Insights into the regulatory mechanisms of Clostridioides difficile biofilm formation

Mucosal biofilms play an important role in intestinal health; however, the mucosal bacterial community has been implicated in persistent infections. Clostridioides difficile is an important nosocomial pathogen, with an unacceptable high rate of recurrence following antibiotic treatment. As C. difficile is a known biofilm producer, a property which may contribute to this suboptimal therapeutic response, we have investigated the transcriptional changes and regulatory pathways during the transition from planktonic to biofilm mode of growth. Widespread metabolic reprogramming during biofilm formation was detected, characterised by an increased usage of glycine metabolic pathways to yield key metabolites, which are used for energy production and synthesis of short chain fatty acids. We detected the expression of 107 small non-coding RNAs that appear to, in some part, regulate these pathways; however, 25 of these small RNAs were specifically expressed during biofilm formation, indicating they may play a role in regulating biofilm-specific genes. Similar to Bacillus subtilis , biofilm formation is a multi-regulatory process and SinR negatively regulates biofilm formation independently of other known mechanisms. This comprehensive analysis furthers our understanding of biofilm formation in C. difficile , identifies potential targets for anti-virulence factors, and provides evidence of the link between metabolism and virulence traits. ### Competing Interest Statement The authors have declared no competing interest.