Prophages contribute to the evolution and virulence of most bacterial pathogens, but their role in Clostridium difficile is unclear. Here we describe the isolation of four Myoviridae phages, fΦMMP01, fΦMMP02, fΦMMP03, and fΦMMP04, that were recovered as free viral particles in the filter-sterilized stool supernatants of patients suffering from C. difficile infection (CDI). Furthermore, identical prophages were found in the chromosomes of C. difficile isolated from the corresponding fecal samples. We therefore provide, for the first time, evidence of in vivo prophage induction during CDI. We completely sequenced the genomes of f{cyrillic}MMP02 and f{cyrillic}MMP04, and bioinformatics analyses did not reveal the presence of virulence factors but underlined the unique character of f{cyrillic}MMP04. We also studied the mobility of fΦMMP02 and fΦ}MMP04 prophages in vitro. Both prophages were spontaneously induced, with 4 to 5 log PFU/ml detected in the culture supernatants of the corresponding lysogens. When lysogens were grown in the presence of subinhibitory concentrations of ciprofloxacin, moxifloxacin, levofloxacin, or mitomycin C, the phage titers further increased, reaching 8 to 9 log PFU/ml in the case of fΦMMP04. In summary, our study highlights the extensive genetic diversity and mobility of C. difficile prophages. Moreover, antibiotics known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobility in vitro and probably in vivo as well, which underscores their potential impact on phage-mediated horizontal gene transfer events and the evolution of C. difficile.
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