We investigated the activity of the supernatant of Clostridium sporogenes, which inactivates the toxins TcdA and TcdB of Clostridioides difficile, with the aim of partially characterizing the effector molecule and its basic physiochemical properties. The inhibitory activity was assessed by measuring the relative concentration of toxins using an ELISA-based method following the application of different experimental approaches, including thermal denaturation (99°C, 1 h), pH modification, EDTA addition, and molecular weight–based filtration with cut-off values of 30 kDa and 3 kDa. Thermal treatment of the supernatant resulted in a complete loss of inhibitory activity, whereas the untreated supernatant caused a statistically significant reduction in the relative concentration of TcdA/TcdB in two independent experiments (p = 0.014 and p = 0.047), indicating that the effector substance is thermolabile. Membrane separation further demonstrated that the inhibitory effect was retained after the removal of molecules larger than 30 kDa and 3 kDa (p = 0.0035 in both cases), while filtration of the control medium (WCAB) had no significant effect on the measured signal (p = 0.14). Taken together, these results suggest that the effector substance is a very small molecule with a molecular weight below 3 kDa, most likely of a peptidic nature. Experiments involving EDTA addition and pH changes did not allow reliable conclusions regarding metal dependence or pH optimum of activity, as pronounced interference with the ELISA method was observed in both cases.
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