Project description: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 PAβN or non-antibiotic efflux substrate acriflavine. The aim was to identify mutations responsible for restoring biofilm formation in populations exposed to PAβN 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 PAβN 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.
Project description:Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections in immunocompromised individuals, such as patients with cystic fibrosis where it commonly forms biofilms. Ciprofloxacin is used extensively to treat P. aeruginosa infections, but its effectiveness can be significantly reduced due to biofilm formation. Although many individual genes associated with biofilm formation have been characterised, the genetic basis of P. aeruginosa biofilm fitness related to antibiotics challenge remain unexplored. In this study we employed a high-density TraDIS-Xpress library of P. aeruginosa PAO1 to assay the impact of gene disruptions or altered gene expression on biofilm formation at different concentrations of ciprofloxacin. Gene fitness was analysed by comparing the biofilm samples to planktonic samples harvested at 12h, 24h and 48h with and without ciprofloxacin. Gene determinants of survival for biofilms at different stages of maturity in the presence and absence of ciprofloxacin were identified.