Karst spring microbiome: Diversity, core taxa, and community response to pathogens and antibiotic resistance gene contamination

Karst aquifers are important water resources for drinking water supplies worldwide. Although they are susceptible to anthropogenic contamination due to their high permeability, there is a lack of detailed knowledge on the stable core microbiome and how contamination may affect these communities. In this study, eight karst springs (distributed across three different regions in Romania) were sampled seasonally for one year. The core microbiota was analysed by 16S rRNA gene amplicon sequencing. To identify bacteria carrying antibiotic resistance genes and mobile genetic ele-ments, an innovative method was applied, consisting of high-throughput antibiotic resistance gene quantification per-formed on potential pathogen colonies cultivated on Compact DryTM plates. A taxonomically stable bacterial community consisting of Pseudomonadota, Bacteroidota, and Actinomycetota was revealed. Core analysis reaffirmed these results and revealed primarily freshwater-dwelling, psychrophilic/psychrotolerant species affiliated to Rhodoferax, Flavobacterium, and Pseudomonas genera. Both sequencing and cultivation methods indicated that more than half of the springs were contaminated with faecal bacteria and pathogens. These samples contained high levels of sulfonamide, macrolide, lincosamide and streptogramins B, and trimethoprim resistance genes spread primarily by transposase and insertion sequences. Differential abundance analysis found Synergistota, Mycoplasmatota, and Chlamydiota as suitable candidates for pollution monitoring in karst springs. This is the first study highlighting the ap-plicability of a combined approach based on high-throughput SmartChipTM antibiotic resistance gene quantification and Compact Dry-pathogen cultivation for estimating microbial contaminants in karst springs and other challenging low biomass environments.