Experimental evolution of Escherichia coli on semi-dry silver, copper, stainless steel, and glass surfaces.

To study bacterial adaptation to antimicrobial metal surfaces in application-relevant conditions, Escherichia coli was exposed to copper and silver surfaces for 30 exposure cycles in low-organic dry or high-organic humid conditions. The evolved populations demonstrated increased metal surface tolerance without concurrent increase in minimal biocidal concentration (MBC) and minimal inhibitory concentration (MIC) values of respective metal ions or selected antibiotics. Mutation analysis did not detect increased mutation accumulation nor mutations in cop, cus, cue, sil, pco, or general efflux genes known to actively maintain copper/silver homeostasis. Instead, during cyclic exposure, mutations in genes related to cellular barrier functions and sulfur metabolism were enriched, potentially suggesting that reducing bioavailability and passively restricting uptake of the toxic metals rather than active efflux is selected for on copper and silver surfaces. The changes detected in the evolved populations did not indicate an increased risk of antibiotic cross-resistance as a result of copper or silver surface exposure. However, rapid emergence of mutations in silS activated the cryptic sil efflux locus during silver ion challenge in liquid MBC assay with the evolved populations. The silS mutants showed no benefit on copper and silver surfaces but demonstrated decreased sensitivity to ampicillin and ciprofloxacin, as well as copper and silver ions in liquid tests, indicating that efflux might be specific to granting heavy metal tolerance in liquid but not surface exposure format. Our findings highlight the critical importance of appropriate exposure conditions not only in efficacy testing but also in risk assessment of antimicrobial surface applications. IMPORTANCE: This study examines the evolutionary adaptations of Escherichia coli after semi-dry exposure to copper and silver surfaces, leading to an increase in surface tolerance but no increase in mutation accumulation or substantially 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 was rapidly selected for 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 scenarios 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.