Antibiotics or Heavy Metals in Livestock Wastewater: Which One Is the Main Driver for the Development and Spread of Antibiotic Resistance under Coexposure?

Antibiotics and heavy metals are widely used in livestock farming to promote animal health and growth, leading to their frequent co-occurrence as contaminants in livestock wastewater. However, their relative contributions to shaping the antibiotic resistome in treatment systems remain unclear. In this study, we simulated an aerobic activated sludge process treating livestock wastewater containing enrofloxacin and heavy metals (Cu²⁺ and Zn²⁺) to evaluate the development of antibiotic resistance using metagenomic and metatranscriptomic approaches. We observed a diverse and transcriptionally active resistome with over half of the detected antibiotic resistance genes (ARGs) showing expression. ARG profiles under coexposure to enrofloxacin and heavy metals more closely resembled those under heavy metal exposure alone than those under enrofloxacin exposure alone. Zn²⁺ exposure resulted in the highest absolute ARG abundance, nearly double that of the control group. Both enrofloxacin and heavy metals significantly altered the abundance and phylogenetic composition of the antibiotic-resistant bacteria (ARB). The exposure to Zn²⁺ enhanced the relative abundance and expression level of both metal resistance genes (MRGs)-carrying ARB and the ARGs-carrying plasmids. Phylogenetic analysis of ARG flanking sequences revealed high homology across various genetic contexts. Among mobile genetic elements, plasmids had a greater influence on ARG profiles than did phages or integrative and conjugative elements (ICEs). Transcriptional profiles of microbial physiological adaptations suggested that modulation of cell membrane permeability, promotion of conjugative transfer, and formation of biofilm might play roles in enhancing antibiotic resistance. These findings suggest at environmentally relevant concentrations, heavy metals such as Zn²⁺ may present a stronger selective pressure than enrofloxacin for the propagation of antibiotic resistance in aerobic activated sludge process treating livestock wastewater.