FakA impacts antiseptic susceptibility in Staphylococcus aureus and Enterococcus faecalis

Biocides are widely used to control and prevent healthcare-associated infections and understanding how nosocomial pathogens respond to biocidal agents is key to improving infection prevention and control products and practices. An evolution model was used to study how Staphylococcus aureus and Enterococcus faecalis responded after repeated exposure to sub-lethal concentrations of chlorhexidine digluconate (CHX) and octenidine dihydrochloride (OCT) when grown planktonically and as biofilms. Both pathogens were able to adapt to grow at concentrations above the MIC of both biocides with planktonic lineages surviving at higher concentrations of both agents than biofilm lineages. Exposure to CHX was linked with lower biofilm biomass production in E. faecalis although biofilm biomass increased for S. aureus isolates after exposure to both agents. Evolved isolates had no major fitness deficit and only low-level changes to susceptibility to antibiotics were observed after biocide exposure. Sequencing of biocide adapted mutants repeatedly identified mutations within fakA encoding a fatty acid kinase in independent lineages of S. aureus after exposure to both biocides in all conditions. Analogous changes were observed within the homologous gene in parallel experiments with Enterococcus faecalis. Further assays to study the mechanistic basis and relationship to phospholipid production showed that evolved isolates with fakA mutations accumulated less ethidium bromide than parent strains, exhibited altered cell envelope morphology and decreased susceptibility to daptomycin. This data shows important pathogens can evolve limited tolerance to two common biocides but that this has collateral impacts on biofilm formation, colony morphology and fitness. FakA appears to play an important role in biocide tolerance.