Whole genome sequencing of Salmonella enterica serovar Typhimurium directed evolution experiment, involved continuous exposure of planktonic cells or biofilms to subinhibitory concentrations of acriflavine or phenylalanine-arginine ß-naphthylamide (PABN)
We previously developed a directed evolution model that passaged biofilm populations of in S. Typhimurium under antibiotic stress, which identified trade-offs between biofilm formation and antibiotic resistance, conferred by increased efflux activity (Trampari et al., 2021). We applied this same model to expose biofilm populations of S. Typhimurium to RND efflux inhibitor PAßN or non-antibiotic efflux substrate acriflavine. The aim was to identify mutations responsible for restoring biofilm formation in populations exposed to PAßN and to identify how cells in the biofilm can upregulate efflux in the presence of acriflavine. We hypothesised that comparing the mutations selected in both conditions would identify intersecting regulatory relationships between efflux activity and biofilm formation.   Parallel linages of S. Typhimurium were grown on glass beads and in planktonic culture in the presence of a subinhibitory concentration of PAßN or acriflavine (or an untreated control) and passaged into new treated media every 48-72 hours. After one, five and ten passages (corresponding to 2, 17 and 35 days of continuous exposure), the populations were stored, DNA was extracted and sequenced to determine mutations selected in these conditions. We compared mutations from conditions treated with efflux substrates to untreated controls in planktonic and biofilm conditions. To explore phenotypic heterogeneity in these populations, we streaked biofilm populations from passages five and ten onto LB agar and picked three single colonies at random to sequence alongside the populations.
We previously developed a directed evolution model that passaged biofilm populations of in S. Typhimurium under antibiotic stress, which identified trade-offs between biofilm formation and antibiotic resistance, conferred by increased efflux activity (Trampari et al., 2021). We applied this same model to expose biofilm populations of S. Typhimurium to RND efflux inhibitor PAßN or non-antibiotic efflux substrate acriflavine. The aim was to identify mutations responsible for restoring biofilm formation in populations exposed to PAßN and to identify how cells in the biofilm can upregulate efflux in the presence of acriflavine. We hypothesised that comparing the mutations selected in both conditions would identify intersecting regulatory relationships between efflux activity and biofilm formation.   Parallel linages of S. Typhimurium were grown on glass beads and in planktonic culture in the presence of a subinhibitory concentration of PAßN or acriflavine (or an untreated control) and passaged into new treated media every 48-72 hours. After one, five and ten passages (corresponding to 2, 17 and 35 days of continuous exposure), the populations were stored, DNA was extracted and sequenced to determine mutations selected in these conditions. We compared mutations from conditions treated with efflux substrates to untreated controls in planktonic and biofilm conditions. To explore phenotypic heterogeneity in these populations, we streaked biofilm populations from passages five and ten onto LB agar and picked three single colonies at random to sequence alongside the populations.
European Bioinformatics Institute