0.1 mm glass beads

Cheese samples (5 grams) were homogenized with 45 mL of a 2% sterile sodium citrate solution and incubated at 37°C for 3 h in the presence of 1 mg mL⁻¹ pronase (Sigma-Aldrich) and 100 μL β-mercaptoethanol (Merck). Total microbial cells were harvested by centrifugation at 5000 ×g for 20 min and disrupted using 0.1 mm glass beads (Sigma-Aldrich) as reported elsewhere [30 (link)]. Genomic DNA was purified by phenol and phenol/chloroform extractions and precipitated with 2-propanol (all chemicals from Sigma-Aldrich). Finally, it was suspended in sterile water containing 5–15 mg mL⁻¹ of RNase (Sigma-Aldrich).
Total DNA was also isolated from pure cultures of control strains carrying known AR genes: Staphylococcus epidermidis SE36 [tet(K)], Enterococcus faecium ET51 [tet(L)], Lactococcus lactis IPLA 31008 [tet(M)], Enterococcus faecalis Jtet [tet(O)], Enterococcus spp. ET15 [tet(S)], Bifidobacterium longum B93 [tet(W)], Lactobacillus johnsonii G41 [erm(B)], and Bacteroides fragilis 79a [erm(F)]. Genomic microbial DNA from these strains was purified from 1 mL of an overnight culture in brain heart infusion broth (BHI; Merck) using the Kit GenElute bacterial genomic DNA (Sigma-Aldrich). The recovered DNA was then stored at −20°C until analysis.

DNA was extracted using the PowerSoil^® DNA Isolation Kit (Cat. No. 12888, MO BIO) according to the manufacturer’s protocol. Bead beating (0.1-mm glass beads; Sigma-Aldrich, St. Louis, MO, USA; bead beater, Bullet Blender Storm 24, Averill Park, NY, USA) at a speed of 4000 rpm for 30 s was used to homogenize the suspension and mechanically disrupt the bacterial cells. The purity and concentration of genomic DNA were measured using a spectrophotometer (Nanodrop 115 1000, Thermo Fisher Scientific Oxoid Ltd., Basingstoke, UK), and the integrity of DNA was verified by agarose (0.7%) gel electrophoresis. DNA samples were stored at −80 °C until further processing. The extracted DNA was further sequenced, processed, and analyzed by Macrogen (Macrogen Inc., Seoul, Korea).

DNA was extracted from 200 mg of feces, which was added to a 2-mL screw cap vial containing 300 mg of 0.1-mm glass beads (Sigma, St. Louis, Missouri) and was maintained on ice until the addition of 1.4 mL ASL buffer using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). The samples were immediately subjected to bead beating (45 s, speed 6.5) twice using a FastPrep-24 machine (MP Biomedicals, Solon, Ohio) before the initial incubation for heat and chemical lysis at 95°C for 5 minutes. Subsequent DNA extraction was performed following the QIAamp kit protocol for pathogen detection.
DNA was isolated from biopsy samples using the QIAamp DNA Mini Kit (Qiagen). Extraction was performed according to the manufacturer’s instructions, with an additional bead-beating step (45 s, speed 6.5, twice) using a FastPrep-24 machine at the beginning of the protocol. The extracted DNA was stored at −80°C until use.