A single clinical Clostridioides difficile RT012/ST54 isolate retains inflammatory activity under glucose-associated toxin-reducing conditions.

INTRODUCTION

Clostridioides difficile infection is primarily attributed to toxin A (TcdA) and toxin B (TcdB), the major mediators of epithelial damage and intestinal inflammation. However, strain-dependent differences in pathogenicity have been reported, suggesting that additional bacterial factors may contribute to host inflammatory responses.

OBJECTIVE

To compare the inflammatory activity of culture supernatants from the single clinical isolate LIBA-5751/NAPCR1/RT012/ST54 and reference C. difficile strains under toxin-containing and glucose-repressed conditions.

METHODS

C. difficile strains, including the clinical isolates LIBA-5751/NAPCR1/RT012/ST54 and LIBA-5758/NAP1/RT027/ST01 and the reference strains VPI10463/RT003/ST046 and 630/RT012/ST54, were grown anaerobically in tryptose-yeast extract-thioglycolate broth with or without 1% glucose. Glucose-associated reduction of toxin activity was assessed by electrophoretic protein profiling, cytotoxicity assays, and Rac1 glucosylation analysis. Biological activity was evaluated in a mouse ligated ileal loop model by macroscopic examination, histopathology, cytokine quantification, and myeloperoxidase activity.

RESULTS

Glucose supplementation did not substantially alter bacterial growth but markedly reduced detectable toxin-associated bands, cytopathic activity, and Rac1 glucosylation. Toxin-containing supernatants from LIBA-5758/NAP1/RT027/ST01 and VPI10463/RT003/ST046 produced the most pronounced epithelial injury. Under the same conditions, LIBA-5758/NAP1/RT027/ST01, VPI10463/RT003/ST046, and LIBA-5751/NAPCR1/RT012/ST54 induced robust inflammatory responses. Under glucose-repressed conditions, epithelial damage was no longer evident, and inflammatory activity was markedly reduced in LIBA-5758/NAP1/RT027/ST01, VPI10463/RT003/ST046, and 630/RT012/ST54. In contrast, supernatants from LIBA-5751/NAPCR1/RT012/ST54 retained substantial inflammatory activity, including increased TNF-α, IL-1β, IL-6, and myeloperoxidase activity, despite the absence of overt epithelial damage.

CONCLUSIONS

The clinical isolate LIBA-5751/NAPCR1/RT012/ST54 retained substantial inflammatory activity under glucose-repressed conditions despite markedly reduced detectable toxin-associated activity. This phenotype was not observed to the same extent in the other strains examined, including the RT012/ST54 reference strain 630. These findings identify a strain-specific inflammatory phenotype observed in LIBA-5751/NAPCR1/RT012/ST54 and support further investigation into the extracellular factors that may contribute to inflammatory responses in C. difficile.