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Microbiology

Genetic Determinants

This section covers molecular methods used to detect specific genes that confer antibiotic resistance. These methods provide rapid and accurate identification of resistance determinants

Molecular Methods

  • Principle: Molecular methods detect the presence of specific genes associated with antibiotic resistance. These methods are based on the principles of nucleic acid amplification and detection
  • Advantages
    • Rapid: Results are typically available within hours, allowing for timely clinical decision-making
    • Specific: Detects the presence of specific resistance genes, providing highly accurate results
    • Sensitive: Can detect resistance genes even in low concentrations
    • Strain-Independent: Can detect resistance genes regardless of the bacterial species
  • Disadvantages
    • Cost: Molecular tests can be more expensive than phenotypic tests
    • Limited Scope: May not detect all resistance mechanisms, especially those caused by mutations in genes not targeted by the test
    • Interpretation: Requires expertise in molecular biology and bioinformatics to interpret the results

Commonly Detected Resistance Genes

  • mecA
    • Organism: Staphylococcus aureus
    • Resistance: Methicillin (oxacillin) resistance, indicating methicillin-resistant Staphylococcus aureus (MRSA)
    • Mechanism: mecA encodes for PBP2a, a penicillin-binding protein that has a low affinity for beta-lactam antibiotics
    • Methods: Polymerase chain reaction (PCR) is the most common method, but real-time PCR is often used for rapid detection
  • vanA
    • Organism: Enterococcus species (e.g., Enterococcus faecalis, Enterococcus faecium)
    • Resistance: Vancomycin resistance
    • Mechanism: vanA encodes for an enzyme that alters the peptidoglycan precursor, reducing the binding affinity of vancomycin
    • Methods: PCR and real-time PCR
  • blaKPC
    • Organism: Klebsiella pneumoniae and other Enterobacteriaceae
    • Resistance: Carbapenem resistance
    • Mechanism: blaKPC encodes for a carbapenemase, an enzyme that hydrolyzes carbapenem antibiotics
    • Methods: PCR and real-time PCR are commonly used. Multiplex PCR assays can detect multiple carbapenemase genes (e.g., blaKPC, blaOXA-48, blaNDM)
  • Other Resistance Genes
    • blaNDM: Carbapenem resistance in Enterobacteriaceae (New Delhi metallo-beta-lactamase)
    • blaOXA-48: Carbapenem resistance in Enterobacteriaceae
    • erm: Macrolide, lincosamide, and streptogramin B (MLS) resistance in Staphylococcus aureus and streptococci
    • tet: Tetracycline resistance in various bacteria
    • sul: Sulfonamide resistance in various bacteria
    • qnr: Quinolone resistance in Enterobacteriaceae
    • AmpC: AmpC beta-lactamase genes in Enterobacteriaceae (e.g., blaCMY)

Molecular Methods in Detail

  • Polymerase Chain Reaction (PCR)
    1. DNA Extraction: Extract bacterial DNA from a pure culture or clinical sample
    2. Primer Design: Design primers that are specific to the target resistance gene
    3. Amplification: PCR amplifies the target DNA sequence using primers, a DNA polymerase, and deoxynucleotide triphosphates (dNTPs)
    4. Detection: The amplified DNA (PCR product) is detected by gel electrophoresis or other methods
  • Real-Time PCR (qPCR)
    1. DNA Extraction: Extract bacterial DNA
    2. Primer and Probe Design: Design primers and a fluorescently labeled probe that binds to the target resistance gene
    3. Amplification and Detection: PCR amplification is performed, and the fluorescent signal is measured in real-time. The cycle threshold (Ct) value is inversely proportional to the amount of target DNA present
    4. Interpretation: The presence of a fluorescent signal indicates the presence of the resistance gene
  • Other Molecular Methods
    • Multiplex PCR: Amplifies multiple target genes simultaneously
    • Microarrays: Detect the presence of multiple resistance genes in a single test
    • Next-Generation Sequencing (NGS): Provides a comprehensive analysis of the bacterial genome, including the identification of resistance genes and mutations

Workflow

  1. Sample Collection: Collect appropriate clinical samples
  2. Culture and Identification: Culture the sample and identify the bacterial species
  3. Phenotypic Susceptibility Testing: Perform routine antimicrobial susceptibility testing (e.g., disk diffusion, microbroth dilution)
  4. Targeted Molecular Testing: If phenotypic testing suggests resistance or if there is a high clinical suspicion, perform targeted molecular testing to detect specific resistance genes
  5. Result Reporting: Report the results of both phenotypic and molecular testing to the clinician
  6. Infection Control: Use molecular results to guide infection control measures, such as patient isolation and contact precautions

Interpretation and Application

  • Positive Result: Detection of a resistance gene indicates the presence of that gene in the organism. This confirms the mechanism of resistance
  • Negative Result: A negative result does not always mean the organism is susceptible. Resistance may be due to other mechanisms not targeted by the test. Phenotypic susceptibility testing is still required
  • Clinical Significance: Molecular results are used to guide antibiotic selection, particularly in cases of severe infections, treatment failures, or the need for rapid results. They are also essential for infection control, helping to identify and prevent the spread of resistant organisms

Key Terms

  • Molecular Methods: Laboratory techniques that use nucleic acid amplification and detection to identify specific genes
  • PCR (Polymerase Chain Reaction): A method to amplify specific DNA sequences
  • Real-Time PCR (qPCR): A PCR method that measures DNA amplification in real-time using fluorescent probes
  • Gene: A segment of DNA that codes for a specific protein or RNA molecule
  • Resistance Gene: A gene that confers resistance to an antibiotic
  • mecA: A gene that confers methicillin resistance in Staphylococcus aureus (MRSA)
  • vanA: A gene that confers vancomycin resistance in Enterococcus species (VRE)
  • blaKPC: A gene that confers carbapenem resistance in Enterobacteriaceae (CRE)
  • Primer: A short DNA sequence used in PCR to initiate DNA amplification
  • Probe: A short, labeled DNA sequence used in real-time PCR to detect the amplified DNA
  • DNA Extraction: The process of isolating DNA from a biological sample
  • Cycle Threshold (Ct) Value: In real-time PCR, the number of cycles required for the fluorescent signal to cross a threshold