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TcdA and TcdB exotoxins are the main virulence factors ofClostridium difficile , one of the most deadly nosocomial pathogens. Recent data suggest that prophages can influence the regulation of toxin expression. Here we present the characterization of φCD38-2, apac -type temperateSiphoviridae phage that stimulates toxin expression when introduced as a prophage intoC. difficile . Host range analysis showed that φCD38-2 was able to infect 99/207 isolates ofC. difficile representing 11 different PCR ribotypes. Of 89 isolates corresponding to the NAP1/027 hypervirulent strain, which recently caused several outbreaks in North America and Europe, 79 (89%) were sensitive to φCD38-2. The complete double-stranded DNA (dsDNA) genome was determined, and a putative function could be assigned to 24 of the 55 open reading frames. No toxins or virulence factors could be identified based on bioinformatics analyses. Our data also suggest that φCD38-2 replicates as a circular plasmid inC. difficile lysogens. Upon introduction of φCD38-2 into a NAP1/027 representative isolate, up to 1.6- and 2.1-fold more TcdA and TcdB, respectively, were detected by immunodot blotting in culture supernatants of the lysogen than in the wild-type strain. In addition, real-time quantitative reverse transcriptase PCR (qRT-PCR) analyses showed that the mRNA levels of all five pathogenicity locus (PaLoc) genes were higher in the CD274 lysogen. Our study provides the first genomic sequence of a newpac -typeSiphoviridae phage family member infectingC. difficile and brings further evidence supporting the role of prophages in toxin production in this important nosocomial pathogen.

Prophage-Stimulated Toxin Production in Clostridium difficile NAP1/027 Lysogens