Rifampicin

The T. asahii YAN0802 strain (wild-type strain) was isolated from giant pandas and stored at −80 °C in SDA medium supplemented with 20% (v/v) glycerol.
E. coli Trelief^TM 5α strains were purchased from TSINGKE (Beijing, China) and cultured with LB medium. Colonies bearing plasmids were selected on LB medium with ampicillin (50 µg/mL). Plasmid extraction from E. coli Trelief^TM 5α strains was performed by choosing transformants in a 2 mL LB liquid medium with the appropriate antibiotics overnight at 37 °C.
A. tumefaciens EHA105, competent for transformation, was purchased from TSINGKE (Beijing, China) and grown on LB plates with rifampicin (50 µg/mL) (Sangon Biotech, Shanghai, China) and kanamycin (50 μg/mL) (Sangon Biotech, Shanghai, China). Transformants were selected and maintained in an induction medium (IM) [40 (link)].
The OE plasmid, pEGFP-N1, was synthesized by Honor Gene (Hunan, China). Selection of T. asahii containing the recombinant plasmids was performed on SDA medium supplemented with G418 (200 µg/mL) (Sangon Biotech, Shanghai, China).

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.