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Microbiology

Phenotypic Resistance

This section explores phenotypic methods, which rely on observable characteristics of bacteria to detect specific resistance mechanisms

Beta-Lactamase Detection

  • Mechanism of Resistance: Beta-lactamases are bacterial enzymes that hydrolyze the beta-lactam ring of beta-lactam antibiotics (penicillins, cephalosporins, carbapenems, and monobactams), rendering them inactive. This is a common mechanism of resistance
  • Methods
    1. Acidimetric Method: This method detects the production of acid by hydrolysis of the beta-lactam ring. A bacterial suspension is mixed with a beta-lactam antibiotic (e.g., penicillin) and a pH indicator (e.g., bromcresol purple). If beta-lactamase is present, the antibiotic is hydrolyzed, producing acid and causing a color change in the indicator
    2. Chromogenic Cephalosporin (Cefinase) Test: This is a rapid and widely used test. A nitrocefin disk is used, which contains a chromogenic cephalosporin. If beta-lactamase is present, the beta-lactam ring is hydrolyzed, producing a red-colored product. A positive result is indicated by a rapid color change
    3. Automated Methods: Some automated systems can detect beta-lactamase production using similar principles
  • Interpretation: A color change in the acidimetric or chromogenic cephalosporin test indicates the presence of beta-lactamase and resistance to beta-lactam antibiotics. The interpretation of the results should be correlated with the susceptibility testing results
  • Application: This test is typically performed on Gram-positive and Gram-negative bacteria to determine resistance to beta-lactam antibiotics. Results are used to guide antibiotic selection

Extended-Spectrum Beta-Lactamase (ESBL) Detection

  • Mechanism of Resistance: ESBLs are a group of beta-lactamases that can hydrolyze extended-spectrum cephalosporins (e.g., ceftazidime, cefotaxime, ceftriaxone) and monobactams (e.g., aztreonam), while retaining susceptibility to cephamycins and carbapenems. They are typically found in Enterobacteriaceae
  • Methods
    1. Screening: Many laboratories screen for ESBL production using susceptibility testing with extended-spectrum cephalosporins. Isolates with reduced susceptibility (e.g., resistance or intermediate susceptibility) to these agents are considered potential ESBL producers
    2. Confirmatory Testing: Confirmatory tests are performed on isolates that screen positive. Common confirmatory tests include:
      • Combination Disk Diffusion Test: This test uses disks containing an extended-spectrum cephalosporin (e.g., ceftazidime, cefotaxime) alone and in combination with a beta-lactamase inhibitor (e.g., clavulanic acid). An increase in the zone of inhibition of ≥5 mm with the combination disk compared to the cephalosporin disk alone suggests ESBL production
      • MIC Testing with and without Beta-Lactamase Inhibitor: This is similar to the combination disk diffusion test, but it uses MIC testing to detect the effect of the beta-lactamase inhibitor. A significant reduction in the MIC of the cephalosporin in the presence of the inhibitor suggests ESBL production
  • Interpretation: A positive confirmatory test (e.g., increased zone diameter or reduced MIC) indicates ESBL production and resistance to extended-spectrum cephalosporins. These isolates are often also resistant to penicillins and may exhibit cross-resistance to other antibiotics
  • Application: ESBL detection is crucial because these enzymes can lead to treatment failures with extended-spectrum cephalosporins. Results are used to guide antibiotic selection and infection control measures

Inducible Clindamycin Resistance Detection

  • Mechanism of Resistance: Some bacteria, particularly Staphylococcus aureus and Streptococcus species, can develop resistance to clindamycin through inducible resistance. This is mediated by the erm gene, which modifies the ribosomal target of macrolides and lincosamides, leading to resistance to both classes of antibiotics
  • Methods
    1. D-Test (or D-Zone Test): This is a phenotypic test to detect inducible clindamycin resistance. Erythromycin and clindamycin disks are placed on a Mueller-Hinton agar plate inoculated with the organism. If the organism is resistant to erythromycin and susceptible to clindamycin, and the erm gene is present, the erythromycin will induce the erm gene, leading to the production of resistance to clindamycin. This results in a flattening of the zone of inhibition around the clindamycin disk, forming a “D” shape
  • Interpretation: A positive D-test (D-shaped zone) indicates inducible clindamycin resistance. The organism should be reported as resistant to clindamycin, even if it appears susceptible in routine susceptibility testing
  • Application: This test is important for Staphylococcus aureus and streptococcal infections. Clindamycin may be ineffective in treating infections caused by organisms with inducible clindamycin resistance. The D-test helps to prevent treatment failures

