Bat guano contamination of karst spring water revealed by an automated microbial source tracking pipeline: Integrating amplicon sequencing and shotgun metagenomics

Karst spring water in Korea is widely consumed untreated, yet its microbiological safety remains unexamined in bat-inhabited limestone cave regions. We developed MST-Pipeline, an open-source platform automating the complete microbial source tracking (MST) workflow from raw 16S rRNA amplicon data to source attribution, featuring automated V4 sub-region extraction for cross-region harmonization. Validation with fecal spike-in experiments confirmed dose-dependent source attribution, and PacBio long-read analysis demonstrated cross-platform compatibility. Application to the Mungyeong karst region (28 amplicon and 9 shotgun metagenomic samples) identified bat guano as the highest-contributing fecal source among the nine host groups tested in cave and stream water, with contributions higher in August (4.76%) than July (1.31%), coinciding with increased summer bat activity and antecedent rainfall. The downstream residential site showed distinct human and pig fecal signatures. Shotgun metagenomics revealed that guano harbors a wildlife-derived antibiotic resistance gene (ARG) reservoir dominated by multidrug efflux systems, while the downstream residential site carries anthropogenic ARG signatures including sul1-qacEΔ1 integron cassettes at 4-fold higher abundance. Genome-resolved analysis identified contigs co-carrying ARGs and virulence factors corresponding to multidrug-resistant uropathogenic Escherichia coli, Salmonella enterica serovar Typhimurium, and Shigella dysenteriae. These findings demonstrate that karst spring water perceived as clean is contaminated with antibiotic-resistant pathogenic bacteria from bat guano, compounded by anthropogenic inputs downstream.