Characterization of the Virulence of a Non-Rt027, Non-Rt078 and Binary Toxin-Positive Clostridium Difficile Strain Associated with Severe Diarrhea

The expression of the Clostridium difficile binary toxin CDT is generally observed in the RT027 (ST1) and RT078 (ST11) C. difficile isolates, which are associated with severe C. difficile infection (CDI). However, we recently reported that the non-RT027 and non-RT078 C. difficile strain LC693 (TcdA+TcdB+ CDT+, ST201) caused severe diarrhea in a patient in Xiangya Hospital in China. C.difficile LC693 is a member of Clade 3, and in this study, we identified LC693 as RT871 and compared its virulence and pathogenicity to those of C.difficile R20291 (TcdA+TcdB+CDT+, ST1/RT027), UK6 (TcdA+TcdB+CDT+, ST35/RT027), CD630 (TcdA+TcdB+CDT-, ST54, RT012), and 1379 (TcdA+TcdB+CDT-, ST54/RT012), with strain 1379 being an epidemic C.difficile isolate from the same hospital. LC693 displayed a higher sporulation rate than R20291, CD630 or strain 1379. LC693 was comparable to R20291 with respect to spore germination, motility, and biofilm formation, but showed a faster germination rate, higher motility and a higher biofilm formation capability compared to CD630 and strain 1379. The adherence of spores to human gut epithelial cells was similar for all strains.The total toxin release of LC693 was lower than that of R20291, but higher than that of CD630 and strain 1379. Finally, in a mouse model of CDI, LC693 was capable of causing moderate to severe disease. Our findings demonstrate the pathogenicity of non-RT027 and non-RT078 binary toxin-positive C. difficile strains. Furthermore, our data indicate that LC693 may be more virulent than strain 1379, an epidemic strain from the same hospital, and provide the first phenotypic characterization of a non-RT027 and non-RT078 binary toxin-positive ST201 isolate.