Carbapenemase Detection

  • Mechanism of Resistance: Carbapenemases are beta-lactamases that hydrolyze carbapenem antibiotics (e.g., imipenem, meropenem, ertapenem), rendering them inactive. Carbapenem resistance is a serious concern due to the limited treatment options for these infections
  • Methods
    1. Screening: Isolates with resistance to carbapenems are screened for carbapenemase production
    2. Confirmatory Testing: Several methods are used to confirm carbapenemase production:
      • Modified Hodge Test (MHT): This is a phenotypic test for carbapenemase production. A carbapenem disk is placed on a Mueller-Hinton agar plate that has been inoculated with a carbapenem-susceptible Escherichia coli or Klebsiella pneumoniae and the test organism. A positive test results in a “cloverleaf” appearance, indicating carbapenem hydrolysis and resistance
      • Carbapenem Inactivation Method (CIM): This method uses a carbapenem disk incubated with a bacterial suspension. After incubation, the disk is placed on a lawn of a carbapenem-susceptible organism. The carbapenemase enzyme in the test organism hydrolyzes the carbapenem in the disk, and if carbapenemase is present, the susceptible organism will grow around the disk
      • Modified Carbapenem Inactivation Method (mCIM): This is a modified version of the CIM, and it is used to detect carbapenemase production. The test organism is incubated with a meropenem disk, and the disk is placed on a lawn of a carbapenem-susceptible organism
      • Carba NP Test: This is a rapid colorimetric test that detects carbapenem hydrolysis. The test organism is incubated with a carbapenem, and the pH is measured. If carbapenemase is present, the carbapenem is hydrolyzed, producing acid and causing a color change in the pH indicator
      • Molecular Methods: Molecular methods, such as PCR, are used to detect the genes that code for carbapenemases (e.g., blaKPC, blaOXA-48, blaNDM)
  • Interpretation: A positive confirmatory test indicates carbapenemase production and resistance to carbapenems. These isolates often exhibit resistance to other antibiotics
  • Application: Carbapenemase detection is crucial for guiding antibiotic selection and infection control measures. Carbapenem-resistant organisms are associated with high mortality rates. The results are used to guide antibiotic therapy and implement infection control strategies to prevent the spread of resistant organisms

General Considerations

  • Quality Control: Perform quality control on all tests using appropriate control organisms (e.g., Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922)
  • Interpretive Criteria: Always refer to the most current CLSI guidelines for the most up-to-date interpretive criteria for each test
  • Reporting: Report the results of these tests to the clinician, along with the results of routine susceptibility testing. These results are essential for guiding antibiotic therapy and infection control measures
  • Limitations: Phenotypic tests may not detect all resistance mechanisms. Molecular methods are often used to confirm the presence of resistance genes
  • Rapid Methods: Rapid tests are available for some resistance mechanisms, allowing for faster identification of resistance and timely reporting to clinicians

Key Terms

  • MIC (Minimum Inhibitory Concentration): The lowest concentration of an antimicrobial agent that completely inhibits the visible growth of a microorganism after incubation
  • Beta-Lactamase: An enzyme produced by some bacteria that inactivates beta-lactam antibiotics (e.g., penicillins, cephalosporins) by breaking the beta-lactam ring
  • ESBL (Extended-Spectrum Beta-Lactamase): A type of beta-lactamase that hydrolyzes extended-spectrum cephalosporins and monobactams, often found in Enterobacteriaceae
  • Carbapenemase: A type of beta-lactamase that hydrolyzes carbapenem antibiotics (e.g., imipenem, meropenem), leading to carbapenem resistance
  • Inducible Resistance: Resistance that is not expressed unless the organism is exposed to an inducing agent, such as an antibiotic
  • Zone of Inhibition: The clear area around an antibiotic disk on an agar plate where bacterial growth is inhibited
  • Susceptible (S): A category indicating that the microorganism is inhibited by the antimicrobial agent at concentrations achievable at the site of infection using the normal dosage
  • Intermediate (I): A category indicating that the microorganism may be inhibited by the antimicrobial agent at a higher concentration, or when the agent is concentrated at the site of infection
  • Resistant (R): A category indicating that the microorganism is not inhibited by the antimicrobial agent at concentrations achievable at the site of infection using the normal dosage
  • Phenotypic Resistance: Resistance determined by observing the characteristics of the organism, such as its ability to produce an enzyme that inactivates an antibiotic