the toxin a- and toxin b - specific recombination vectors pjir3051 and pjir3050 were constructed by cloning a 566 bp tcda ( accession m30307 ) pcr product ( nt 10711637 ) and the equivalent tcdb ( accession x53138 ) region , respectively , into the shuttle vector pjir1456 ( accession u90554 ) .
these plasmids were transferred by conjugation to c. difficile strain jir8094 and the resultant chromosomal mutants analyzed by pcr , southern blotting and plasmid rescue13 .
filtered supernatants of 2ty broth cultures ( 72 h ) were used for western blots , cytotoxicity and toxin neutralization assays .
serial two - fold dilutions of these supernatants were used in vero and ht-29 cell cytotoxicity assays , with the end - point scored as the last dilution yielding complete cpe .
toxins were neutralized with affinity purified polyclonal antibodies specific for toxin a and toxin b ; neutralization was carried out at the last dilution giving complete cpe .
partially purified ammonium sulphate - precipitated toxin proteins were western blotted using toxin a - specific affinity purified polyclonal goat antibodies .
clindamycin was administered orogastrically to render the hamsters susceptible to infection ( day zero ) . on day
fecal pellets were collected daily for 12 days , then weekly until day 30 and inoculated onto selective tccfa plates to monitor colonization .
fecal pellets were analyzed for the presence of toxin a using a toxin a - specific elisa and for the presence of toxin b using a human foreskin fibroblast cytotoxicity assay .
full methods and any associated references are available in the online version of the paper at www.nature.com/nature .
 clostridium difficile is the leading cause of infectious diarrhea in hospitals worldwide , because of its virulence , spore - forming ability and persistence1,2 . 
 c. difficile - associated diseases ( cdad ) are induced by antibiotic treatment or disruption of the normal gastrointestinal flora3,4 . 
 recently , morbidity and mortality resulting from cdad have increased significantly due to changes in the virulence of the causative strains and antibiotic usage patterns1,2,5,6 . since 2002 , 
 epidemic toxinotype iii nap1/027 strains1,2 , which produce high levels of the major virulence factors , toxin a and toxin b , have emerged . 
 these toxins have 63% amino acid sequence similarity7 and are members of the large clostridial glucosylating toxin family , which are monoglucosyltransferases that are proinflammatory , cytotoxic and enterotoxic in the human colon810 . inside host cells , both toxins 
 catalyze the transfer of glucose onto the rho family of gtpases , leading to cell death8 , 11 . however , the role of these toxins in the context of a c. difficile infection is unknown . here 
 we describe the construction of isogenic tcda and tcdb mutants of a virulent c. difficile strain and their use in the hamster disease model to show that toxin b is a key virulence determinant . 
 previous studies showed that purified toxin a alone can induce most of the pathology observed following infection of hamsters with c. difficile8,9 , 12 and that toxin b is not toxic in animals unless it is co - administered with toxin a , suggesting that the toxins act synergistically12 . 
 our work provides evidence that toxin b , not toxin a , is essential for virulence , which represents a major paradigm shift . 
 furthermore , it is clear that the importance of these toxins in the context of infection can not be predicted exclusively from studies using purified toxins , reinforcing the importance of using the natural infection process to dissect the role of toxins in disease . 
METHODS SUMMARY
Supplementary Material