Adaptive Evolution of Escherichia coli to an α-Peptide/β-Peptoid Peptidomimetic Induces Stable Resistance

Abstract

Antimicrobial peptides (AMPs) and synthetic analogues thereof target conserved structures of bacterial cell envelopes and hence, development of resistance has been considered an unlikely event. However, recently bacterial resistance to AMPs has been observed, and the aim of the present study was to determine whether bacterial resistance may also evolve against synthetic AMP analogues, e.g. α-peptide/β-peptoid peptidomimetics. E. coli ATCC 25922 was exposed to increasing concentrations of a peptidomimetic (10 lineages), polymyxin B (10 lineages), or MilliQ water (4 lineages) in a re-inoculation culturing setup covering approx. 500 generations. All 10 lineages exposed to the peptidomimetic adapted to 32×MIC while this occurred for 8 out of 10 of the polymyxin B-exposed lineages. All lineages exposed to 32×MIC of either the peptidomimetic or polymyxin B had a significantly increased MIC (16–32×) to the selection agent. Five transfers (∼35 generations) in unsupplemented media did not abolish resistance indicating that resistance was heritable. Single isolates from peptidomimetic-exposed lineage populations displayed MICs against the peptidomimetic from wild-type MIC to 32×MIC revealing heterogeneous populations. Resistant isolates showed no cross-resistance against a panel of membrane-active AMPs. These isolates were highly susceptible to blood plasma antibacterial activity and were killed when plasma concentrations exceeded ∼30%. Notably, MIC of the peptidomimetic against resistant isolates returned to wild-type level upon addition of 25% plasma. Whole-genome sequencing of twenty isolates from four resistant lineages revealed mutations, in murein transglycosylase D (mltD) and outer-membrane proteins, which were conserved within and between lineages. However, no common resistance-conferring mutation was identified. We hypothesise that alterations in cell envelope structure result in peptidomimetic resistance, and that this may occur via several distinct mechanisms. Interestingly, this type of resistance result in a concomitant high susceptibility towards plasma, and therefore the present study does not infer additional concern for peptidomimetics as future therapeutics.

Experimental Induction of Bacterial Resistance to the Antimicrobial Peptide Tachyplesin I and Investigation of the Resistance Mechanisms

“…These results are consistent with previous reports that resistance may evolve from consistent long-term exposure to increasing levels of peptides (3) and synthetic AMP analogues, e.g., ␣-peptide/ ␤-peptoid peptidomimetics (25). However, they contradict our previous report indicating no tachyplesin I-resistant mutants in bacteria exposed to fewer than 20 serial passages with higher-concentration tachyplesin I induction (17), possibly because of differences in induction time and induction method.…”

“…For example, the disappearance of the metallic luster of the E. coli ATCC 25922 resistant strains grown on eosin methylene blue agar medium might be associated with changes in the charge of the cell membrane. The biological char- (25). In short, AMP resistance might compromise bacteria in other ways, for example, by reducing their growth rate or causing a longer lag phase.…”

Vibrio anguillarum Is Genetically and Phenotypically Unaffected by Long-Term Continuous Exposure to the Antibacterial Compound Tropodithietic Acid

“…This function is of course essential and highly conserved, which likely explains why resistance or tolerance does not develop, since mutational changes in the PMF apparatus would be lethal to the bacteria. It could be speculated that multiple cooccurring mutations could result in resistance, as has been shown for antimicrobial peptides (30,31). It was assumed that resistance to AMPs could not develop; however, several adaptive laboratory evolution (ALE) experiments have demonstrated that long-term exposure does indeed result in the evolution of resistant strains (27,30).…”

“…It could be speculated that multiple cooccurring mutations could result in resistance, as has been shown for antimicrobial peptides (30,31). It was assumed that resistance to AMPs could not develop; however, several adaptive laboratory evolution (ALE) experiments have demonstrated that long-term exposure does indeed result in the evolution of resistant strains (27,30). Using this method, the MIC of the AMP pexiganan for Pseudomonas fluorescens increased to 128 times the wild-type MIC after approximately 700 generations (27), and the MICs of the synthetic AMP analogues ␣-peptide/␤-peptoid peptidomimetics against E. coli increased to 16 to 32 times the wild-type MIC after approximately 500 generations (30).…”

“…It was assumed that resistance to AMPs could not develop; however, several adaptive laboratory evolution (ALE) experiments have demonstrated that long-term exposure does indeed result in the evolution of resistant strains (27,30). Using this method, the MIC of the AMP pexiganan for Pseudomonas fluorescens increased to 128 times the wild-type MIC after approximately 700 generations (27), and the MICs of the synthetic AMP analogues ␣-peptide/␤-peptoid peptidomimetics against E. coli increased to 16 to 32 times the wild-type MIC after approximately 500 generations (30). However, our ALE experiments did not result in TDA-resistant strains, and the experiment was terminated after two failed attempts to obtain growth in the presence of increased TDA concentrations.…”