Fig. 4: L28R-specific 4′-DTMP slows down evolution of antibiotic resistance in the morbidostat.

a Overlaid changes in resistance levels (IC₅₀) over time for E. coli populations evolved in parallel under inhibition by 4′-DTMP (teal, n = 8 replicates) and TMP (gray, n = 7 replicates), demonstrating an increase in resistance by ~100-fold and ~2000-fold, respectively. Day 0 corresponds to parent strain. Light lines represent individual cultures; dark lines with filled circles represent mean values. b A log-logistic function was used to estimate final resistance levels and rate of evolution for evolved E. coli cultures. Representative example showing log-logistic function fits in red lines. c E. coli cultures acquired lower resistance levels (p = 2.10e⁻⁸) at slower rates (p = 0.0016) when evolved in 4′-DTMP compared to cultures evolved in TMP. Student’s t-test (two-tailed) is used to quantify significance of differences in all panels (*p < 0.05, **p < 0.01, and ***p < 0.001, error bars show the standard deviation, center of the error bars corresponds to the mean value of the measurements). d, Representative examples of time-resolved changes in DHFR as measured by deep sequencing of the folA gene throughout evolution in the morbidostat (top row for TMP- and bottom row for 4′-DTMP-evolved cultures). E. coli cultures develop TMP resistance by acquiring the L28R mutation (6 out of 7 cultures) whereas E. coli cultures become 4′-DTMP resistant without acquiring L28R (only 1 out of 8 cultures acquired L28R). e Final frequencies of observed resistance-conferring DHFR mutations under TMP and 4′-DTMP selection on day 21 were compared. The frequency of L28R mutation (magenta) was drastically reduced from ~80% (in TMP-evolved group) to less than 15% (4′-DTMP-evolved group). f Observed evolutionary trajectories leading to resistance in E. coli. The observed DHFR mutations are indicated by circles, filled with colors. The solid arrows represent the mutations acquired by E. coli evolved under TMP selection (left, gray background) whereas the dashed arrows represent the mutations acquired by E. coli evolved under 4′-DTMP selection (right, teal background). The color of the arrows indicate the specific mutations observed. The width of the arrow corresponds to the observed frequency of that mutation. The number on a circle indicates the culture number (gray for TMP and teal for 4′-DTMP evolved cultures, f).