Co-exposure to heavy metals and antibiotics enhances resistance and transcriptional adaptation in enteric pathogens from poultry

• Poultry gut acts as a reservoir for emerging MDR bacteria under metal pressure. • Cloacal isolates tolerate high doses of heavy metals commonly present in poultry environments. • Prolonged exposure to heavy metals increased the antibiotic resistance of enteric pathogens. • Heavy metal significantly reduces the ZOI for multiple antibiotics. • Metal exposure upregulates efflux pumps and resistance genes, contributing to cross-resistance to antibiotics. Heavy metals and antibiotics are widely used in poultry production for growth promotion and infection control. This co-exposure affects antibiotic susceptibility patterns and transcriptional responses through co-resistance and cross-resistance mechanisms. This study investigated the impact of heavy metals on antibiotic susceptibility in Escherichia coli and Salmonella enterica serovar Typhimurium, isolated from cloacal swabs of broiler chickens in Sylhet. Dose–response assays were used to determine the minimum inhibitory concentrations (MICs) for arsenic (As), zinc (Zn), copper (Cu), and chromium (Cr). Changes in antibiotic susceptibility were evaluated by exposing bacteria to different concentrations of heavy metals, followed by the Kirby-Bauer disk diffusion method. Growth kinetics were measured under various combinations of stress conditions. PCR and RT-qPCR were performed to detect resistant determinants and to quantify mRNA copy numbers. Both bacteria tolerated high concentrations of heavy metals. Antimicrobial susceptibility testing showed a significant reduction in zones of inhibition after AsO₄³⁻ and Cu 2+ pre-exposure for ciprofloxacin, chloramphenicol, meropenem, imipenem, and tetracycline ( p < 0.02). Growth kinetics demonstrated that co-exposure to AsO₄³⁻, Zn 2+ , or Cu 2+ with tetracycline significantly enhanced bacterial resistance. Prolonged exposure to AsO₄³⁻ or Cu 2+ increased the MIC of tetracycline by approximately 60%, accompanied by transcriptional upregulation of multidrug efflux pumps ( acrB, mdtA, tolC ), global regulators ( marA, soxS, baeS ), and metal-detoxification operons ( ars, cop ). Sub-inhibitory heavy-metal exposure remodeled antibiotic susceptibility and promoted tetracycline tolerance in poultry-derived E. coli and S. Typhimurium, in part via efflux activation and metal-stress regulons. Species-specific transcriptional reprograming under AsO₄³⁻ and Cu 2+ highlighted complex co-selection dynamics in poultry environments.