Fig. 4

Reversing resistance due to conjugation-assisted persistence. a Combining inhibition of conjugation and promotion of plasmid loss to reverse resistance. This strategy is expected to increase \({\eta _{\rm Crit}}\) and decrease \({\eta _{\rm C}}\), potentially destabilizing the plasmid (Eq. (1)). b Evaluating conjugation inhibitor linoleic acid (Lin) and plasmid loss rate promoter phenothiazine (Pheno). Left: B^K and R⁰ were grown overnight with or without 3.25 mM Lin to quantify conjugation efficiency (see Methods). Right: B^K− was propagated daily in the presence of 50 μg/mL Kan, nothing, or 120 μM of Pheno. Kan was used as a control. Y-axis is the fraction of B^K− without antibiotic normalized by that treated with Kan quantified via flow cytometry. Pheno significantly increased the rate of plasmid loss by ~four-fold (see Supplementary Fig. 6B, right panel). c, d Inhibition of R^K and R⁴¹. R⁰ and R^K or R⁴¹ were mixed in equal fractions and diluted 10,000× daily for 11 days. Y-axis is fraction of plasmid-carrying cells and x-axis is days. Green shading indicates the treatments from dark to light: control, Pheno, Lin, and combined. Both plasmids were successfully reversed; when Lin was sufficient alone, Pheno had minimal effect (K). If Lin alone was insufficient, Lin with Pheno synergistically destabilized the plasmid. e Combination treatment with Lin and Pheno suppressed or reversed resistance. The same strains and protocol were used as in Fig. 2d, except media was supplemented with 3.25 mM Lin and 120 μM Pheno fresh daily (see Methods). The plasmids used are (i) #168, (ii) #193, (iii) R388, (iv) C, (v) #41, (vi) RP4, (vii) K, (viii) PCU1, and (ix) R6K (see Supplementary Tables 1 and 3). All CFU measurements were done in replicates of four-to-six plates, and repeated at least twice for reproducibility. All flow measurements were propagated with at least eight well replicates and repeated at least twice for reproducibility. Error bars represent the standard deviation of the plate or well replicates