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The rising number of antibiotic‐resistant bacteria requires alternative antibacterial substances as therapeutics. However, besides a strong effect against bacteria, they should not exhibit cytotoxicity towards human cells. Typically, both properties are tested in separate in vitro experiments that do not take into account naturally occurring interactions. To analyze whether the bacteria‐cell interaction influences the results of antibacterial and cytotoxic testing, surfactant‐free antibacterial silver‐gold alloy nanoparticles produced by laser ablation in liquid were examined both separately and in a straightforward coculture setup. Whereas different commensal bacteria and human skin cells exhibited reduced metabolic activity at comparable concentrations when treated separately, their response completely differed when analyzed in a combined coculture. For the combination of oral keratinocytes and Staphylococcus aureus, reduced cytotoxicity with increasing cell numbers, but a similar antibacterial effect quantified by standard plate counting could be observed. In contrast, coculture of keratinocytes and Porphyromonas gingivalis resulted in a complete absence of cytotoxicity and antibacterial effect, probably due to bacterial invasion of cells. These results clearly showed that the bacteria‐cell interaction greatly influences the results of antibacterial and cytotoxic testing and highlight the importance of more complex in vitro experiments to reliably characterize novel antibacterial substances for efficient clinical translation. [ABSTRACT FROM AUTHOR]

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