cell Efflux: pumps antibiotic out of cell Antimicrobial Susceptibility Testing - Antibiotics

Microbiology 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 Antimicrobial Susceptibility Testing

Microbiology 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 Antimicrobial Susceptibility Testing

Microbiology Standardization of Testing Components Bacterial inoculum purity & size –Direct method: 4-5 colonies same morphology, hrs old, non-selective agar, emulsify in saline or supplied media Antimicrobial Susceptibility Testing

Microbiology Standardization of Testing Components Bacterial inoculum purity & size –Adjust to 0.5 McFarland density standard (1.5 X 10 8 CFU/ml) –150,000,000 CFU/ml Antimicrobial Susceptibility Testing

Microbiology 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 Antimicrobial Susceptibility Testing

Microbiology Standardization of Testing Components Incubation –35°C, ambient air hours –If organism requires CO 2, specific interpretative criteria Antimicrobial concentrations used for testing Interpretation of results Antimicrobial Susceptibility Testing

Microbiology Testing Methods – Diffusion Tests Kirby-Bauer Qualitative Result –S, I, R Antimicrobial Susceptibility Testing

Microbiology 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” Antimicrobial Susceptibility Testing - Diffusion

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

Microbiology 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 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)

Microbiology 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. Antimicrobial Susceptibility Testing

Microbiology 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” Susceptibility Testing: Enterococcus sp. Antimicrobial Susceptibility Testing

Microbiology 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 Susceptibility Testing: Enterococcus sp. 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 Antimicrobial Susceptibility Testing

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

Microbiology 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 Antimicrobial Susceptibility Testing

Microbiology 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