All LAB strains phenotypically resistant to the tested antimicrobial agents were examined by PCR for the presence of selected AMR genes. The following genes were detected: bla gene (ampicillin-resistant strains); the erm(A), erm(B), erm(C), msr genes, and the lnu(A) gene (erythromycin and/or clindamycin-resistant strains); genes encoding ribosomal protection proteins (universal primer set and subsequently, specific primers for tet(W) and tet(M) genes for positive strains) and the tet(K) and tet(L) genes encoding a tetracycline efflux pump (tetracycline-resistant strains); the cat gene (chloramphenicol-resistant strains); the aph(3″)-IIIa gene (kanamycin-resistant strains); the ant(6), str(A)/str(B) and aad(A) genes (streptomycin-resistant strains); the aac(6′)-aph(2″) gene (aminoglycosides-resistant strains). In case of the detection of resistance genes, the cut-off values given in the previous EFSA guidance (25) were additionally used for a results analysis.

The characteristics of the primers used in the study and appropriate references (2636) are shown in Supplementary Table 2. The primer set for msr(C) detection was designed using the PCR Primer Design Tool (https://eurofinsgenomics.eu/en/ecom/tools/pcr-primer-design) and checked using an Oligo Analysis Tool (https://eurofinsgenomics.eu/en/ecom/tools/oligo-analysis). PCR reactions were performed in a total volume of 25 μL containing 1 μL of each primer (10 pmol/μL), 12.5 μL of DreamTaq PCR Master Mix (2×) (ThermoFisher Scientific) or JumpStart REDTaq ReadyMix Reaction Mix (2×) (Sigma-Aldrich) and 50 ng of DNA template. A template bacterial genomic DNA was purified using GenElute™ Bacterial Genomic DNA Kits (Sigma-Aldrich) following the manufacturer's instruction for Gram-positive bacteria cells (pre-incubation with lysozyme). The amount and quality of DNA was determined using the Thermo Scientific NanoDropTM 1000 Spectrophotometer.

PCR products were separated by electrophoresis on a 1% agarose gel (Sigma-Aldrich), stained with ethidium bromide, in TBE buffer (100 V). The O'RangeRulerTM 200bp DNA Ladder, GeneRulerTM 100 bp DNA Ladder or GeneRulerTM 100 bp Plus DNA Ladder (ThermoFisher Scientific) were used as size standard markers. Additionally, PCR products were purified and sequenced (Genomed S.A.). The obtained DNA sequences were analyzed using BLASTn (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/Blast.cgi) and compared with sequences available in GenBank (National Center for Biotechnology Information) and CARD database (The Comprehensive Antibiotic Resistance Database, https://card.mcmaster.ca) (Supplementary Table 3).

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