Multi-targeting antibiotics, i.e., single compounds capable of inhibiting two or more bacterial targets
are generally considered as a promising therapeutic strategy against resistance evolution. The
rationale for this theory is that multi-targeting antibiotics demand the simultaneous acquisition of
multiple mutations at their respective target genes to achieve significant resistance. The theory
presumes that individual mutations provide little or no benefit to the bacterial host. Here we propose
that such individual, stepping-stone mutations can be prevalent in clinical bacterial isolates, as they
provide significant resistance to other antimicrobial agents. To test this possibility, we focused on
gepotidacin, an antibiotic candidate that selectively inhibits both bacterial DNA gyrase and
topoisomerase IV. In a susceptible organism, Klebsiella pneumoniae, a combination of two specific
mutations in these target proteins provide an over 2000-fold reduction in susceptibility, while
individually none of these mutations affect resistance significantly. Alarmingly, strains with decreased
susceptibility against gepotidacin are found to be as virulent as the wild-type Klebsiella pneumoniae
strain in a murine model. Moreover, numerous pathogenic isolates carry mutations which could
promote the evolution of clinically significant reduction of susceptibility against gepotidacin in the
future. As might be expected, prolonged exposure to ciprofloxacin, a clinically widely employed
gyrase inhibitor, co-selected for reduced susceptibility against gepotidacin. We conclude that
extensive antibiotic usage could select for mutations that serve as stepping-stones towards resistance
against antimicrobial compounds still under development. Our research indicates that even balanced
multi-targeting antibiotics are prone to resistance evolution