The Integrity of Heme Is Essential for Reproducible Detection of Metronidazole-Resistant Clostridioides difficile by Agar Dilution Susceptibility Tests

J Clin Microbiol. 2021 Aug 18;59(9):e0058521. doi: 10.1128/JCM.00585-21. Epub 2021 Aug 18.

Abstract

Metronidazole resistance in clinical Clostridioides difficile is often described as unstable, since resistant strains reportedly appear susceptible following freezer storage or brief passage. This has presented a conundrum for adopting susceptibility testing to accurately evaluate the connection between metronidazole resistance and decreased clinical efficacy of metronidazole in patients with C. difficile infections (CDIs). We discovered that supplementation of microbiological media with the metalloporphyrin heme is crucial for detection of metronidazole-resistant C. difficile using the agar dilution susceptibility testing method. Known metronidazole-resistant strains appeared susceptible to metronidazole in media lacking heme. Similarly, these resistant strains exhibited increased susceptibility to metronidazole when tested on heme-containing agars that were exposed to room light for more than 1 day, likely due to heme photodecomposition. In parallel experiments, resistance was reproducibly detected when heme-containing agars were either prepared and used on the same day or protected from light and then used on subsequent days. Notably, heme did not influence the susceptibilities of drug-susceptible strains that were of the same ribotype as the resistant strains. These findings firmly show that the consistent detection of metronidazole-resistant C. difficile is dependent upon heme and its protection from light. Studies are warranted to determine the extent to which this heme-associated metronidazole-resistant phenotype affects the clinical efficacy of metronidazole in CDI and the underlying genetic and biochemical mechanisms.

Keywords: antibiotic resistance; photodecomposition; porphyrin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Agar
  • Anti-Bacterial Agents / pharmacology
  • Anti-Bacterial Agents / therapeutic use
  • Clostridioides
  • Clostridioides difficile* / genetics
  • Clostridium Infections* / drug therapy
  • Heme
  • Humans
  • Metronidazole / pharmacology
  • Microbial Sensitivity Tests

Substances

  • Anti-Bacterial Agents
  • Metronidazole
  • Heme
  • Agar