Doxycycline induces the rebound of three tetracycline resistance genes during maturation of laying hen manure composting by increasing the abundance of potential host bacteria

Livestock manure is considered as an important reservoir of antibiotic resistance genes (ARGs). Antibiotics used in livestock production can stress microorganisms and lead to the spread of ARGs, yet composting is viewed as an effective means of reducing ARGs. However, it remains unclear why ARGs typically decline at high composting temperatures but rebound during compost maturation with varied concentrations of antibiotics. Two concentrations of doxycycline (5 and 20 mg/kg) were added to manure of laying hen mixed with sawdust for composting to evaluate changes in ten tetracycline resistance genes (TRGs) and two mobile genetic elements (MGEs), and to investigate the rebound of TRGs during compost maturation. The results showed that the total absolute abundances of TRGs continuously decreased in all compost treatments. Compared with the start (day 0), the relative abundance of tetA, tetG and tetX in the three compost treatments significantly decreased at thermophilic stage (day 3-day 8), but rebounded in the maturation stage (day 9-day 25) (P < 0.01). 16S rRNA sequencing showed that the relative abundance of Proteobacteria and Bacteroidetes in the three groups decreased significantly at thermophilic stage (day 3-day 8) compared with the mesophilic stage (day 0-day 2) and then increased significantly during the maturation stage (day 9-day 25) (P < 0.05), while Firmicutes showed the opposite trend. Pearson correlation analysis showed the strong correlation between the rebound TRGs and major bacteria, such as Proteobacteria and Bacteroidetes, under doxycycline stress. It suggested that the potential host bacteria caused the rebound of TRGs during compost maturation under doxycycline stress. RDA and VPA analysis showed that Marinimicrobium, Pusillimonas and Sphingobacterium contributed to the rebound of tetA, tetG and tetX. This study provides insights into the rebound of ARGs typically seen during the compost maturation stage and a reference for evaluating the risk of spreading ARGs in the process of manure composting treatment.