Inner-membrane transporter genotype shapes susceptibility to non-lytic antimicrobials and bacterial fitness in Escherichia coli

Intracellularly acting antibacterials must cross the bacterial inner membrane to reach their targets in the cytoplasm. Using a panel of isogenic Escherichia coli strains with single, double and triple deletions of sbmA, ygdD and mdtM the susceptibility to bleomycin, Oncocin, Api88 and Bac7(1-17), was assessed together with bacterial fitness in vitro and in vivo during mouse intestinal colonization. SbmA was the dominant susceptibility determinant, while YgdD contributed strongly to bleomycin and Api88 susceptibility and more modestly to Oncocin and Bac7(1-17) susceptibility. Simultaneous loss of YgdD and SbmA produced both a 128-fold increase in the bleomycin MIC and a 37% increase in doubling time, indicating that the two proteins share partly overlapping functions. Single mutants showed no detectable growth defect, but the {Delta}ygdD {Delta}sbmA, {Delta}ygdD {Delta}mdtM and {Delta}ygdD {Delta}sbmA{Delta}mdtM mutants showed significant growth defects. In mice, {Delta}sbmA and {Delta}mdtM showed reduced late competitive persistence. These findings support a compound-specific network of inner-membrane susceptibility determinants with context-dependent fitness costs.