| Summary: | The Gram positive anaerobic bacterium, Clostridioides difficile, is one of the leading causes of hospital associated diarrhoea world-wide. An opportunistic pathogen, C. difficile colonises the gut during intestinal microbial dysbiosis, causing Clostridioides difficile infection (CDI). Treatment of CDI is complicated by the increasing numbers of recurrent infections. C. difficile has demonstrated its ability to produce composite, adherent multicellular communities, or biofilms, in vitro. In vitro biofilms offer the bacteria within increased resistance to a range of environmental stresses including antibiotics and oxygen stress. However the mechanisms underlying C. difficile community formation are poorly understood. In other bacteria, quorum sensing, a process mediated by small signalling molecules that accumulate in the extracellular environment, coordinates biofilm formation. In several bacteria, the metabolic enzyme LuxS, produces the signalling molecule autoinducer-2 (AI-2), which plays a key role in quorum sensing. AI-2 is considered to be a cross-species signalling molecule and for many bacterial species AI-2 has been shown to have a signalling role during biofilm formation and development. Here we show C. difficile luxS mutants (LuxS) are defective in biofilm formation and demonstrate the ability for chemically synthesised AI-2 to partially restore the biofilm defect of LuxS. Through RNA-seq analysis we show that LuxS/AI-2 quorum sensing likely influences C. difficile prophage expression, affecting levels of extracellular DNA present within the biofilm. Additionally we show that Bacterioides fragilis has an inhibitory effect on C. difficile, with increased levels of inhibition observed in WT compared to LuxS. By utilising dual species RNA-sequencing we propose a number of possible mechanisms responsible for the observed inhibition.
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