Denitrifiers Make Great Contribution to Antibiotic Resistance Genes Dissemination in the Gut of Earthworms

Antibiotic resistance genes (ARGs) pose a serious threat to the environment worldwide. The guts of soil animals are a hotspot for ARGs and denitrification in soils. However, it is unclear how denitrification affects the spread of ARG in the earthworm's gut. In this study, the typical soil earthworm <i>Pheretima guillelmi</i> was employed, and was used for performing anoxic incubation with gut content amended with nitrate and nitrite. To analyze the data, a combination of chemical analysis, 16S rRNA-based Illumina sequencing, and high-throughput qPCR were employed. Nitrate treatments, particularly at 5 mM, caused substantial reductions in nitrate concentrations, with a corresponding increase in nitrite, nitrous oxide (N₂O), and nitric oxide (NO) emissions compared to the treatments with the addition of 1 and 2 mM nitrate. Nitrite (0.2, 0.5 and 1 mM) amendments also enhanced the accumulation of nitrogen intermediates. Organic acid production, including acetate and pyruvate, was the highest under the 5 mM nitrate treatment. This treatment also promoted the highest level of glucose utilization, suggesting that glucose metabolism supports enhanced organic acid production. Both nitrate and nitrite treatments exhibited the pronounced enrichment in ARGs, particularly for beta-lactam and multidrug resistance genes. Denitrifying bacteria such as <i>Aeromonas</i>, <i>Bacillus</i>, <i>Raoultella</i>, and <i>Enterobacter</i> were identified as key hosts for these ARGs. These results emphasized that denitrifying bacteria play a pivotal role in the horizontal transfer of ARGs, underscoring the need for careful nitrogen management in agricultural practices to control the spread of antibiotic resistance in natural environments.