| Summary: | <i>Leuconostoc </i>spp. are environmental microorganisms commonly associated with fermented foods. Absence of antibiotic resistance (AR) in bacteria is a critical issue for global food safety. Herein, we updated the occurrence of AR genes in the <i>Leuconostoc</i> genus through in silico analyses of the genomes of 17 type strains. A total of 131 putative AR traits associated with the main clinically relevant antibiotics were detected. We found, for the first time, the <i>lsa</i>A gene in <i>L. fallax</i> ATCC 700006<sup>T</sup> and <i>L. pseudomesenteroides</i> NCDO 768<sup>T</sup>. Their amino acid sequences displayed high similarities (59.07% and 52.21%) with LsaA of <i>Enterococcus</i><i>faecalis</i> V583, involved in clindamycin (CLI) and quinupristin-dalfopristin (QUD) resistance. This trait has different distribution patterns in <i>Leuconostoc</i> nontype strains—i.e., <i>L. pseudomesenteroides</i>, <i>L. lactis</i> and <i>L. falkenbergense</i> isolates from fermented vegetables, cheeses, and starters. To better explore the role of <i>lsa</i>A, MIC for CLI and QUD were assessed in ATCC 700006<sup>T</sup> and NCDO 768<sup>T</sup>; both strains were resistant towards CLI, potentially linking <i>lsa</i>A to their resistant phenotype. Contrarily, NCDO 768<sup>T</sup> was sensitive towards QUD; however, expression of <i>lsa</i>A increased in presence of this antibiotic, indicating an active involvement of this trait and thus suggesting a revision of the QUD thresholds for this species.
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