Evidence of in vivo prophage induction during Clostridium difficile infection 1 2 3

27 28 Prophages contribute to the evolution and virulence of most bacterial pathogens, but their role in 29 C. difficile is unclear. Here we describe the isolation of four Myoviridae phages: φMMP01, 30 φMMP02, φMMP03, and φMMP04, that were recovered as free viral particles in filter-sterilized 31 stool supernatants of patients suffering from C. difficile infection (CDI). Furthermore, identical 32 prophages were found in the chromosome of C. difficile isolated from the corresponding fecal 33 samples. We therefore provide for the first time evidence of in vivo prophage induction during 34 CDI. We completely sequenced the genomes of φMMP02 and φMMP04, and bioinformatics 35 analyses did not reveal the presence of virulence factors, but underlined the unique character of 36 φMMP04. We also studied the mobility of φMMP02 and φMMP04 prophages in vitro. Both 37 prophages were spontaneously induced, with 4-5 log PFU/mL detected in culture supernatants of 38 the corresponding lysogens. When lysogens were grown in the presence of sub-inhibitory 39 concentrations of ciprofloxaxin, moxifloxacin, levofloxacin, or mitomycin C, phage titers further 40 increased, reaching 8-9 log PFU/mL in the case of φMMP04. In summary, our study highlights 41 the extensive genetic diversity and mobility of C. difficile prophages. Moreover, antibiotics 42 known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobility 43 in vitro and probably in vivo as well, which underscores their potential impact on phage44 mediated horizontal gene transfer events and the evolution of C. difficile. 45 46 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom