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.
Highlights
Resistance of human bacterial pathogens to conventional antibiotics has increased drastically worldwide within the last decades [1]
The aim of this study was to determine whether bacterial resistance could develop following exposure to a synthetic antimicrobial peptides (AMPs) analogue belonging to the class of antibacterial a-peptide/bpeptoid peptidomimetics
In this study it was demonstrated that Escherichia coli may develop resistance to a synthetic antimicrobial peptide analogue during prolonged continuous exposure to increasing concentrations (Table 1), and this was consistently observed in all lineages
Summary
Resistance of human bacterial pathogens to conventional antibiotics has increased drastically worldwide within the last decades [1] This has led to an intensified search for safer alternatives for which resistance is less likely to evolve [2,3]. These include novel natural compounds with antimicrobial activity [4], inhibitors of quorum sensing [5,6], and antimicrobial peptides (AMPs) [7,8]. There has been increased focus on the characterisation of natural AMPs [13,14] and on semi-synthetic [15,16,17] and synthetic analogues [18,19,20], as well as on the development of these leads into future antibiotics against human bacterial pathogens
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