Small colony variants are more susceptible to copper-mediated contact killing for Pseudomonas aeruginosa and Staphylococcus aureus

Applying self-sanitizing copper surfaces to commonly touched places within hospital facilities is an emerging strategy to prevent healthcare-associated infections. This is due to the fact that bacterial pathogens are rapidly killed on copper, a process termed contact killing. However, the mechanisms of contact killing are not fully understood, and the potential of bacterial pathogens to develop resistance has rarely been explored. Here, we hypothesize that bacteria are predominantly killed by a burst release of toxic copper ions, resulting from chemical reactions between bacterial cell surface components and metallic copper. To test this, we isolated and characterized small colony variants (SCVs) derived from Pseudomonas aeruginosa and Staphylococcus aureus. SCVs overproduce extracellular polymeric substances (EPS), which will enhance copper ion release, causing more rapid death on copper. Indeed, all 13 SCVs tested were more rapidly killed than wild-types on the surfaces of both pure copper and brass (63.5 % Cu). Next, using the non-pathogenic Pseudomonas fluorescens SBW25 as a model, we examined the roles of specific cell surface components in contact killing, including EPS, LPS, capsule, flagella and pili. We also subjected P. fluorescens SBW25 to daily serial passage of sub-lethal conditions on brass. After 100 transfers, there was a slight increase of survival rate, but ~97 % of cells can still be killed within 60 min on brass. Together, our data implicate that the rate of contact killing on copper is largely determined by the cell surface components, and bacteria have limited ability to evolve resistance to metallic copper.