Freshwater beach sandy sediments harbour latent β-lactamases with deep phylogenetic roots in Chitinophagaceae

Background : Antibiotic resistance genes (ARGs) of environmental origin represent an underexplored but potentially important source of future clinical resistance. Here, we characterized the resistome of wet beach sandy sediments from Lake Maggiore using a metagenomic approach to assess the diversity, origin, and potential horizontal mobility of ARGs in this transitional environment. Results : Despite significant spatial variation in resistome β-diversity between beaches and sampling depths, the dominant resistance classes were consistent across sites, with aminoglycoside, polyketide, quinolone, and tetracycline resistance genes prevailing. In contrast, at the metagenome-assembled genome (MAG) level, β-lactamases were the most abundant ARGs, primarily associated with members of the family Chitinophagaceae (phylum Bacteroidetes). Within this group, the genera Ferruginibacter and Lacibacter carried the majority of β-lactamase genes, belonging to classes A, B1, and D. None of the detected ARGs were associated with mobile genetic elements, and all β-lactamase genes were chromosomal. Phylogenetic analyses of our MAGs and all deposited Chitinophagaceae β-lactamase gene sequences revealed that this family contributed substantial novel diversity to β-lactamase phylogenies, with strong phylogenetic clustering at the genus level indicating lineage-specific conservation. The presence of multiple β-lactamase classes within single strains further suggests convergent evolution and potential functional roles beyond antibiotic resistance. Conclusions : Collectively, these findings identify Chitinophagaceae as a major environmental reservoir of chromosomal β-lactamases and point to freshwater sandy sediments as overlooked hotspots of latent resistance diversity. These results highlight the need to consider the environment not as a passive reservoir for ARGs, but as potentially active sources of their development and evolution.