Antibiotic Susceptibility Testing Methods

Antibiotic Susceptibility Testing (AST) Methods

• Phenotypic Methods: These methods involve observing the growth of the organism when exposed to antibiotics.
  • Broth Microdilution: Doubling dilutions of an antibiotic are placed in individual wells of a microtiter plate. A standard bacterial inoculum is added to each well. The plate is incubated and examined for growth. The minimum inhibitory concentration (MIC) is the lowest antibiotic concentration that inhibits growth. The MIC is interpreted according to organism-specific and drug-specific criteria, typically categorized as Susceptible (S), Intermediate (I), or Resistant (R).
    • Automated, high-throughput formats assist with clinical testing by using multiwell plates with indicators for bacterial growth. Instruments automate the reading process.
  • Gradient Strips (Etests): Bacteria form a lawn on an agar plate. Strips containing a gradient of the antibiotic are placed on the plate. The minimum inhibitory concentration (MIC) is determined by where the elliptical zone of growth inhibition intersects with the strip.
  • Disk Diffusion: Bacteria form a lawn on the plate. Antibiotic-containing disks are placed on the lawn. The antibiotic diffuses into the agar. The zone of inhibition is measured. A larger zone indicates susceptibility to the antibiotic. Conversely, a small or nonexistent zone suggests resistance.

Genotypic Methods

• These methods directly identify resistance genes.
  • Polymerase Chain Reaction (PCR) with sequencing: PCR amplifies specific resistance genes. Sequencing identifies the presence or absence of these genes. This method correlates well with phenotypic results (observed susceptibility). This assists with identifying resistances like vancomycin-resistant Enterococcus (VRE) – vanA or vanB genes, methicillin-resistant Staphylococcus aureus (MRSA) – mecA gene, or rifampin-resistant Mycobacterium tuberculosis – rpoB gene mutations.
  • Genotypic methods sometimes struggle to predict MIC (minimum inhibitory concentration) and phenotypic resistance. Gram-negative bacteria, in particular, often have several complex resistance mechanisms, including alterations in membrane permeability, making genotype-phenotype correlation less straightforward.

Other Methods

• Antibody-Based Detection: Rapid detection of resistance factors, like PBP2a from Staphylococcus aureus (mecA gene product), can be performed via lateral flow immunoassays.
• Advanced Diagnostics: Digital microscopy combined with machine learning may correlate bacterial growth patterns with susceptibility or resistance characteristics.

Selecting the Appropriate Method

• Factors to Consider: Turnaround time, throughput, accuracy, cost, and desired information (e.g., MIC).

Standardization

• Critical for Accurate AST: The FDA, CLSI (Clinical and Laboratory Standards Institute), and the European Committee on Antimicrobial Susceptibility Testing determine standards for interpretative MIC and zone size criteria. Reproducibility between labs requires strict quality control standards and clearly defined guidelines for media, drug concentration, incubation, and other variables. Ranges are developed through pharmacodynamics, site of infection, clinical outcome studies, and distributions of wild-type MICs.

Additional Concepts

• Antibiotic Misuse: Inappropriate prescribing and incomplete adherence, agricultural overuse, and climate change negatively impact efficacy.
• Antibiotic Stewardship: Rational and judicious use of antibiotics is essential to prevent resistance, improve patient outcomes, and maintain antibiotic effectiveness in the clinical setting and society. Infection prevention and control programs track hospital-associated infections and follow standard precautions to prevent transmission. Local antibiograms can guide prescribing, tracking historical susceptibility, but require caution due to differences in patients, and should be combined with appropriate data.
• Drivers of Resistance: Resistance arises through environmental factors such as waste products from healthcare and manufacturing, misuse/overuse in agriculture and hospitals, poor water/sanitation conditions, and others.
• Treatment Considerations: Broad-spectrum antibiotics are frequently used initially in unsure situations. Further treatment is targeted by laboratory results, de-escalating and elevating coverage as needed. Source control interventions may be necessary along with treatment to help with outcomes of infections. Consider alternative therapies or no antibiotic use, if warranted. Treatment might involve therapeutic drug monitoring, interactions with other drugs, side effects, route of administration, allergies, and infections involving multiple organisms/sites, especially for conditions within (and outside) the body as certain drugs are effective only at certain sites such as brain or lung treatment.

Case Studies (Examples)

• Case Study 1: A patient with spinal osteomyelitis was treated with broad-spectrum antibiotics initially, then modified to target the specific bacterial pathogen (Staphylococcus aureus) identified by laboratory testing.
• Case Study 2: A patient with a multi-drug resistant Pseudomonas aeruginosa infection required targeted therapy, including susceptibility testing, as well as possibly contact precautions and other standard infection control practices.