Reconsidering the sporulation characteristics of hypervirulent Clostridium difficile BI/NAP1/027

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and a major burden to healthcare services worldwide. In recent years, C. difficile strains belonging to the BI/NAP1/027 type have become highly represented among clinical isolates. These so-called 'hypervirulent' strains are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Spores, formed during sporulation, play a pivotal role in disease transmission and it has been suggested that BI/NAP1/027 strains are more prolific in terms of sporulation in vitro than 'non-epidemic' C. difficile types. Work in our laboratory has since provided credible evidence to the contrary suggesting that the strain-to-strain variation in C. difficile sporulation characteristics is not type-associated. However, the BI/NAP1/027 type is still widely stated to have an increased rate of sporulation. In this study, we analysed the sporulation rates of 53 C. difficile strains, the largest sample size used to-date in such a study, including 28 BI/NAP1/027 isolates. Our data confirm that significant variation exists in the rate at which different C. difficile strains form spores. However, we clearly show that the sporulation rate of the BI/NAP1/027 type was no higher than that of non-BI/NAP1/027 strains. In addition, we observed substantial variation in sporulation characteristics within the BI/NAP1/027 type. This work highlights the danger of assuming that all strains of one type behave similarly without studying adequate sample sizes. Furthermore, we stress the need for more rigorous experimental procedures in order to quantify C. difficile sporulation more accurately in the future.