Response of microbial nitrogen transformation processes to antibiotic stress in a drinking water reservoir

Effects of antibiotics on microbial nitrogen transformation processes in natural aquatic ecosystems are largely unknown. In this study, we utilized the ¹⁵N stable isotope tracers and metagenomic sequencing to identify how antibiotics drive nitrogen transformation processes in Danjiangkou Reservoir, which is the largest artificial drinking water reservoir in China. We retrieved 51 nitrogen functional genes, and found that the highest abundances of nitrate reduction and denitrification-related genes occurred in dissimilatory nitrogen transformation pathways. ¹⁵N-labelling analysis substantiated that denitrification was the main pathway for nitrogen removal, accounting for 57.1% of nitrogen loss. Nitrogen functional genes and antibiotic resistance genes co-occurred in Danjiangkou Reservoir, and they were mainly carried by the denitrifying bacteria such as Rhodoferax, Polaromonas, Limnohabitans, Pararheinheimera, Desulfobulbus, and Pseudopelobacter. Genome annotation revealed that antibiotic deactivation, Resistance-Nodulation-Division and facilitator superfamily efflux pumps were responsible for the multiple-resistance to antibiotics in these bacteria. Moreover, antibiotics showed non-significant effects on nitrogen transformation processes. It is speculated that denitrifying bacteria harboring ARGs played crucial roles in protecting nitrogen transformation from low-level antibiotics stress in the reservoir. Our results highlight that denitrifying bacteria are important hosts of ARGs, which provides a novel perspective for evaluating the effects of antibiotics on nitrogen cycle in natural aquatic ecosystems.