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 ...

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.

Fidaxomicin, a nonabsorbed macrocyclic compound, is the first antimicrobial agent approved by the FDA for the treatment of Clostridium difficile infection (CDI) in adults over the last 25 years. It is bactericidal, and its mechanism of action relates to inhibition of a RNA polymerase at a site distinct from where rifamycins interact. Fidaxomicin, 200 milligrams by mouth twice daily, is not inferior to vancomycin, 125 milligrams by mouth 4 times daily, for treatment of CDI as determined by clinical response after 10 days of treatment and is superior to vancomycin for sustained response without recurrence 25 days after treatment completion. These results are a significant advance in the treatment of CDI and herald the development of narrow-spectrum anti-C. difficile agents that relatively spare the indigenous fecal microbiota. Continued vigilance for the development of resistance and unanticipated side affects will be important as the drug is introduced into clinical practice.