Clostridioides difficile is a bacterium that causes severe intestinal illness, but can be difficult to study and treat, and represents a significant public health issue. Studies in mice and in children carried out by researchers at Children’s Hospital of Philadelphia (CHOP) have now found that an opportunistic, antibiotic-resistant Enterococcus pathogen works together with C. difficile to reshape and enhance the metabolic environment in the gut so that C. difficile can thrive.

“When we talk about bacterial infections, we often just think of the pathogen itself, but the ‘bystanders’ in the gut can have a huge impact on the course of infection,” said Joseph P. Zackular, PhD, Investigator and Assistant Professor of Pathology and Laboratory Medicine at Children’s Hospital of Philadelphia. “This study reveals that the coincidence of two pathogenic organisms—Enterococcus and C. difficile—is more than a coincidence; they truly take advantage of each other. Understanding this relationship, as well as other factors that contribute to clinical outcomes of C. difficile infection, is essential for combating this urgent public health challenge.”

Zackular is senior author of the team’s published paper in Nature, which is titled “Enterococci enhance Clostridioides difficile pathogenesis.” In their report the authors concluded, “These findings have implications for our understanding of the variables affecting CDI, the risk of recurrence and the factors that influence treatment outcomes.”

Approximately 1 in 6 patients infected with C. difficile will be reinfected within two months. Yet scientists have not figured out why C. difficile infection (CDI) is more difficult to treat in some patients than it is in others. “Understanding the factors that contribute to the clinical outcomes of CDI is essential for combating this urgent public health challenge,” the scientists wrote. The human gut is filled with trillions of microbes, and these microbes influence the virulence of various pathogens, but until now, scientists have also had little understanding of how C. difficile cooperates with the rich collection of microorganisms in the gastrointestinal tract. “The study of pathogen–microbiota interactions during infection is central to our ability to understand and treat enteric infections,” the team continued. “One of the most significant enteric pathogens globally is Clostridioides difficile, but little is known about how C. difficile cooperates with the rich collection of microorganisms in the gastrointestinal tract.”

Prior studies have shown that adults infected with C. difficile have high levels of Enterococcus in their gut and that vancomycin-resistant Enterococcus (VRE) frequently coinfects patients with C. difficile. “However, the effect of Enterococcus on susceptibility to C. difficile infection (CDI) and clinical outcomes remains unknown,” the scientists continued.

To further define the association between Enterococcus and C. difficile during infection, Zackular and colleagues analyzed stool samples from 54 pediatric patients infected with C. difficile. Consistent with studies in adults, the researchers found that stool samples from these pediatric patients had high levels of Enterococcus, and there was a positive correlation between enterococcal and C. difficile burdens. “These data confirm that enterococci are highly abundant in the CDI gut and positively correlate with C. difficile burden,” they wrote.

Having confirmation that enterococci are highly abundant in the gut of children with a C. difficile infection and that this positively correlates with C. difficile burden, the researchers then validated the mechanism of how these two pathogens work together. Using both in vitro and in vivo experimental models, they found that enterococci increase C. difficile virulence by enhancing its production of toxins.

Then, using transcriptomic and metabolomics data relating to the pathogens, the researchers discovered that enterococci reshape the gut metabolic environment, making it more conducive for the C. difficile pathogen to thrive. They found that enterococci use arginine, an amino acid, for energy and that in the process of doing so, the pathogen exports ornithine, another amino acid. Further analysis showed that enterococci modulate levels of arginine and ornithine in the gut during C.difficile infection and that arginine depletion plays a central role in C. difficile virulence. As the authors explained, “Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence.”

Finally, the researchers explored whether their findings in the lab correlated with findings in human patients. Analyzing the microbiomes of children with C. difficile infection and inflammatory bowel disease (IBD), they found that these children had high levels of fermentable amino acids, including ornithine. The team also observed a positive correlation between C. difficile burden and ornithine, supporting a key role for this amino acid in C. difficile infection. “Collectively, these data suggest that enterococci and C. difficile interact during CDI through metabolic cross-talk to support increased colonization, pathogenesis, and persistence in the gut,” the authors concluded. “Together, our work demonstrates the supportive role of pathogenic microbiota in the outcome of CDI and highlights the importance of integration of metabolic signals from the microbiota in pathogen virulence.” Zackular suggested that “Future research should explore targeting enterococcal metabolism —and the resulting amino acid landscape in the gut—as a way of altering the pathogenesis of C. difficile.”

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