Summary In the ongoing fight against drug resistant bacterial infections it will be critical to identify novel antibiotic chemotypes that are less likely to induce resistance. We have used synthetic molecular evolution (SME), iterative library design and screening, to identify novel antimicrobial peptides that have potent broad-spectrum sterilizing activity against all ESKAPE pathogens tested, including drug-resistant and biofilm-forming pathogens, in vitro and in vivo. These peptides are highly soluble, protease resistant, have low cytotoxicity, and are active in the presence of concentrated host cells and/or serum proteins. Most importantly, they do not induce resistance in P. aeruginosa under conditions that rapidly enable development of resistance to conventional antibiotics and some AMPs. Resistance to some other AMPs has been reported, and it has been experimentally selected. However, avoidance or delay in resistance to AMPs have also been reported. Here we seek to understand the mechanism of resistance-avoidance. The most commonly observed AMP resistance mechanism, by far, is the modification of Lipid A phosphates with cationic moieties, which dramatically reduces the potential for AMP interactions with the outer membrane(OM). We have previously shown that some AMPs bind strongly and accumulate significantly on OM LPS. In this work we will test the hypothesis that peptides requiring large scale accumulation on the outer membrane LPS, including Lipid A, before they can cross it will be able to drive selection for resistance using this mechanism. AMPs that can bypass the OM to reach the inner membrane target without requiring large scale accumulation on the OM will be slower or unable to elicit resistance. To test this hypothesis, we have identified two related AMPs, one that rapidly invokes resistance in Pseudomonas aeruginosa, and one that does not invoke any resistance at all in multiple passages under the same conditions. We will use these two peptides, and others, to study resistance and resistance-avoidance mechanisms in P. aeruginosa and other Gram-negative bacteria using i) direct measurements of AMP-bacteria binding, ii) genetic assessment of resistance pathways, iii) direct assessment of Lipid A modifications. We will also test for resistance avoidance in a panel of other Gram-negative pathogens to test for the generality of the mechanism.