Response of antibiotic resistance genes expression and mediating role of viral community to antibiotics and heavy metals in anaerobic digestion

This study investigated the effects of chlortetracycline (CTC) and copper (Cu), individually and in combination, on the transfer and expression of antibiotic resistance genes (ARGs) in anaerobic digestion (AD). Utilizing metagenomics and high-throughput quantitative PCR (HT-qPCR), we found that high concentrations of Cu (400 mg/L) and CTC (80 mg/L) significantly enriched the relative abundance of ARGs attributed to the changes of bacterial community (p < 0.05), whereas lower concentrations (Cu: 40 mg/L; CTC: 8 mg/L) had minimal effects. The species abundance distribution and neutral models indicated that antibiotic resistome is determined by stochastic process, and the abundance of ARGs is determined by a limited number of core ARGs, showing the resilient to the selection of antibiotics and heavy metals. Reverse transcription HT-qPCR indicated that most ARGs were silent or expressed at low levels; however, regardless of the concentration, CTC enhanced the expression of ARGs, particularly those linked to tetracycline ribosome protection. In contrast, high Cu levels inhibited ARGs expression due to its non-selective toxicity, which was evidenced by a failure to produce methane. CTC and Cu both showed limited impacts on the potential mobility of ARGs shown by metagenomics, although TET significantly increased the conjugation frequency reflected by conjugation assays (p < 0.05). ARGs carried by virus only accounted for 0.26 % ± 0.10 % of the total, with no evidence of phage-mediated transduction, and phage lysis significantly contributed to ARGs reduction. While CTC and Cu showed limited effects on phage-carrying ARGs, they notably inhibited phage lytic activity, as indicated by virus-host ratios and phage lysis tests, further leading to the enrichment of ARGs in AD system. Our findings provide novel insights into the ARGs transfer under selective pressure of antibiotics and heavy metals in AD.