Competition analysis of strains carrying different <i>tet</i>(A) alleles in presence of increasing antibiotic concentrations.

(A) Schematic of head-to-head competition experiments. Isolates to be competed are labelled with fluorescent markers and mixed at equal proportions. After competition growth, cells are counted using a flow cytometer to determine the cell ratio. (B) Example of ratios of cells measured over a period incubation (left). The slope of the change in ratio (allele X/allele Y) gives the selection coefficient, which is also plotted on the right. Green: positive selection coefficient (allele X has a fitness advantage over allele Y). Grey: neutral selection (neither allele has a fitness advantage). Orange: negative selection coefficient (allele Y has a fitness advantage over allele X). (C) Competitions of isogenic strains carrying either tet(A)wt or tet(A)ΔtetR, showing the selection coefficient (tet(A)wt over tet(A)ΔtetR) at different concentrations of TC, TGC, DO, MI, OT, and CT (Wilcoxon rank sum test). X-axis shows the antibiotic concentration in mg/L. The highest concentrations used correspond to 1/2 the MIC of the tet(A)wt-carrying strain. Missing values for TGC represent concentrations that were removed as they strongly affected growth of both alleles. Green circles: tet(A)wt has a fitness advantage over tet(A)ΔtetR. Grey circles: no discernible fitness advantage for either allele. Orange circles: tet(A)ΔtetR has a fitness advantage over tet(A)wt. A total of 18 to 25 biological replicates per antibiotic concentration and competition, mean, and SD shown here. The underlying data can be found in S1 Data. (D) The maximum fitness advantage of tet(A)ΔtetR, given as the selection coefficient of tet(A)ΔtetR, is indicated as well as the concentration at which the advantage was observed. CT, chlortetracycline; DO, doxycycline; MI, minocycline; OT, oxytetracycline; TC, tetracycline; TGC, tigecycline.