This study employed high-throughput quantitative PCR and 16S rRNA sequencing to evaluate the effect of temperature and residual antibiotics on the dynamics of antibiotic resistance genes (ARGs) and microbial communities during anaerobic digestion of swine manure. The abundances of total ARGs and 16S rRNA genes significantly decreased in all of four treatments (25°C, 37°C, and 37°C with 50 mg of wet weight antibiotics of body weight, and 55°C). The abundances of most ARG types were significantly correlated with those of the 16S rRNA gene and transposase gene (P < 0.01). However, the abundances of total ARGs at 55°C were much higher than those of other treatments. Meanwhile, the microbial communities at 55°C, where the Streptococcus pathogen remained at a relatively high abundance and cellulose degraders and hydrogen producers, such as Ethanoligenens and Coprococcus bacteria, increased, were markedly different from those of other treatments. Redundancy analysis indicates that temperature, pH, and the genus Streptococcus had the highest explanation for ARG variation among experimental factors, chemical properties, and representative genera, respectively. Network analysis further showed that the genus Streptococcus contributed greatly to the higher ARG abundance at 55°C. The moderate antibiotic residue only caused a slight and transitory inhibition for microbially diverse populations and promotion for ARG abundance, probably due to the degradation of antibiotics and microbial adaptability. Our results clarify the cooperativity of gene transfer-related items on ARG variation and intensively prove that higher temperature cannot always achieve better ARG removal in anaerobic digestion unless pathogens and gene transfer elements are more efficiently inhibited.

IMPORTANCE
Antibiotic resistance genes (ARGs) are frequently detected with high abundance in manure-applied soils. Anaerobic digestion is one of widely used processes for animal waste treatment. Thus, it is critical to understand the potential of anaerobic digestion to attenuate ARGs. Although some previous studies recommended thermophilic digestion for ARG removal, they did not get sufficient evidence to support this view. The antibiotics applied to animals are mostly excreted through feces and urine because of incomplete metabolism. It is indispensable to know whether residual antibiotics in manure will hinder ARG attenuation in anaerobic digesters. The significance of our research is in comprehensively understanding the evolution and mechanism of ARGs in anaerobic digestion of swine manure affected by temperature and residual antibiotics, which will allow the development of an ARG elimination strategy before their release into the environment.

Network analysis showing the significant correlation between 64 microbial genera (source) and 35 ARGs (target) (P < 0.01). The genera with frequency of >0.01% in at least one treatment, and the ARGs with minimum CT value of ≤21 and higher relative abundance in T55 and T37 than in T25 (Table S3) were involved in network analysis. The size of nodes is proportioned to the number of connections (the degree). The ARGs with connections more than 20 are labeled with primer name. The genera with more than 10 connections are labeled with code number and characterized in Table S4.