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Microbiology

Susceptibility Testing

This section covers the essential components of antimicrobial susceptibility testing and antibiotic resistance, covering the methods used to determine bacterial susceptibility, the mechanisms of resistance, and how to apply this information in clinical practice

Methods of Antimicrobial Susceptibility Testing

  • Microbroth Dilution
    • Method: Quantitative method using microtiter plates. Serial dilutions of antibiotics are prepared, and a standardized bacterial inoculum is added. The MIC (Minimum Inhibitory Concentration) is determined by observing the lowest concentration that inhibits growth
    • Theory: Based on exposing bacteria to varying concentrations of antibiotics, allowing for precise measurement of susceptibility
    • Interpretation: MIC values are interpreted using CLSI guidelines to categorize the organism as susceptible (S), intermediate (I), or resistant (R)
    • Application: Primary method for determining MICs, used for guiding antibiotic selection, monitoring resistance trends, and dosage optimization
  • Disk Diffusion (Kirby-Bauer)
    • Method: Qualitative/semi-quantitative method. Antibiotic-impregnated disks are placed on an agar plate inoculated with the bacteria. The diameter of the zone of inhibition is measured
    • Theory: Antibiotics diffuse from the disks, inhibiting bacterial growth. Zone size correlates inversely with the MIC
    • Interpretation: Zone diameters are compared to CLSI interpretive criteria to determine S, I, or R
    • Application: Simple, cost-effective, and widely used for routine susceptibility testing
  • Gradient Diffusion (Etest)
    • Method: Semi-quantitative method. A test strip with a pre-defined antibiotic gradient is placed on an agar plate inoculated with the bacteria. The MIC is read from the strip where the zone of inhibition intersects the strip
    • Theory: The antibiotic diffuses from the strip, creating a concentration gradient
    • Interpretation: MIC value is read directly from the strip and interpreted using CLSI criteria to determine S, I, or R
    • Application: Provides a quantitative MIC value, particularly useful for fastidious organisms and for more accurate results, especially in the intermediate range

Phenotypic Detection of Resistance

  • Beta-Lactamase Detection
    • Mechanism: Detects enzymes that break down beta-lactam antibiotics
    • Methods: Acidimetric, chromogenic cephalosporin (Cefinase) test
    • Interpretation: Positive test indicates resistance to beta-lactam antibiotics
    • Application: Rapid identification of beta-lactamase-producing organisms
  • ESBL (Extended-Spectrum Beta-Lactamase) Detection
    • Mechanism: Detects enzymes that inactivate extended-spectrum cephalosporins
    • Methods: Combination disk diffusion test, MIC testing with and without beta-lactamase inhibitor
    • Interpretation: Positive test indicates resistance to extended-spectrum cephalosporins
    • Application: Identification of ESBL-producing organisms to guide therapy and infection control
  • Inducible Clindamycin Resistance Detection: (D-Test)
    • Mechanism: Detects inducible resistance to clindamycin mediated by the erm gene
    • Method: D-test (D-zone test) - Erythromycin and clindamycin disks are placed on an agar plate
    • Interpretation: D-shaped zone around clindamycin disk indicates inducible clindamycin resistance
    • Application: Prevents treatment failures with clindamycin
  • Carbapenemase Detection
    • Mechanism: Detects enzymes that inactivate carbapenem antibiotics
    • Methods: Modified Hodge Test (MHT), Carbapenem Inactivation Method (CIM), Modified Carbapenem Inactivation Method (mCIM), Carba NP Test, Molecular methods (PCR)
    • Interpretation: Positive test indicates resistance to carbapenems
    • Application: Identification of carbapenem-resistant organisms to guide therapy and prevent spread

Mechanisms of Action of Major Antibiotic Classes

  • Cell Wall Synthesis Inhibitors: Beta-lactams, glycopeptides, fosfomycin
  • Protein Synthesis Inhibitors: Aminoglycosides, tetracyclines, macrolides, chloramphenicol, oxazolidinones, glycylcyclines
  • DNA Synthesis Inhibitors: Quinolones, nitroimidazoles, rifamycins
  • Folate Synthesis Inhibitors: Sulfonamides, trimethoprim
  • Cell Membrane Disruptors: Polymyxins, daptomycin

Detection of Genetic Determinants of Resistance

  • Molecular Methods: PCR, real-time PCR, multiplex PCR, microarrays, Next-Generation Sequencing (NGS)
  • Common Resistance Genes: mecA (MRSA), vanA (VRE), blaKPC (CRE)
  • Application: Rapid, specific, and sensitive detection of resistance genes. Essential for guiding therapy and infection control

Intrinsic Resistance Patterns for Common Species

  • Understanding Intrinsic Resistance: Inherent resistance patterns that dictate antibiotic choices
  • Examples
    • E. coli: Intrinsic resistance to ampicillin, amoxicillin-clavulanate, and some first-generation cephalosporins
    • P. aeruginosa: Intrinsic resistance to many antibiotics
    • Bacteroides fragilis: Intrinsic resistance to aminoglycosides, trimethoprim-sulfamethoxazole, and clindamycin

Antimicrobials Appropriate for Reporting by Species and Body Site

  • Principles: Select antimicrobials for reporting based on CLSI guidelines, likely pathogens, body site, and local resistance patterns
  • Examples
    • S. aureus: Report oxacillin, vancomycin, linezolid, clindamycin (with D-test)
    • E. coli: Report ampicillin (if susceptible), amoxicillin-clavulanate, cefazolin, ceftriaxone, gentamicin, trimethoprim-sulfamethoxazole, nitrofurantoin (urine)
    • P. aeruginosa: Report piperacillin-tazobactam, ceftazidime, gentamicin, ciprofloxacin
  • Body Site Considerations: Account for the ability of the antibiotic to reach effective concentrations at the site of infection
  • Reporting Practices: Report susceptibility categories (S, I, R), MICs, and relevant comments