Distinct sub-MIC kill kinetics of Cu and Ag in Escherichia coli

ABSTRACT 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) is widespread in natural, clinical, and built environments, and may influence microbial survival dynamics that could have implications for tolerance or resistance development. 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 the 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 sustained growth inhibition from transient killing followed by regrowth may be important for interpreting how sub-MIC exposures relate to tolerance and resistance development. Our results suggest that, under these conditions, silver primarily permits regrowth of surviving cells after an initial killing phase, whereas copper imposes sustained growth inhibition. The findings highlight new and known challenges in antimicrobial characterization and risk assessment of metal-based formulations by using widespread non-kinetic endpoint assays such as MIC. IMPORTANCE Copper and silver are widely used metals with antimicrobial properties that are often considered to employ diverse, overlapping biocidal mechanisms of action, and corresponding bacterial defense responses. Here, we show that sub-minimal inhibitory concentration (sub-MIC) copper causes lasting, dose-dependent growth inhibition of Escherichia coli , while silver seemingly delays otherwise normal growth kinetics. The latter is primarily not caused by growth inhibition, but partial killing by silver, followed by normal regrowth of the survivors. These previously under-recognized differences in sub-MIC toxicity kinetics suggest that copper and silver create distinct short-term survival and growth regimes, which may have implications for future studies of tolerance and resistance development. Under the conditions tested, silver-associated growth delay was explained by survival and regrowth of a reduced subpopulation, whereas copper exposure required continued growth under inhibitory conditions. This difference imposes new challenges for the design and application-relevant risk assessment of metal-based antimicrobial formulations.