Columbia agar medium

The strains were stored at −80 °C. The cultures were grown on solid Columbia agar medium with 5% sheep blood (bioMérieux) under aerobic conditions at 32 °C. Thereafter, several colonies were transferred in sterile conditions into 1 L of liquid Tryptic soy broth medium (Sigma-Aldrich, France), which was incubated for 3 days for Bacillus spp. and for 14 days for Streptomyces spp. at 32 °C and 110 rpm in a shaker incubator under aerobic conditions.
The supernatant-pellet separation was carried out by centrifugation at 8000× g for 20 min at 4 °C using an A98813 J-Lite PP bottle assembly with a JLA-8.1000 rotor (Beckman Coulter, Villepinte, France). After centrifugation, the supernatant was immediately filtered through a 0.45 μm filter and placed into 75 mL flasks, after which it was frozen horizontally overnight at −80°C and then lyophilized the next day. The lyophilizate was stored at −20°C prior to the assays.

The effects of BBR on the motility of C. difficile were investigated in 12 strains. Among them, 3 strains were used as a control: motile 630, non-motile M120, and motility ATCC 9689 [23 (link)]. The 9 other strains consisted of clinical isolates and toxigenic strains belonging to the PCR-ribotype (RT027). After thawing, all strains were seeded on Columbia agar medium (bioMérieux, Marcy l’Etoile, France) and incubated for 48 h in an anaerobic atmosphere. For each strain, a single colony calibrated with 1 μL of C. difficile culture was plated onto pre-reduced 0.4% agar plates with BHI medium without BBR (as a control of motility) and with different concentrations of BBR according to the 1/2 MIC value of all the individual strains (512, 450, 320, or 128 mg/L), followed by incubating at 37 °C under anaerobic conditions. Readings were carried out after 24 h and 48 h. The inhibition of motility was assessed by measuring the diameter of the spot on the soft agar and comparing the results with the control plates (without BBR). The readings were performed independently by two separate individuals.