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.