Toxigenic Clostridium difficile strains produce two toxins (TcdA and TcdB) during the stationary phase of growth and are the leading cause of antibiotic-associated diarrhea. C. difficile isolates of the molecular type NAP1/027/BI have been associated with severe disease and hospital outbreaks worldwide. It has been suggested that these "hypervirulent" strains produce larger amounts of toxin and that a mutation in a putative negative regulator (TcdC) allows toxin production at all growth phases. To rigorously explore this possibility, we conducted a quantitative examination of the toxin production of multiple hypervirulent and nonhypervirulent C. difficile strains. Toxin gene (tcdA and tcdB) and toxin gene regulator (tcdR and tcdC) expression was also monitored. To obtain additional correlates for the hypervirulence phenotype, sporulation kinetics and efficiency were measured. In the exponential phase, low basal levels of tcdA, tcdB, and tcdR expression were evident in both hypervirulent and nonhypervirulent strains, but contrary to previous assumptions, toxin levels were below the detectable thresholds. While hypervirulent strains displayed robust toxin production during the stationary phase of growth, the amounts were not significantly different from those of the nonhypervirulent strains tested; further, total toxin amounts were directly proportional to tcdA, tcdB, and tcdR gene expression. Interestingly, tcdC expression did not diminish in stationary phase, suggesting that TcdC may have a modulatory rather than a strictly repressive role. Comparative genomic analyses of the closely related nonhypervirulent strains VPI 10463 (the highest toxin producer) and 630 (the lowest toxin producer) revealed polymorphisms in the tcdR ribosome binding site and the tcdR-tcdB intergenic region, suggesting that a mechanistic basis for increased toxin production in VPI 10463 could be increased TcdR translation and read-through transcription of the tcdA and tcdB genes. Hypervirulent isolates produced significantly more spores, and did so earlier, than all other isolates. Increased sporulation, potentially in synergy with robust toxin production, may therefore contribute to the widespread disease now associated with hypervirulent C. difficile strains.Clostridium difficile is a leading bacterial nosocomial pathogen. Antibiotic treatment alters and suppresses commensal microbiota, allowing ingested C. difficile spores to germinate and colonize the gut. If the infecting strain is of the toxinproducing lineage of C. difficile (toxigenic), the resulting infection (CDI) can range from mild diarrhea to potentially fatal pseudomembranous colitis. Since 2000, highly virulent variants of toxigenic C. difficile have caused epidemics of CDI characterized by greater incidence, severity, and fatality (12, 25, 29). These "hypervirulent" (HV) strains cluster into a distinct phylogenetic group (38), as assessed by several different molecular methods (21) [33,41]). BI/ NAP1/027 strains have spread rapidly and widely in the past 10 years and have ...

Studies suggest that asymptomatic colonization with Clostridium difficile (CD) decreases the risk of CD-associated disease (CDAD) in humans. A hamster model was used to test the efficacy of colonization with 3 nontoxigenic CD strains for preventing CDAD after exposure to toxigenic CD. Groups of 10 hamsters were given 10(6) nontoxigenic CD spores 2 days after receiving a single dose of clindamycin. Five days later, the hamsters were given 100 spores of 1 of 3 toxigenic CD strains previously shown to cause mortality within 48 h. Each nontoxigenic strain prevented disease in 87%-97% of hamsters that were challenged with toxigenic strains. Failure to prevent CDAD was associated with failure of colonization with nontoxigenic CD. Colonization with nontoxigenic CD strains is highly effective in preventing CDAD in hamsters challenged with toxigenic CD strains, which suggests that use of a probiotic strategy for CDAD prevention in humans receiving antibiotics might be beneficial.

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.

Five different toxigenic strains of Clostridium difficile of known human epidemiologic importance were tested for virulence in hamsters. Three strains-types B1, J9, and K14-have caused hospital outbreaks. Type Y2 is associated with a high rate of asymptomatic colonization in patients. The fifth strain, type CF2, is a toxin A-negative, toxin B-positive strain implicated in multiple human cases of C. difficile-associated diarrhea. Groups of 10 hamsters per strain were given 1 dose of clindamycin, followed 5 days later with gastric inoculation of 100 cfu of C. difficile. Hamsters given types B1, J9, K14, or Y2 showed 90%-100% colonization (albeit at a slower rate with type Y2) and 100% mortality of colonized animals. Hamsters challenged with type CF2 showed 60% (P= .01) colonization and 30% mortality (P= .0003). The hamster model demonstrated pathogenicity differences between a toxin variant strain and standard toxigenic strains but no significant differences among the standard strains.

Toxin A variant strains of C. difficile cause serious disease and are undetectable in clinical laboratories that use only toxin A immunoassays for C. difficile testing.