Growth and metabolism of Clostridioides difficile in hungate-style media

Authors: LAKES, JOURDAN - Oak Ridge Institute For Science And Education (ORISE); Ferrell, Jessica; Flythe, Michael

Submitted to: Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/25/2023
Publication Date: 12/30/2023
Citation: Lakes, J.E., Ferrell, J.L., Flythe, M.D. 2023. Growth and metabolism of Clostridioides difficile in hungate-style media. Applied Microbiology. 4(1):85-95. https://doi.org/10.3390/applmicrobiol4010006.
DOI: https://doi.org/10.3390/applmicrobiol4010006

Interpretive Summary: Clostridioides difficile, commonly called C. difficile or "C. diff", is well know for causing diarrhea in people who undergo prolonged antibiotic treatment. Horses and other livestock can also get sick from this bacterium. C. difficile has close relatives on the tree of life that normally grow in the rumen (first "stomach") of ruminant animals. These latter bacteria do not cause disease, but they decrease the amount of amino acids that the animal host gets from the diet, converting the amino acids to ammonia. The close relationship makes C. difficile an important comparison to the ammonia-producing rumen bacteria. However, the comparisons have not been possible because of how C. difficile is typically grown in the laboratory. Medical microbiologists use commercially available culture media. As gastrointestinal microbiologists with a focus on ruminant nutrition, we make our own media. Our custom media types enable us tell exactly what resources bacteria are using and what they are making. We determined that C. difficile will grow in our custom media types and that it will use sugar, amino acids, and make its typical products. These results will allow direct comparison of C. difficile to its close relatives from ruminants, horses, and other livestock. The impact of this work is better understanding of both C. difficile and related, nutritionally important bacteria.

Technical Abstract: Objective: Clostridioides difficile is a clinically and agriculturally important organism with diverse metabolic capabilities. Commercially available media types used to grow C. difficile typically include multiple growth substrates and often selective agents. Under these conditions it is difficult to determine what the bacteria utilized, and which products derived from which substrates. Methods: These experiments compared a commercial broth (Reinforced Clostridium Medium/RCM) to simpler, defined, carbonate-based media types influenced by Robert Hungate. Peptides (tryptone peptone, T), amino acids (casamino acids, CAA) and/or glucose (G) were added to evaluate the growth and metabolism of C. difficile strain ATCC 9689. Results: C. difficile grew to the greatest optical density (3.0 OD600) in the rich RCM broth but produced less ammonia than the tryptone-containing media types (p < 0.05). C. difficile utilized all glucose in RCM and T+G media in addition to performing amino acid fermentations (measured by ammonia production), though the volatile fatty acids produced, and the concentrations thereof, were not necessarily consistent across media type. In T, T+G, and CAA+G media 9689 produced acetate (8, 16, and 3 mM, respectively) but its production was below the limit of quantitation in RCM media. Generally, when cultured in CAA-containing media, 9689 performed very little metabolism and did not grow regardless of supplementation with glucose. Conclusions: These data demonstrated that C. difficile 9689 could metabolize substrates and grow in defined, anaerobic, carbonate-buffered media. Hungate-style media appear to be an acceptable choice for reliable in vitro culture of C. difficile 9689.