Quantifying C. difficile Toxin Production

A previously published method was used to quantify toxin production from C. difficile isolates (16 (link)). Briefly, C. difficile strains were grown in 5 mL BHI broth for 72 h. The cultures were then centrifuged, and cell-free supernatants clarified using 0.22 μm filters (Argos Technologies, Elgin, IL, United States). Toxin amounts in 50 μL of the supernatants were quantified using the Techlab^®C. difficile Tox A/B II™ EIA kit (Techlab, Blacksburg, VA, United States) per manufacturer’s instructions, with absorbance measured at 450 nm. Total protein concentration was quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, United States), and toxin levels reported as absorbance at 450 nm/mg total protein. All data were collected in biological triplicate. Toxin status of the isolates was derived from the genotype (i.e., inferred from ribotype) or actual quantitation of toxin.