Microbial community structure and antibiotic resistance genes in Dinaric karst groundwater under contrasting...

Karst aquifers are vulnerable drinking water resources, yet microbiome-resistome studies remain scarce. We investigated microbial community composition and antibiotic resistance gene (ARG) profiles in five karst groundwater sources along an anthropogenic gradient in coastal Croatia under contrasting hydrological conditions. 16S rRNA amplicon sequencing showed that hydrological conditions were associated with community composition. Distance-based redundancy analysis identified total organic carbon as the variable associated with community structure, while ammonium and Escherichia coli were associated with dry hydrological conditions. High-throughput qPCR (96 gene targets) revealed distinct resistome profiles across sites and conditions. Sites with greater anthropogenic influence showed higher ARG abundances than the reference site, particularly under wet hydrological conditions. Efflux pump genes were prevalent across sites and conditions, while aminoglycoside and trimethoprim ARGs were particularly prominent at the most impacted site under dry conditions. Escherichia/Shigella and Klebsiella showed the broadest ranges of significant ARG correlations under wet and dry hydrological conditions, respectively, although several associations were sensitive to individual sampling locations. Clinically relevant ARGs, including vancomycin and colistin resistance genes, were detected at low relative abundances. These findings demonstrate associations between hydrological conditions, anthropogenic influence, and karst groundwater microbiomes and resistomes, highlighting the potential of molecular AMR surveillance in drinking water sources.