ALEE-AMC: a standardizable method to determine evolutionary adaptation of bacteria on non-porous surfaces with antibacterial activity

Evolutionary adaptation of bacteria to antimicrobial materials and coatings (AMCs) threatens their efficacy and may lead to cross-resistance to antibiotics. We develop an adaptive laboratory evolution experiment to be performed on AMCs (ALEE-AMC) based on the efficacy standard ISO 22196:2011, using metallic copper as a reference AMC and Escherichia coli as a model microorganism. The method consists of three steps: (i) determination of a timepoint at which viable cells are reduced upon AMC exposure by ~1000-fold, (ii) repeated cycles of exposure to AMCs and re-growth of surviving cells, and (iii) isolation of clones from the evolved populations and phenotypic testing. Repeated exposure to copper consistently selects for increased survival of evolved populations across participants of a ring trial. We find that laboratory identity did neither have a statistically significant effect on the average adapted antibacterial activity nor on the number of replicate populations that increased their survival to >100-fold. Antimicrobial susceptibility testing of isolated clones and whole genome sequencing showed that ALEE-AMC can detect changes in susceptibility including those above the clinical breakpoint related to mutations in known resistance determinants; specifically, cross-resistance to fosfomycin was observed in several copper-evolved clones. ALEE-AMC offers a standardizable platform to assess the risk of bacterial adaptation to AMCs.