Clostridium difficile is a major cause of healthcare-associated infection and inflicts a considerable financial burden on healthcare systems worldwide. Disease symptoms range from self-limiting diarrhoea to fatal pseudomembranous colitis. Whilst C. difficile has two major virulence factors, toxin A and B, it is generally accepted that other virulence components of the bacterium contribute to disease. C. difficile colonises the gut of humans and animals and hence the processes of adherence and colonisation are essential for disease onset. Previously it has been suggested that flagella might be implicated in colonisation. Here we tested this hypothesis by comparing flagellated parental strains to strains in which flagella genes were inactivated using ClosTron technology. Our focus was on a UK-outbreak, PCR-ribotype 027 (B1/NAP1) strain, R20291. We compared the flagellated wild-type to a mutant with a paralyzed flagellum and also to mutants (fliC, fliD and flgE) that no longer produce flagella in vitro and in vivo. Our results with R20291 provide the first strong evidence that by disabling the motor of the flagellum, the structural components of the flagellum rather than active motility, is needed for adherence and colonisation of the intestinal epithelium during infection. Comparison to published data on 630Δerm and our own data on that strain revealed major differences between the strains: the R20291 flagellar mutants adhered less than the parental strain in vitro, whereas we saw the opposite in 630Δerm. We also showed that flagella and motility are not needed for successful colonisation in vivo using strain 630Δerm. Finally we demonstrated that in strain R20291, flagella do play a role in colonisation and adherence and that there are striking differences between C. difficile strains. The latter emphasises the overriding need to characterize more than just one strain before drawing general conclusions concerning specific mechanisms of pathogenesis.

EA 4043, USC INRA 'Ecosystè me microbien digestif et santé ', Faculté de Pharmacie, Université Paris-Sud 11, Châ tenay-Malabry, FranceClostridium difficile is a frequent cause of severe, recurrent, post-antibiotic diarrhoea and pseudomembranous colitis. Its pathogenicity is mediated mainly by two toxins, TcdA and TcdB. However, different adhesins have also been described as important colonization factors which are implicated in the first step of the intestinal infection. In this study, we focused our interest on one of these adhesins, fibronectin-binding protein A (FbpA), and on its role in the intestinal colonization process. A mutant of FbpA (CDDFbpA) was constructed in C. difficile strain 630Derm by using ClosTron technology. This mutant was characterized in vitro and in vivo and compared to the isogenic wild-type strain. Adhesion of the CDDFbpA mutant to the human colonic epithelial cell line Caco-2 and to mucus-secreting HT29-MTX cells was examined. Surprisingly, the CDDFbpA mutant adhered more than the wild-type parental strain. The CDDFbpA mutant was also analysed in three different mouse models by following the intestinal implantation kinetics (faecal shedding) and caecal colonization (7 days post-challenge). We showed that in monoxenic mice, CDDFbpA shed C. difficile in faeces at the same rate as that of the isogenic wild-type strain but its colonization of the caecal wall was significantly reduced. In dixenic mice, the shedding rate was slower for the CDDFbpA mutant than for the isogenic wildtype strain during the first days of infection, but no significant difference was observed in caecal colonization. Similar rates of intestinal implantation and caecal colonization were observed for both strains in assays performed in human microbiota-associated mice. Taken together, our data suggest that FbpA plays a role in intestinal colonization by C. difficile. INTRODUCTIONClostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence, especially with the appearance of hypervirulent strains in the last few years. It is the major cause of pseudomembranous colitis and causes 15 -20 % of antibiotic-associated diarrhoea cases associated with the use of antibiotic treatment (Cartman et al., 2010;Poxton et al., 2001). Antibiotics disrupt the normal intestinal microbiota, allowing C. difficile to colonize the gut. The pathogenesis of C. difficile infections has been attributed to two toxins, TcdA and TcdB, which act as glycosyltransferases and modify small GTPases of the Rho protein family within the host cell, resulting in alterations in the cytoskeleton (Genth et al., 2008;Voth & Ballard, 2005). Apart from these two toxins, a binary toxin is also produced by a few strains but little is known about the other virulence factors which are involved in the colonization process. Presently, only a few cell surface proteins have been identified and characterized. These proteins include the S-layer proteins (Calabi et al., 2002;Cerquetti et al., 2000), the flagellum and its components (Tasteyr...