Experimental evolution ofEscherichia colion solid silver, copper, stainless steel, and glass surfaces

Abstract

To study bacterial adaptation to antimicrobial metal surfaces in application-relevant semi-dry environmental conditions,Escherichia coliATCC 8739 was exposed to copper, silver, stainless steel, and glass surfaces for thirty consecutive cycles in exposure conditions designed to select for either survival or growth. Subsequent mutation analysis did not find changes in overall mutation frequency that could lead to enhanced adaptation potential nor mutations incop, cus, cue, sil, pcoor general efflux genes known to actively maintain copper/silver homeostasis in copper or silver exposed populations compared to populations evolved on steel and glass control surfaces. However, mutations insilSrapidly activated the crypticsilefflux locus during silver ion challenge in liquid assay with the evolved populations and caused collateral increase in ampicillin and ciprofloxacin tolerance, indicating that efflux might be specific to granting heavy metal tolerance in liquid but not surface exposure format. Instead, during cyclic surface exposure mutations in genes related to cellular barrier functions and sulfur metabolism were enriched on copper and silver surfaces. This increased metal surface tolerance but had less to no protective effect in subsequent conventional MBC and MIC tests indicating that reducing bioavailability and passively restricting uptake of the toxic metals rather than active efflux contributes to increased viability on copper and silver surfaces compared to liquid format. Our findings highlight the critical importance of appropriate exposure conditions not only in efficacy assessment but also risk assessment of antimicrobial surfaces rendering results acquired via liquid tests not applicable for dry-use surfaces.ImportanceThis study examines the evolutionary adaptations ofEscherichia coliunder sublethal exposure to copper and silver surfaces, revealing a moderate increase in surface tolerance but no heightened mutation frequency or enhanced metal ion tolerance in standard tests. Notably, enriched mutations indicate a shift toward more energy-passive mechanisms of metal tolerance. Additionally, while enhanced silver efflux—with a collateral increase in antibiotic tolerance—was rapidly selected in a single round of silver exposure in liquid tests and substantially increased copper and silver ion tolerance in conventional test formats, the causal mutations did not improve viability on silver and copper surfaces, underscoring the different fitness dynamics of tolerance mechanisms dependent on exposure conditions. These findings emphasize the need for appropriate exposure conditions in evaluating of both efficacy and the potential risks of using antimicrobial surfaces, as the results from conventional liquid-based tests may not apply in solid contexts.