Genetically diverse Clostridium difficile strains harboring abundant prophages in an estuarine environment

Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in health care settings across the world. Despite its pathogenic capacity, it can be carried asymptomatically and has been found in terrestrial and marine ecosystems outside hospital environments. Little is known about these environmental strains, and few studies have been conducted on estuarine systems. Although prophage abundance and diversity are known to occur within clinical strains, prophage carriage within environmental strains of C. difficile has not previously been explored. In this study, we isolated C. difficile from sites sampled in two consecutive years in an English estuarine system. Isolates were characterized by PCR ribotype, antibiotic resistance, and motility. The prevalence and diversity of prophages were detected by transmission electron microscopy (TEM) and a phage-specific PCR assay. We show that a dynamic and diverse population of C. difficile exists within these sediments and that it includes isolates of ribotypes which are associated with severe clinical infections and those which are more frequently isolated from outside the hospital environment. Prophage carriage was found to be high (75%), demonstrating that phages play a role in the biology of these strains.