Microplastics and Copper Co-Exposure Induces Intestinal Damage, Gut Dysbiosis, and Antimicrobial Resistance in Zebrafish (Danio rerio)

Microplastics (MPs) and metals frequently co-occur in aquatic environments, yet their combined effects on gut health and antimicrobial resistance in fish remain poorly understood. This study investigated the chronic effects of polyethylene (PE) and polystyrene (PS) microplastics, alone or combined with copper (Cu), on intestinal integrity, the gut-associated Gram-negative cultivable fraction, and phenotypic antimicrobial resistance in adult zebrafish (Danio rerio). Fish were exposed for 21 days to MPs (1 mg/L), Cu (25 µg/L), or their combinations. Histopathological analysis revealed that Cu-containing treatments induced more severe intestinal alterations, including edema, villus degeneration, and necrosis, whereas MPs-only exposures produced milder and heterogeneous responses. The composition of the Gram-negative cultivable fraction varied among treatments, with Cu, particularly in combination with MPs, associated with a broader occurrence of opportunistic and potentially pathogenic taxa. Antimicrobial susceptibility testing showed a high prevalence of multidrug resistance across treatments, with broader resistance spectra observed in Cu-containing exposures, consistent with metal-driven co-selection. In contrast, MPs alone did not systematically increase resistance and, for some antibiotics, showed resistance levels comparable to or lower than controls. Integrated multivariate analyses indicated that intestinal pathology and antimicrobial resistance co-varied along gradients of overall stress severity and stressor type, with Cu acting as the dominant driver and MPs exerting a modulatory, context-dependent influence. Overall, these findings highlight the importance of integrated assessments of gut pathology, microbial composition, and antimicrobial resistance to better understand the ecological and One Health implications of combined microplastic–metal exposure in aquatic systems.