Antibiotics Help Clostridium Difficile Compete for Nutrients to Grow

Casey Theriot, PhD, assistant professor of infectious disease at North Carolina State University

Casey Theriot, PhD

Researchers studying how antibiotic use affects the growth of Clostridium difficile (C. difficile) bacteria in the gut of mice may have taken a step toward developing strategies for preventing the infection.

“If we can figure out what C. difficile requires for growth in the gastrointestinal tract, we can use this information against C. difficile to design novel therapeutics to restore colonization resistance against C. difficile in the gut,” Casey Theriot, PhD, assistant professor of infectious disease at North Carolina State University, told MD Magazine.

Such therapeutics might include new strains of targeted probiotics, said Theriot, corresponding author of a paper published in mSphere that describes the research.

“Our results also further highlight the importance of the inverse relationship between the indigenous gut microbiota and nutrient levels, including many nutrients that are essential for C. difficile colonization,” researchers wrote in the paper.

Theriot and Joshua Fletcher, a postdoctoral fellow at NC State, used a mouse model to show that antibiotic use alters existing gut bacteria that normally compete with C. difficile for nutrients.

Mice were exposed to the C. difficile pathogen, a major cause of antibiotic-associated diarrhea that can lead to fatal infections in the gut. The bacteria are found throughout the environment, but cause infections mainly in patients who are taking, or have recently taken antibiotics.

The researchers then conducted metabolomic and RNA sequencing analysis of the gut contents and C. difficile at 4 time points, 0, 12, 24, and 30 hours, to find out which nutrients the bacteria were "eating."

The 2-tiered approach reinforced the idea that C. difficile uses certain amino acids and carbohydrates early in the process of colonization of a susceptible host, the researchers wrote.

“We already know that in order for C. difficile to produce toxin, which mediates disease in the gut, it has to grow to high levels,” Theriot said. “In order to grow to high levels, it requires nutrients, specifically proline and other amino acids.’’

Proline is used in the biosynthesis of proteins.

“What we found is that after antibiotics, proline and other proline-rich metabolites were increased in the mouse gut,” she said. “When C. difficile was introduced to the mouse it was able to grow to high levels and produce toxin.’’

Theriot noted that the main byproduct produced throughout the C. difficile infection was 5-aminovalerate, which is a byproduct of proline metabolism. C. difficile gene expression was consistent with their utilization by the bacterium in vivo, she said.

“This finding is interesting because we now have a metabolic target to go after, so we can design a targeted probiotic strain that is able to outcompete C. difficile in the gut for amino acids, specifically proline,’’ Theriot said.

Asked how the findings might translate to C. difficile infections in people, Theriot replied: "The clinical implications of this study are still to be determined at this point, since this study was done in mice and not humans.’’

However, she noted, the findings suggest that proline and proline-rich metabolites such as trans-4-hydroxyproline are very important for growth during the early stages of C. difficile colonization.

"We see evidence of this based on the production of 5-aminovalerate throughout colonization and infection in this mouse model of C. difficile, which is a byproduct of proline utilization,’’ she said.

Theriot and her team are moving ahead with new research.

“We have been working on the next part of this story for the last year,’’ she said.

They plan more mechanistic studies to really prove that proline is the “smoking gun” and hope to figure out where the proline is coming from.

"There are many factors that could be supplying proline to C. difficile in the gut, including other members of the gut microbiota, diet, and the host," Theriot said. “Now we need to figure out where it is coming from and shut it down.”

The paper, "Shifts in the Gut Metabolome and Clostridium difficile Transcriptome throughout Colonization and Infection in a Mouse Model" appears in mSphere.