Low-toxin Clostridioides difficile RT027 strains exhibit robust virulence

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. Currently, there is a lack of consensus for an optimal diagnostic method for C. difficile infection (CDI). Multi-step diagnostic algorithms use enzyme immunosorbent analysis (EIA)-based detection of C. difficile toxins TcdA/TcdB in stool, premised on the rationale that EIA toxin-negative (Tox⁻) patients have less severe disease and shorter diarrhoea duration. The aim of this study was to characterize toxigenic (i.e. tcdA/tcdB-positive) C. difficile strains isolated from diarrheic patient stool with an EIA Tox⁻ (i.e. “discrepant”) CDI diagnostic test result. Recovered strains were DNA fingerprinted (ribotyped), subjected to multiple toxin, genome and proteome evaluations, and assessed for virulence. Overall, of 1243 C. difficile-positive patient stool specimens from Southern Arizona hospitals, 31% were discrepant. For RT027 (the most prevalent ribotype)-containing specimens, 34% were discrepant; the corresponding RT027 isolates were cytotoxic to cultured fibroblasts, but their total toxin levels were comparable to, or lower than, the historic low-toxin-producing C. difficile strain CD630. Nevertheless, these low-toxin RT027 strains (LT-027) exhibited similar lethality to a clade-matched high-toxin RT027 strain in Golden Syrian hamsters, and heightened colonization and persistence in mice. Genomics and proteomics analyses of LT-027 strains identified unique genes and altered protein abundances, respectively, relative to high-toxin RT027 strains. Collectively, our data highlight the robust virulence of LT-027 C. difficile, provide a strong argument for reconsidering the clinical significance of a Tox⁻ EIA result, and underscore the potential limitations of current diagnostic protocols.