Environmental resistome of culturable gram-negative bacilli from the Yaque del Norte River: baseline genomic evidence for one health surveillance.

Antimicrobial resistance (AMR) represents a significant global public health threat. Aquatic ecosystems are increasingly recognized as critical reservoirs of resistance determinants within the One Health framework. Freshwater systems, in particular, facilitate the persistence and dissemination of antimicrobial resistance genes (ARGs) at the human-animal-environment interface. This study characterized the environmental resistome of culturable Gram-negative bacilli isolated from the Yaque del Norte River, the largest river in the Dominican Republic. Water samples were collected along the river basin to assess physicochemical parameters and recover bacterial isolates. Isolates were identified using MALDI-TOF MS and JspeciesWS. Whole-genome sequencing and bioinformatic analyses with CARD and the Galaxy staramr toolkit were performed to identify ARGs and plasmid replicons. Pathogenic potential was evaluated using PathogenFinder v1.1. Six bacterial strains were recovered: Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Enterobacter mori, Acinetobacter pittii, and Acinetobacter schindleri. A total of 112 ARGs were identified, with P. aeruginosa harboring the most extensive resistome, comprising 51 genes. ARG identification was based on sequence identity ≥ 90% and coverage ≥ 80% according to CARD and ResFinder criteria. Multidrug resistance-associated determinants were predominant, particularly efflux pump systems and transcriptional regulators. Additional resistance mechanisms included genes conferring resistance to fluoroquinolones, β-lactams, bacitracin, fosfomycin, and aminoglycosides. Plasmid replicons were detected exclusively in Klebsiella spp. Both species carried IncFIB(K), and one isolate harbored IncY, a replicon rarely reported outside Escherichia coli and Salmonella serovar Typhi, suggesting potential interspecies dissemination. Phenotypic resistance was observed only in a single K. pneumoniae isolate. Nevertheless, the presence of diverse ARGs in this environmentally impacted river underscores the potential for future emergence and spread of AMR. These findings provide baseline genomic evidence to support environmental AMR surveillance and highlight the need to strengthen antibiotic stewardship and One Health-oriented monitoring strategies in the Dominican Republic.