Herein we describe a real-time quantitative PCR assay for evaluating the adherence of Clostridium difficile to differentiated human intestinal Caco-2 cells. Our investigations demonstrated that the method, employing the C. difficile-specific triose-phosphate isomerase gene, is as reliable but less time-consuming than counting c.f.u. We conclude that the method will be useful for evaluating the role of host cell adherence in the pathogenesis of C. difficile infection.
INTRODUCTIONClostridium difficile is a Gram-positive anaerobic microorganism that is the most important cause of antibioticassociated diarrhoea, now generally referred to as C. difficile infection (CDI), in humans. To cause clinical illness, C. difficile first penetrates the mucus layer covering the epithelial surface of the gastrointestinal (GI) tract; a strategy that is most likely facilitated by flagella and proteases (Deneve et al., 2009;Tasteyre et al., 2001). Once established in the GI tract, C. difficile expresses two large exotoxins, toxin A (TcdA) and toxin B (TcdB) (Jank & Aktories, 2008;Voth & Ballard, 2005). Delivery of TcdA and TcdB to the cytoplasmic compartment of epithelial cells then leads to glucosylation of Rho family GTPases, resulting in disorganization of the host cell cytoskeleton (Deneve et al., 2009). This results in the loosening of tight junctions between adjacent intestinal epithelial cells ultimately leading to inflammation and diarrhoea.Although TcdA and TcdB represent primary virulence factors, whether adherence to host epithelial cells is also important in the C. difficile pathogenic strategy is less clear. However, C. difficile adherence to epithelial cells in vitro has been reported to be mediated by several colonization factors, including the low-and high-molecular-mass Slayer proteins which are derived by post-translational cleavage of the S-layer precursor protein, SlpA (Calabi et al., 2001). The high-molecular-mass S-layer protein is largely responsible for C. difficile binding to both human GI tissues and to components of the extracellular matrix (Calabi et al., 2002). In addition, Cwp84, a putative colonization factor and cysteine protease, has recently been implicated in a process whereby SlpA is cleaved into lowand high-molecular-mass counterparts (Janoir et al., 2007;Kirby et al., 2009). Several other cell-surface colonization factors including Cwp66, Fbp68 and GroEL, may also all play a role in C. difficile adherence to intestinal epithelial cells (Hennequin et al., , 2003Waligora et al., 2001). Moreover, the flagellar proteins FliC (flagellin) and FliD (flagellar cap protein) bind to axenic mouse mucus possibly facilitating the initial penetration of the intestinal mucus layer (Tasteyre et al., 2001). CDI patients are known to produce serum antibodies to one or more of these cellsurface proteins during infection, emphasizing their potential role in pathogenesis and generating a protective host immune response (Pechine et al., 2005;Wright et al., 2008).Several in vitro C. difficile adherence assays have be...