| Antibiotics and heavy metals are used widely to treat disease and promote growth in intensive livestock farming, thereby leading to high concentrations of antibiotics, heavy metals, and antibiotic resistance genes(ARGs) in animal manure. As an emerging pollutant, ARGs may be transferred to human pathogens via horizontal gene transfer, which could make antibiotic therapies ineffective and pose a great health risk to humans. Aerobic composting is employed widely to reuse animal manure and the agricultural application of the compost product to soil is an important pathway for transmitting ARGs to the natural environment. This study investigated the diversity and abundance of ARGs in intensive livestock manure, as well as the effects of composting on ARGs in various animal manures. In addition, laboratory composting monitoring experiments were established to study the effects of antibiotics, heavy metals, metal passivators, and temperature on ARGs. The relationships among ARGs, metal resistance genes(MRGs), mobile gene elements(MGEs), and bacterial communities were also analyzed. The aims of this study were to explore the conditions that affect the reductions in ARGs during aerobic composting and to elucidate the underlying microbiological mechanisms. The main results and conclusions are as follows.(1) Diverse and abundant ARGs were detected in livestock manure and the compost produced from this manure, thereby demonstrating that they are both important reservoirs for ARGs. There were significant differences in the ARGs present in manure from various animals. In general, chicken manure and pig manure contained a greater abundance of ARGs than cattle manure. Network analysis showed that many ARGs and MGEs co-occurred in the manure and compost. Equations were established using regression analysis to predict the total abundance of ARGs in manure and compost environments. Aerobic composting had inconsistent effects on the ARGs in manure from various animals, where it reduced the total abundance of ARGs in chicken manure and pig manure, but the reductions were greater in chicken manure. Aerobic composting slightly decreased the total abundance of ARGs in manure from beef cattle whereas it increased the total abundance in manure from dairy cattle. Redundancy analysis shown that the abundance of transposons, Cu concentration, and total nitrogen content contributed most to the differences in the ARG profiles of manure form various animal species.(2) The ARGs profiles and bacterial communities were grouped according to the composting time under different treatments, where a high concentration(200 mg/kg) of oxytetracycline had more persistent effects on the ARG profiles and bacterial communities. A high concentration of oxytetracycline increased the relative and absolute abundances of 5/8 ARGs and int1. The variations in ARGs during composting were affected more by the succession of the bacterial community than the presence of oxytetracycline. Aerobic composting was not effective in reducing most of the ARGs from dairy manure, and diverse ARGs and antibiotic-resistant bacteria were found in the compost product. In addition to the relative and absolute abundances of ARGs, the status and hosts of ARGs should be considered when evaluating the environmental risk of compost products.(3) The Cu-L(500 mg/kg Cu), Cu-H(2000 mg/kg Cu), and Zn-L(2000 mg/kg Zn) treatments significantly increased the abundances of five ARGs, where the Cu-H treatment also enhanced the abundances of tetG and tetQ in the compost product. The ARGs co-occurred with MRGs, and they were also linked with integrons and transposons. The Zn-H(15000 mg/kg Zn) treatment decreased the total abundance of ARGs, and the abundances of sul1, sul2, and tetX were 0.51–1.87 logs lower than those in the control. High toxicity due to the high concentration of Zn may have killed the ARG hosts. The effects of Cu and Zn on ARGs varied among the different composting stages, which could have been related to differences in the bacterial communities at various stages. Redundancy analysis demonstrated that the changes in the ARGs were affected by MRGs, MGEs, and bacterial communities, where they had different effects on ARGs in various stages. These results demonstrate that the influence of Cu and Zn on ARGs was due to a combined effect via multiple mechanisms, such as co-selection, SOSr, and a cytotoxic effect. Overall, the presence of heavy metals in animal manure increased the abundances of ARGs and enhanced their transfer ability, thereby increasing the ecological risk of ARGs.(4) The addition of three passivators reduced the abundances of at least six ARGs in the compost products. The total abundances of ARGs in the compost products under the BC, ZE, and FA treatments were 81%, 83%, and 74% lower than that in the control, respectively. There was no significant correlation between DTPA-Cu and MRGs, and the passivator treatments did not reduce the MRGs, thereby demonstrating that the reduction of ARGs with the passivator treatments was not attributable to a decrease in co-selection pressure due to Cu. Network and redundancy analyses revealed that the addition of passivators reduced the abundance of ARG hosts, which decreased the abundances of ARGs in the compost products. The abundances of integrons and transposons were reduced to various degrees under the three passivator treatments, which indicates that passivators can decrease the DTPA-Cu concentration to inhibit horizontal gene transfer among bacterial communities.(5) The thermophilic and mature phases played important roles in reducing the abundances of ARGs during composting. A sufficiently high temperature led to greater decreases in 5/10 ARGs and two integrons, and it was necessary for reducing the abundances of tetC, tetG, tetQ, and int1. Bacterial succession and horizontal gene transfer were responsible for variations in the ARG profiles. The lower abundance of ARGs after composting for a longer duration at a high temperature may have been related to reductions in ARG host bacteria(including some human pathogenic bacteria), as well as the inhibition of horizontal gene transfer. Continuous thermophilic composting performed better at reducing the abundances of ARGs, integrons, and human pathogenic bacteria; thus, it is recommended for recycling animal manure to decrease the environmental risk due to ARGs. |
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