Umerov, S. | bioRxiv Science

bioRxiv2025

Sub-MIC kill kinetics of copper and silver in Escherichia coli

Copper and silver are well-known and widely used antimicrobial metals that are often considered to employ diverse overlapping biocidal mechanisms of action and induce corresponding bacterial defense responses. Exposure to antimicrobial metals at concentrations below the minimal inhibitory concentration (sub-MIC) are widespread in natural, clinical and built environments and can shape evolutionary trajectories of the affected microbes that could lead to antimicrobial tolerance and/or resistance. By analyzing growth and kill kinetics of Escherichia coli under copper or silver exposure we observed that sub-MIC copper concentrations resulted in a lasting dose-dependent slowing of exponential growth with reduced yield while silver seemed to cause dose-dependent growth delay without substantially affecting exponential growth or yield. Time-kill experiments revealed minimal loss of viability in early copper exposure while in case of silver a rapid dose-dependent transient killing followed by normal exponential regrowth of the survivors was observed, underlying the seemingly dose-dependently extended lag phase durations. Distinguishing conditions that select for antimicrobial resistance (sustained growth) versus tolerance (survival without growth) is essential for antimicrobial stewardship as acquiring tolerance is considered a steppingstone towards developing resistance. Our results suggest that short-term survival of the initial killing by silver is sufficient for selective advantage while maintaining energy-intensive enhanced growth in the presence of copper is needed to gain competitive benefit over the general population. The findings highlight new and known challenges in antimicrobial characterization and risk assessment of metal-based formulations by using wide-spread non-kinetic endpoint assays such as MIC.

microbiology↗

bioRxiv2024

Experimental evolution of Escherichia coli on solid 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 thirty exposure cycles in low-organic dry or high-organic humid conditions. The evolved populations demonstrated increased metal surface tolerance without concurrent increase in MBC and 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 assessment but also risk assessment of antimicrobial surface applications. ImportanceThis 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.

microbiology↗