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Microbiology

Resistance Markers

This section focuses on the direct detection of resistance markers in Mycobacteria and Nocardia. This approach uses molecular methods to identify specific genetic mutations associated with resistance to antimicrobial agents, directly from clinical specimens or cultures. This bypasses the need for phenotypic susceptibility testing in some cases and provides rapid results

General Principles

  • Molecular Detection of Resistance: Direct detection of resistance markers involves using molecular techniques to identify specific genetic mutations or sequences that are known to confer resistance to antimicrobial agents
  • Rapid Results: This approach provides rapid results compared to traditional phenotypic susceptibility testing methods, which can take days or weeks
  • Targeted Approach: Direct detection of resistance markers is a targeted approach that focuses on detecting specific known resistance mechanisms
  • Complementary to Phenotypic Testing: Direct detection of resistance markers is often used in conjunction with phenotypic susceptibility testing to provide a comprehensive assessment of antimicrobial resistance
  • Clinical Significance: The detection of specific resistance markers can guide antimicrobial therapy and infection control measures
  • Limited Scope: Only detects known resistance mechanisms, so novel mechanisms or resistance due to other factors may be missed

Target Genes and Mutations

  • Mycobacterium tuberculosis
    • rpoB gene: Mutations in the rpoB gene are associated with resistance to rifampin (RIF)
    • katG gene: Mutations in the katG gene are associated with resistance to isoniazid (INH)
    • inhA promoter region: Mutations in the inhA promoter region are associated with resistance to isoniazid (INH)
    • gyrA gene: Mutations in the gyrA gene are associated with resistance to fluoroquinolones
    • rrs gene: Mutations in the rrs gene are associated with resistance to aminoglycosides
    • eis promoter region: Mutations in the eis promoter region are associated with resistance to kanamycin and amikacin
  • Mycobacterium avium complex (MAC)
    • rrl gene: Mutations in the rrl gene are associated with resistance to macrolides (clarithromycin and azithromycin)
  • Rapidly Growing Mycobacteria (RGM)
    • rrl gene: Mutations in the rrl gene are associated with resistance to macrolides
    • erm(41) gene: The presence and sequence of the erm(41) gene determines inducible macrolide resistance in Mycobacterium abscessus
  • Nocardia spp.
    • sul1 and sul2 genes: Presence of sul1 and sul2 genes are associated with sulfonamide resistance
    • gyrA and gyrB genes: Mutations in the gyrA and gyrB genes are associated with resistance to fluoroquinolones

Molecular Methods

  • Nucleic Acid Amplification Tests (NAATs)
    • PCR (Polymerase Chain Reaction): PCR is used to amplify specific regions of the target genes
    • Real-Time PCR (qPCR): qPCR allows for real-time monitoring of the amplification process, providing quantitative results and rapid detection of resistance markers
    • Multiplex PCR: Multiplex PCR assays can detect multiple resistance markers simultaneously
  • DNA Sequencing
    • Sanger Sequencing: Sanger sequencing is used to determine the nucleotide sequence of the target genes and identify specific mutations
    • Next-Generation Sequencing (NGS): NGS allows for rapid sequencing of multiple genes or entire genomes, providing comprehensive detection of resistance markers
  • Hybridization Assays
    • Line Probe Assays (LPAs): LPAs use labeled probes that hybridize to specific DNA sequences, allowing for the detection of common resistance mutations
  • Other Methods
    • Mass spectrometry: Can be used to detect specific mutations based on mass differences
    • Microarrays: Can be used to detect multiple resistance markers simultaneously

Specimen Considerations

  • Specimen Type: The choice of specimen type depends on the site of infection and the target organism. Sputum, bronchoalveolar lavage fluid, tissue biopsies, and cultures can be used
  • Specimen Collection: Collect specimens using appropriate techniques to minimize contamination and ensure adequate sample volume
  • Specimen Processing: Process specimens according to established protocols to optimize DNA extraction and amplification

Advantages

  • Rapid Results: Provides rapid detection of resistance markers, allowing for timely initiation of appropriate therapy
  • Detection of Resistance Mechanisms: Identifies specific genetic mutations associated with resistance
  • Culture-Independent Detection: Can be performed directly on clinical specimens, bypassing the need for culture in some cases
  • High Sensitivity and Specificity: Molecular methods are generally highly sensitive and specific

