Comparative antibacterial, synergistic, and anti-biofilm efficacy of metal ions against multidrug-resistant pathogens on dairy equipment surfaces.

UNLABELLED: This study evaluated the antibacterial and anti-biofilm activities of selected metal ions (Al3+, Cu2+, Au3+, Ag+, and Zn2+) against extended-spectrum β-lactamase-producing Escherichia coli (ESBL-E. coli), Methicillin-Resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus faecalis (VRE). The rapid emergence of antimicrobial resistance (AMR) has intensified the threat posed by multidrug-resistant (MDR) pathogens, particularly biofilm-forming bacteria associated with dairy equipment surfaces. Among the tested metals, silver ions showed the strongest antibacterial activity, producing inhibition zones up to 19.00 ± 1.15 mm at 0.3125 mM and a minimum inhibitory concentration (MIC) of 0.078 mM, followed by gold ions (MIC 2.5 mM). Zinc and copper exhibited moderate activity at higher concentrations, whereas aluminium showed the least effect. Checkerboard assays demonstrated synergistic or additive interactions, particularly for Cu-Au, Zn-Cu, and Zn-Ag combinations [Fractional inhibitory concentration index (FICI) 0.5-0.75], while a ternary Cu-Zn-Ag combination showed notable additive interaction (FICI 0.512). Propidium iodide uptake assays suggested membrane damage and disruption of cell integrity. Silver also exhibited the highest biofilm eradication activity (50% biofilm eradication concentration (BEC₅₀ 0.156 mM), followed by gold and copper. On stainless steel dairy equipment surfaces, silver and gold reduced biofilm biomass by > 60% and > 40%, respectively, as confirmed by confocal laser scanning microscopy and scanning electron microscopy. Silver additionally induced the highest intracellular reactive oxygen species generation (florescence intensity 93854.92 ± 2616.2 to 165407.18 ± 148.3), indicating oxidative stress-mediated antibacterial action than other metals. This study provides a comparative evaluation of metal ions and their synergistic combinations against MDR pathogens and biofilms associated with dairy equipment surfaces and dairy-processing environments.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s13205-026-05084-4.