Low-Toxin Clostridioides difficile RT027 Strains Exhibit Robust Virulence

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. Currently, there is lack of consensus on a single optimal diagnostic method for C. difficile infection (CDI). Nucleic acid amplification tests (NAAT) that detect toxin genes are highly sensitive, but their specificity limitations could inflate CDI rates. Alternate multi-step diagnostic algorithms emphasize the detection of C. difficile toxins TcdA/TcdB, and are premised on the rationale that stool toxin-negative (Tox^-) CDI patients have less severe disease, shorter diarrhea duration, and fewer disease complications. There have been no systematic assessments, however, of the virulence of C. difficile strains from Tox^-/NAAT⁺ (discrepant) specimens. In our prospective analysis of 1243 C. difficile-positive patient stool specimens from three Southern Arizona hospitals, 31% were discrepant. Ribotype 027 (RT027) strains were recovered from 221 specimens; of these, 23% were discrepant. Post-culture, RT027 strains produced a range of toxin amounts including levels lower than that of the non-epidemic strain CD630. These low-toxin RT027 (LT-027) strains harbored both tcdA and tcdB genes, and their culture supernatants were cytotoxic to cultured fibroblasts. We confirmed robust colonization and virulence of a subset of LT-027 strains using multiple rodent models; lethality in animals infected with LT-027 strains was comparable to that potentiated by a high-toxin RT027 strain. Comparative genomics and proteomics analyses of several LT-027 strains identified unique genes and altered protein abundances relative to closely-related high-toxin strains. Collectively our data highlight the robust virulence and clinical relevance of low-toxin-producing, RT027 C. difficile isolates.