Manure application increases soil doxycycline and zinc levels and resistance gene abundance.

Doxycycline (DOX) and zinc (Zn), are frequently detected in livestock manure. Untreated excrement carries a sizable load of DOX and Zn into the soil, exacerbating agricultural nonpoint source pollution. However, research on the effects of DOX and Zn on soil microbial diversity and the prevalence of resistance genes is limited. Through an 8-year field experiment coupled with 16S rRNA sequence analysis, we explored the dynamics of DOX and Zn contamination and their influence on soil microbial diversity and resistance gene expression. Our findings revealed a pronounced positive correlation between pig manure application and residual DOX and Zn levels in the soil, with the high pig manure) treatment resulting in increases of 16.42- and 1.14-fold in soil DOX and Zn levels, respectively, compared to those of the control. Soil DOX levels increased with pig and chicken manure application, whereas soil Zn levels showed divergent trends, increasing with pig manure application but decreasing with chicken manure application. As soil depth increased, the contamination risks from DOX and Zn in the soil decreased. Notably, soil DOX and Zn levels exhibited a significant positive correlation with Bacteroidota, Firmicutes, and Proteobacteria. The application of livestock and poultry manures amplified the abundance of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) in the soil, with higher ARG levels in pig manure-treated soil than in soil treated with chicken manure. Conversely, a greater abundance of MRGs was found in the soil treated with chicken manure than in the soil treated with pig manure. The results of this study on the DOX and Zn levels, microbial composition, and resistance genes with the continuous application of livestock and poultry manure in China will be highly valuable for the surveillance, maintenance, and biological removal of DOX and Zn composite pollution in manure.