De novo evolution of antibiotic resistance to Oct-TriA₁

The rise of antimicrobial resistance as a global health concern has led to a strong interest in compounds able to inhibit the growth of bacteria without detectable levels of resistance evolution. A number of these compounds have been reported in recent years, including the tridecaptins, a small family of lipopeptides typified by the synthetic analogue octyl-tridecaptin A₁. Hypothesizing that prior reports of negligible resistance evolution have been due in part to limitations in the laboratory evolution systems used, we have attempted to select for resistant mutants using a soft agar gradient evolution (SAGE) system developed by our lab. Following optimization of the media conditions by incorporation of the anti-synaeresis agent xanthan gum into the agar matrix, we successfully evolved high-level resistance to both octyl-tridecaptin A₁ as well as the challenging lipopeptide antibiotic polymyxin B. Decreased tridecaptin susceptibility was linked to mutations in outer membrane proteins ompC, lptD and mlaA, with the effect of these genes confirmed through a mix of allelic replacement and knockout studies. Overall, this work demonstrates the robust evolutionary potential of bacteria, even in the face of challenging antimicrobial agents.