Intriguingly, D22 was found to be highly active against all nine tested clinical CRAB strains (MICs 0.06 to 0.5 µg/mL) (Table ), displaying significantly higher potency than the previous frontrunner D9 (and DA) and nearly equipotent antibacterial activity with colistin plus showing much better activity than other clinically used antibiotics (Table ). In particular, the in vitro potency of D22 against CRAB was at least one order of magnitude better than for D9. This marked improvement is even more impressive as D9 displays already four-fold improved activity compared to native DA. In addition, time-kill kinetics of DA and D22 at 2x, 4x and 8x MIC in E. coli MG1655 (Figure shows rapid bacterial killing of E. coli MG1655 at 4x and 8x MIC after an early static phase of up to 2 hours followed by bactericidal activity with more than 3-log killing exhibited in the reduced colony-forming units (CFU/mL) within 2 to 4 h. The observed initial bacteriostatic effect can possibly be explained by Ritzmann et al., which demonstrated that binding of darobactin on the lateral gate leads to an increased stability by "freezing" BamA and consequently no immediate bactericidal effect. However, DA and D22 at 2x, 4x and 8x MIC acted bactericidal over the first 8 h in time-kill experiments using E. coli MG1655, and the extent of bactericidal activity was concentration-dependent. Nevertheless, re-growth of bacteria was observed at 2x and 4x MIC conditions after 24 h, while this was not the case at 8x MIC (MIC values reported for DA and D22 were 2 µg/mL and 1 µg/mL, respectively). Imai et al. did not report re-growth of E. coli after DA treatment, however, the timeframe in their reported experiment was limited to 8 h with 16x MIC, making direct comparison challenging. They also determined a minimum bactericidal concentration (MBC) of 8 µg/mL in E. coli, which is fully in line with our findings for DA and D22 having MBCs of 16 and 8 µg/mL, respectively. Encouragingly, upon repeating the time-kill experiment using high inoculum conditions (~5x10 8 CFU/mL) we were able to show that surviving bacteria are likely persisters and did not gain resistance, as representative colonies from this experiment showed no MIC shifts (Table ). In a comparison of time-kill kinetics of both darobactins in A. baumannii NCTC13301, D22 shows its clear superiority over DA (Figure 4 c, d). For the latter, we determined a lasting bacteriostatic effect at 2x MIC, and regrowth was observed at both initially bactericidal concentrations (4x and 8x MIC). In contrast, D22 showed bactericidal activity and concentrations as low as 4x MIC and fully prevented re-growth at 24 h post-treatment (MBC of 32 µg/mL).