Limitations

  • Limited Scope: Only detects known resistance mechanisms, so novel mechanisms or resistance due to other factors may be missed
  • Cost: Molecular methods can be more expensive than traditional phenotypic methods
  • Technical Expertise: Requires specialized equipment and technical expertise
  • Interpretation Challenges: Interpretation of results can be complex, especially with NGS data
  • Detection of Non-Viable Organisms: May detect DNA from non-viable organisms, which may not represent active infection
  • Does not replace phenotypic methods: Does not detect novel resistance mechanisms

Interpretation of Results

  • Detection of Resistance Marker: Indicates resistance to the corresponding antimicrobial agent
  • Absence of Resistance Marker: Does not necessarily indicate susceptibility, as other resistance mechanisms may be present
  • Discordant Results: Discordant results between molecular and phenotypic testing may occur due to various factors, such as:
    • Heteroresistance: The presence of both susceptible and resistant subpopulations within a bacterial population
    • Novel Resistance Mechanisms: Resistance mechanisms that are not detected by the molecular assay
    • Technical Errors: Errors in the performance or interpretation of the tests
  • Clinical Correlation: Results should always be interpreted in conjunction with clinical findings and other laboratory data

Key Terms

  • Resistance Marker: A specific genetic mutation or sequence that is associated with resistance to an antimicrobial agent
  • NAAT (Nucleic Acid Amplification Test): A molecular test that amplifies specific DNA or RNA sequences from a target organism
  • PCR (Polymerase Chain Reaction): A molecular biology technique used to amplify specific DNA sequences
  • Real-Time PCR (qPCR): A type of PCR that allows for real-time monitoring of the amplification process
  • Multiplex PCR: A PCR assay that can detect multiple targets simultaneously
  • DNA Sequencing: Determining the precise order of nucleotide bases (A, T, C, G) in a DNA molecule
  • Sanger Sequencing: A method for determining the nucleotide sequence of DNA
  • Next-Generation Sequencing (NGS): A technology that allows for rapid sequencing of multiple DNA or RNA molecules simultaneously
  • Hybridization Assay: A molecular test that uses labeled probes to bind to specific DNA or RNA sequences
  • Line Probe Assay (LPA): A hybridization assay that uses labeled probes attached to a solid support to detect specific DNA sequences
  • rpoB gene: A gene encoding the beta subunit of RNA polymerase, mutations in which are associated with rifampin resistance in M. tuberculosis
  • katG gene: A gene encoding catalase-peroxidase, mutations in which are associated with isoniazid resistance in M. tuberculosis
  • inhA gene: A gene encoding an enoyl-ACP reductase, mutations in the promoter region of which are associated with isoniazid resistance in M. tuberculosis
  • gyrA gene: A gene encoding DNA gyrase subunit A, mutations in which are associated with fluoroquinolone resistance
  • rrs gene: A gene encoding 16S ribosomal RNA, mutations in which are associated with aminoglycoside resistance
  • erm(41) gene: A gene encoding an rRNA methyltransferase, the presence and sequence of which determines inducible macrolide resistance in Mycobacterium abscessus
  • sul1 and sul2 genes: Genes encoding dihydropteroate synthase, mutations in which are associated with sulfonamide resistance in Nocardia
  • Phenotypic Susceptibility Testing: Methods for determining drug susceptibility based on observable characteristics of the organism, such as growth in the presence of the drug
  • Genotypic Susceptibility Testing: Methods for determining drug susceptibility based on the detection of specific resistance mutations in the organism’s DNA
  • Heteroresistance: The presence of both susceptible and resistant subpopulations within a bacterial population
  • Clinical Correlation: The relationship between laboratory results and the patient’s clinical condition
  • Mass Spectrometry: An analytical technique used to measure the mass-to-charge ratio of ions
  • Microarray: A collection of DNA probes attached to a solid surface used to detect multiple targets simultaneously
  • High-throughput: The ability to process a large number of samples quickly