A novel and validated 3D-printed method for the consistent and reproducible dry transfer of microorganisms for the determination of antimicrobial surface efficacy

Chareyre, Emma ORCID: https://orcid.org/0009-0001-0958-0182, Cunliffe, Alexander J ORCID: https://orcid.org/0000-0002-7099-0121, Askew, Peter, Iredale, Gillian, Marchant, Abby, Dean, Andrew P ORCID: https://orcid.org/0000-0001-6893-5118 and Redfern, James ORCID: https://orcid.org/0000-0003-0958-683X (2025) A novel and validated 3D-printed method for the consistent and reproducible dry transfer of microorganisms for the determination of antimicrobial surface efficacy. Applied and Environmental Microbiology, 91 (8). e00802-25. ISSN 0099-2240

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Abstract

Transfer of microorganisms by touch is one way microorganisms can move within an environment. Antibacterial materials offer a potential solution, but currently, methods that use a dry inoculum, which are, therefore, realistic to many examples of touch transfer, are lacking. This study developed a novel, reproducible method for dry transfer of bacteria, using novel 3D-printed apparatuses, and applied the method to antibacterial copper surfaces. Method validation was carried out by transferring MRSA to stainless steel coupons under varying parameters and conditions (bacterial concentration, donor surface water content, bacterial inoculum drying time on donor surface). Additionally, the method was used to quantify the bacterial transfer to copper coupons under varying relative humidity environments, while comparing to the widely adopted standard ISO22196. The method demonstrated reproducible transfer of MRSA to both stainless steel and copper coupons. Antibacterial efficacy of copper following dry deposition was reduced in drier relative humidity environments, with the greatest population reduction after 2 h observed at higher relative humidity environments (>60%, 2.32-log), compared to the mid (40%-60%, 1.29-log) and low (<30%, 0.66-log) relative humidity environments. In contrast, copper demonstrated significant antibacterial activity under ISO 22196 methodology (i.e., wet inoculum and humid). These results provide a validated, novel protocol for dry transfer of bacteria for antibacterial efficacy assessment and highlight that copper-containing surfaces require greater environmental moisture for enhanced antibacterial effects. This study demonstrates the need for new standards more analogous to the end-use of an antibacterial material.IMPORTANCEThe transmission of microorganisms between surfaces by touch contributes to an increased healthcare burden due to secondary infection and mortality rates. Antibacterial materials can help to reduce the transmission of bacteria between surfaces and form part of a wider infection control system. However, testing the efficacy of antibacterial materials often uses unrealistic conditions (e.g., using large volumes of liquid) that may provide data overemphasizing their antibacterial action when in use. Additionally, there is no current standard method for assessing antibacterial surfaces contaminated via touch. This paper describes and validates a novel method to reproducibly transfer microorganisms to a surface enabling realistic deposition. Furthermore, the validated method was applied to antibacterial copper surfaces that are capable of passing current standards due to the availability of liquid (that copper surfaces require to be antibacterial) and found a reduced antibacterial effect under more realistic conditions.