Clindamycin

Hemolysin production was detected using Columbia agar plates supplemented with 5% of sheep blood (Amersco, Solon, OH, USA). The presence of α or β-hemolysis was assessed by the formation of clear or greenish zones around the colonies, respectively.
Enzyme activity was measured using the commercially-available, semi-quantitative API-ZYM system (BioMérieux, Montreal, QC) as previously described. According to the manufacturer’s instructions, cell suspension was adjusted to McFarland standards 5. Then 65 μL of cell suspension were added into each well of the API-ZYM strip and were incubated at 37 °C for 4 h in anaerobic conditions. The results were graded based on the amount of from substrate hydrolyzed on a scale from 0 (no activity) to 40 (or ≥ 40 nM).
For antibiotic susceptibility testing, LAB strains (10⁸ CFU/mL) were inoculated onto MRS soft agar. Commercial antibiotic discs (amoxicillin, penicillin, tetracycline, erythromycin, gentamicin, clindamycin, and ofloxacin, provided by Sangon Biotech (Shanghai) Co., Ltd) were placed onto the agar and incubated at 37 °C for 24 h. Resistance or sensitivity was assessed according to the CLSI/NCCLS standard.

The predicted antibiotic resistance gene information in the genome was obtained by comparing the amino acid sequences of the strains with the comprehensive antibiotic research database (CARD, http://arpcard.mcmaster.ca, accessed on 24 May 2021) [51 (link)]. The strains were clustered using HemI software [50 (link)].
The microbroth dilution method was used to determine antibiotic resistance of L. sakei according to ISO 10932:2010 [52 ]. The following 11 antibiotics were detected: chloramphenicol, rifampicin, streptomycin, kanamycin, gentamycin, tetracycline, clindamycin, neomycin, erythromycin, ciprofloxacin, and vancomycin (all purchased from Sangon Biotech Co., Ltd., Shanghai, China). OD₆₂₅ was determined using an enzyme-labeled instrument (Varioskan Lux, Thermo, Waltham, MA, USA) to determine the MIC of strain to antibiotics.

B. bifidum genomes were aligned against sequences from the latest version of the Comprehensive Antibiotic Resistance Database [39 (link)], and a conservative threshold (amino acid identity ≥ 30%, comparison hit-bit score ≥ 37.0) was used to predict putative ARGs.
The antibiotic susceptibility of the B. bifidum strains was evaluated using the broth microdilution method, according to ISO 10932:2010 [40 ]. The following 10 antibiotics were tested: tetracycline, erythromycin, clindamycin, ampicillin, amoxicillin, trimethoprim, ciprofloxacin, chloramphenicol, rifampicin, and vancomycin (all purchased from Sangon Biotech Co., Ltd., Shanghai, China). The microbiological breakpoints of Bifidobacterium recommended by the European Food Safety Authority were used to distinguish susceptible strains from resistant strains.