A multilocus sequence analysis of ten virulence-associated genes was performed to study the genetic relationships between 29 Clostridium difficile isolates of various origins, hosts and clinical presentations, and selected from the main lineages previously defined by multilocus sequence typing (MLST) of housekeeping genes. Colonization-factor-encoding genes (cwp66, cwp84, fbp68, fliC, fliD, groEL and slpA), toxin A and B genes (tcdA and tcdB), and the toxin A and B positive regulator gene (tcdD) were investigated. Binary toxin genes (cdtA and cdtB) were also detected, and internal fragments were sequenced for positive isolates. Virulence-associated genes exhibited a moderate polymorphism, comparable to the polymorphism of housekeeping genes, whereas cwp66 and slpA genes appeared highly polymorphic. Isolates recovered from human pseudomembranous colitis cases did not define a specific lineage. The presence of binary toxin genes, detected in five of the 29 isolates (17 %), was also not linked to clinical presentation. Conversely, toxigenic A"B+ isolates defined a very homogeneous lineage, which is distantly related to other isolates. By clustering analysis, animal isolates were intermixed with human isolates. Multilocus sequence analysis of virulence-associated genes is consistent with a clonal population structure for C. difficile and with the lack of host specificity. The data suggest a co-evolution of several of the virulence-associated genes studied (including toxins A and B and the binary toxin genes) with housekeeping genes, reflecting the genetic background of C. difficile, whereas flagellin, cwp66 and slpA genes may undergo recombination events and/or environmental selective pressure. INTRODUCTIONSince its recognition as the main cause of pseudomembranous colitis (PMC) in 1978, Clostridium difficile has emerged as an important enteropathogen (Bartlett et al., 1978;Johnson & Gerding, 1998;Larson et al., 1978). C. difficile is currently responsible for virtually all cases of PMC and for 10-25 % of antibiotic-associated diarrhoea (AAD) (Bartlett, 1994;Kelly et al., 1994). It is also the leading cause of nosocomial diarrhoea, and many hospital outbreaks have been described throughout the world (Barbut et al., 1994; Cartmill et al., 1994; McEllistrem et al., 2005). The two main virulence factors are exotoxins, toxins A (enterotoxin) and B (cytotoxin), both of which damage the human colonic mucosa and are potent tissue-damaging enzymes (Bongaerts & Lyerly, 1994;Borriello, 1998). Until recently, it was presumed that a strain must produce both toxins to be considered fully pathogenic. However, it is still unclear (i) why strains that do not produce toxin A (A2B+ variant strains) are yet responsible for diarrhoea and even PMC (Alfa et al., 2000;Toyokawa et al., 2003), and (ii) why some patients infected by toxinogenic strains will present a mild diarrhoea, whereas others will present a fulminant PMC.Abbreviations: AAD, antibiotic-associated diarrhoea; MLST, multilocus sequence typing; PaLoc, pathogenicity locus; P...