Clostridium difficile is a major cause of nosocomial infections. Bacterial persistence in the gut is responsible for infection relapse; sporulation and other unidentified mechanisms contribute to this process. Intestinal bile salts cholate and deoxycholate stimulate spore germination, while deoxycholate kills vegetative cells. Here, we report that sub-lethal concentrations of deoxycholate stimulate biofilm formation, which protects C . difficile from antimicrobial compounds. The biofilm matrix is composed of extracellular DNA and proteinaceous factors that promote biofilm stability. Transcriptomic analysis indicates that deoxycholate induces metabolic pathways and cell envelope reorganization, and represses toxin and spore production. In support of the transcriptomic analysis, we show that global metabolic regulators and an uncharacterized lipoprotein contribute to deoxycholate-induced biofilm formation. Finally, Clostridium scindens enhances biofilm formation of C. difficile by converting cholate into deoxycholate. Together, our results suggest that deoxycholate is an intestinal signal that induces C. difficile persistence and may increase the risk of relapse.

Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.bacterial ecology | biofilm disruption | motile subpopulations | antimicrobials | time-lapse confocal imaging