Long-term application of organic fertilization causes the accumulation of antibiotic resistome in earthworm gut microbiota

Antibiotic resistance genes (ARGs), prevalent across multiple environmental media, threaten human health worldwide and are considered emerging environmental contaminants. Earthworm gut, a niche for bacteria to survive, represents a potential reservoir for ARGs in soil. However, the compositions of ARGs in the earthworm gut microbiota remain elusive, especially under field conditions. In this study, we applied high-throughput quantitative PCR to profile the ARGs in the gut microbiota of earthworms after chronic exposure to fertilizers. To elucidate the factors that impact the ARGs composition, the bacterial community of gut microbiota, mobile genetic elements (MGEs), soil (nutrients, heavy metals, and antibiotics) and the properties of gut content (pH and nutrients) were analyzed. A total of 98 subtypes among 9 major types of ARGs, and 3 different MGEs were detected in the gut microbiota of earthworms. Organic fertilizer (sewage sludge and chicken manure) application significantly increased the diversity and abundance of ARGs. Of the 1123 identified operational taxonomic units (OTUs) at 97% similarity cutoff, most of them were assigned to Firmicutes (55.5%) and Proteobacteria (33.6%) in earthworm gut microbiota. Long-term organic fertilization slightly changed the microbiota composition, but did not impact the diversity. Partial redundancy analysis (pRDA) revealed that bacterial community, combined with environmental factors (soil and gut content properties) and MGEs, explained 72% of the variations of ARGs in the earthworm gut. Furthermore, the co-occurrence pattern between ARGs and MGEs indicated that horizontal gene transfer via MGEs may occur in the earthworm gut. These findings improve the current understanding of the dynamics of soil fauna-associated ARGs and the gut microbiota of earthworms may be an underappreciated hotspot for ARGs in the environment.