<i>Klebsiella pneumoniae</i> Cross‐feeds <i>Clostridioides difficile</i> and Enhances Colonic Pro‐inflammatory Responses

Recent in-depth analysis of the human gut microbiota demonstrates that many intestinal disorders are polymicrobial in origin and manifestation. Select microbes can interact synergistically with pathobionts to induce virulence traits, modify environmental niches, and modulate host responses. In the setting of Clostridioides difficile infection, polymicrobial interactions are incompletely understood. C. difficile is an intestinal pathogen responsible for 500,000 infections and 30,000 deaths annually in the US. Using stool-seeded bioreactors with mucus-coated inserts, we identified Klebsiella pneumoniae in the mucus-associated microbiota along with C. difficile.We hypothesized that K. pneumoniaemay enhance C. difficile traits required for colonization.To test this hypothesis, we cultured 11 K. pneumoniae strains with four C. difficile strains (representing 4 different ribotypes) in a fully defined bacteria media ZMB1. We observed that cell-free supernatant from all K. pneumoniae strains enhanced the growth of all C. difficile strains, indicating that metabolites produced by K. pneumoniae enhanced C. difficile growth. Importantly, C. difficile grew well with K. pneumoniae bacteria and both bacteria were identified in co-cultures by gram staining. Non-targeted metabolomics revealed that K. pneumoniae shifted the metabolism of C. difficile away from amino acid utilization. K. pneumoniae metabolites also suppressed toxin production in an in vitro model of nutrient starvation as assessed by live-imaging of LifeAct expressing Vero cell rounding. These findings further confirm the ability of K. pneumoniae to cross-feed C. difficile. Although K. pneumoniae is well known for robust biofilm production, K. pneumoniae did not significantly enhance C. difficile biofilm formation, indicating that biofilm synthesis is not a significant component of C. difficile-Klebsiella interactions. RNA sequencing of mouse colonic inside-out organoids incubated with live C. difficile and/or K. pneumoniae revealed increased pro-inflammatory responses when the two microbes were added together compared to either microbe alone or media controls.These results suggest that K. pneumoniae can cross-feed and shift the metabolism of C. difficile, as well as enhance the pro-inflammatory capacity of C. difficile. We speculate that interspecies interactions could be among the earliest events that occur as C. difficile colonizes the host. As a result, we believe that an improved understanding of how mucus associated microbes like K. pneumoniae interact with C. difficile will continue to clarify the mechanisms of C. difficile infection.