Thioglycolate broth medium

Antimicrobial susceptibility testing (AST) was performed using the Etest technique, which determines the minimum inhibitory concentration (MIC), i.e., the minimal antibiotic concentration that inhibits the growth of bacteria under specific conditions. To this end, C. difficile colonies were suspended in Thioglycolate broth medium (Becton Dickinson, Heidelberg, Germany) until 1.0 McFarland turbidity. The inoculum was then seeded on Brucella blood agar growth medium (Hy Laboratories, Rehovot, Israel). Gradient Etest strips (bioMérieux, Durham, NC) of vancomycin and metronidazole were added to the plates. Following incubation under anaerobic conditions, at 37°C, for 24 h, the MIC was determined for each antibiotic. Test results (susceptible/with reduced susceptibility) were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations (EUCAST, 2021 ), that determined the following epidemiological cut-offs- reduced susceptibility for vancomycin is defined with an MIC above 2 µg/mL; reduced susceptibility to metronidazole is defined with an MIC above 2 µg/mL.
Table S1 presents all the collected data regarding the study’s isolates (antibiotic susceptibility, ST strain and biofilm production capacity). Table S2 presents a comparison of MIC between Etest and micro broth dilution, for the validation of Etest.

The current experimental protocol was previously established by Calefi et al. [21 (link)]. A pathogenic strain of C. perfringens type A (strain CP8.2, genotype for productions of Tpel and $\alpha$ toxins, NetB negative) has been maintained in our laboratory in glycerol at −80 °C. Briefly, the inoculum was prepared by two alternate cultures in thioglycolate broth medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with 2% yeast extract (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and cooked meat medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Bacteria were given to broilers from ED15 to ED19 in a final concentration of approximately 1 × 10⁴ cfu/g of feed. From ED1 to ED23, animals received feed with 24% crude protein without antimicrobial agent. Starting at ED19, the groups (Cp, Cp + Ei, Cp/HS34, and Cp + Ei/HS34) received the culture medium containing a pathogenic strain of C. perfringens type A mixed with the animal feed at a ratio of 1:1 (v/v). The control groups (C and C/HS34) received only feed without any pathogen mixed with brain heart infusion culture medium (BHI). The infection occurred through voluntary feed intake containing the BHI with C. perfringens. The volume of the inoculum was standardized to be completely consumed within a maximum period of 6 h.