Environmental factors and antimicrobial efficacy: the impact of temperature and humidity on material surfaces

Current International Standards Organization (ISO) for testing antimicrobial materials often lack regulatory approval for clinical use because they do not accurately reflect real-world environmental conditions. In this study, we systematically evaluated the effects of temperature (4°C-37°C) and humidity (15%-100% relative humidity) on microbial survival (<i>Staphylococcus aureus</i>, <i>Escherichia coli</i>, HCoV-OC43, and influenza virus) and the efficacy of various antimicrobial surfaces (stainless steel, copper, polyethylene terephthalate [PET], and Cu₂O@ZrP-modified PET) using established ISO standards, including ISO 21702, 18184, 22196, and 20743. We found that pathogen survival declined sharply with increasing temperature, while the effects of humidity varied by material. Copper and Cu₂O@ZrP-PET surfaces achieved greater than 99% viral and bacterial inactivation within 60 min; however, their activity was delayed by two to threefold at 4°C. The effects of humidity were material-dependent. Non-porous copper maintained consistent efficacy across humidity levels, whereas PET/Cu₂O@ZrP exhibited enhanced antibacterial activity under low humidity. Mechanistically, reactive oxygen species generation correlated with efficacy changes across conditions. We advocate revising ISO protocols to include dynamic environmental parameters and propose a tiered classification of antimicrobial efficiency that aligns with clinical demands. This framework addresses the discrepancy between laboratory tests and real-world performance, enabling the robust evaluation of antimicrobial materials for clinical and public health applications.IMPORTANCEThis study addresses a critical gap in our understanding of how real-world environmental conditions affect the performance of antimicrobial materials. Current International Standards Organization (ISO) testing standards fail to adequately account for temperature and humidity variations, leading to discrepancies between laboratory results and real-world effectiveness. Our findings demonstrate that both temperature and humidity significantly impact pathogen survival and antimicrobial efficacy, with important implications for material selection in healthcare, public spaces, and pandemic preparedness. By systematically evaluating these environmental factors across different material types, we provide evidence-based recommendations for revising international testing protocols. This work is essential for ensuring that antimicrobial materials perform as expected when deployed in actual environments, potentially saving lives by improving the reliability of these critical defense mechanisms against infectious diseases.