KLEBSIELLA PNEUMONIAE IN THE COLONIC MUCUS LAYER INFLUENCESCLOSTRIDIODES DIFFICILE PATHOGENESIS

Abstract

Abstract BACKGROUND Clostridoides difficile is an intestinal pathogen responsible for 500,000 infections and 30,000 deaths annually in the US. Alarmingly, C. difficile infection is increased in patients with inflammatory bowel disease (IBD). When C. difficile infection is superimposed on colitis, it synergistically worsens both conditions. C. difficile infection is initiated by disruption of the gut microbiota; and it is well appreciated that IBD patients already have an altered fecal and mucus-associated microbiota. At present, it is not fully understood how gut microbes, particularly microbes found in IBD, influence C. difficile. Using stool-seeded bioreactors with mucus-coated inserts, we identified Klebsiella pneumoniae in the mucus-associated microbiota with C. difficile. K. pneumoniae is present at increased levels in IBD patients and we hypothesized that K. pneumoniae may promote C. difficile intestinal colonization. METHODS & RESULTS To test this hypothesis, we cultured 12 strains of K. pneumoniae with four ribotypes of C. difficile in a fully defined bacteria media (ZMB1). Growth curve analysis of 24 hour cultures revealed that cell-free supernatant from all K. pneumoniae strains enhanced the growth of all C. difficile strains, suggesting that K. pneumoniae could produce metabolites that enhanced C. difficile growth. Importantly, C. difficile grew well with K. pneumoniae bacteria and both bacteria were present in the cultures by gram stain. Non-targeted metabolomics revealed that the presence of K. pneumonia shifted the metabolism of C. difficile away from carbohydrate utilization and toward alternative fuel sources. K. pneumoniae is well known for its ability to create biofilms. To examine biofilm production, we grew C. difficile alone or with K. pneumoniae and examined biofilm by crystal violet staining. Although biofilms were nominally enhanced with K. pneumoniae compared to C. difficile alone, these biofilms were significantly lower than K. pneumoniae alone; indicating that biofilm synthesis is not a significant component of C. difficile-Klebsiella interactions. Finally, we examined how K. pneumoniae impacts C. difficile infection in vitro using colonic organoids by RNA sequencing. Sequencing revealed significantly elevated pro-inflammatory pathways when C. difficile and K. pneumonia were incubated together than either pathogen alone. CONCLUSIONS These results suggest that K. pneumoniae can influence C. difficile metabolism and exacerbate inflammation.