Fast bacterial growth reduces antibiotic accumulation and efficacy

This is the final version. Available from eLife Sciences Publications via the DOI in this record. Data availability: All data acquired for this study are presented within the manuscript, the supplementary information and the source data files.Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here, we hypothesise that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast-growing variants, including efflux that can reduce macrolide accumulation. We used this new knowledge to eradicate variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures.Engineering and Physical Sciences Research Council (EPSRC)Biotechnology & Biological Sciences Research Council (BBSRC)Medical Research CouncilGordon and Betty Moore FoundationEngineering and Physical Sciences Research CouncilWellcome TrustRoyal SocietyH2020 Marie Skłodowska-Curie Action