Differential selection for survival and for growth in adaptive laboratory evolution experiments with benzalkonium chloride

Biocides are used to control bacteria across different applications, but emerging resistance may pose risks of for those applications. Resistance to biocides has commonly been studied using adaptive laboratory evolution (ALE) experiments with growth at subinhibitory concentrations linked to serial subculturing. It has been shown recently that E. coli adapts to repeated lethal stress imposed by the biocide benzalkonium chloride (BAC) by increased survival (i.e. tolerance) and not by evolving the ability to grow at increased concentrations (i.e. resistance). Here, we investigate the contributions of evolution for tolerance as opposed to resistance in ALE experiments with E. coli exposed to BAC. We find that BAC concentrations close to the half maximal effective concentration (EC50, 4.36 mug mL-1) show initial killing (~40%) before the population resumes growth. This indicates that cells face a two-fold selection pressure: one for increased survival and one for increased growth. To disentangle the effects of both selection pressures, we conducted two ALE experiments: (i) one with initial killing and continued stress close to the EC50 during growth and (ii) another with initial killing and no stress during the growth. Phenotypic characterization of both treatmentsof adapted strains showed that growth at higher BAC concentrations was only selected for when BAC was present during growth. Whole genome sequencing revealed distinct differences in mutated genes across treatments: Treatments selecting for survival only led to mutations in genes for metabolic regulation (cyaA) and cellular structure (flagella fliJ), while treatments selecting for growth and survival led to mutations in genes related to stress response (hslO and tufA). Taken together, our findings show that selection for growth or survival leads to distinct phenotypic and genotypic outcomes and that commonly used serial subculture ALE experiments with the biocide BAC at EC50 select for both. This should be considered in the design of ALE experiments to adjust match the experimentally imposed selection pressure to the different applications for which resistance risks should be assessed. While selection for survival assesses resistance risks emerging during use of the biocide as a disinfectant, selection for growth in the presence of low concentrations assesses the use of BAC as a preservative or selective effects imposed by BAC pollution in the environment.