Evolutionary Biology, Institut für Biologie, Freie Universität Berlin, Berlin, Germany.
Institute for Animal Hygiene and Environmental Health, Centre for Infection Medicine, Freie Universität Berlin, Berlin, Germany.
Sci Rep. 2018 Oct 18;8(1):15359. doi: 10.1038/s41598-018-33593-7.
Antimicrobial peptides (AMP) are highly conserved immune effectors across the tree of life and are employed as combinations. In the beetle Tenebrio molitor, a defensin and a coleoptericin are highly expressed in vivo after inoculation with S. aureus. The defensin displays strong in vitro activity but no survival benefit in vivo. The coleoptericin provides a survival benefit in vivo, but no activity in vitro. This suggests a potentiating effect in vivo, and here we wanted to investigate the effects of this combination on resistance evolution using a bottom-approach in vitro starting with a combination of two abundant AMPs only. We experimentally evolved S. aureus in the presence of the defensin and a combination of the defensin and coleoptericin. Genome re-sequencing showed that resistance was associated with mutations in either the pmt or nsa operons. Strains with these mutations show longer lag phases, slower Vmax, and nsa mutants reach lower final population sizes. Mutations in the rpo operon showed a further increase in the lag phase in nsa mutants but not in pmt mutants. In contrast, final MICs (minimum inhibitory concentrations) do not differ according to mutation. All resistant lines display AMP but not antibiotic cross-resistance. Costly resistance against AMPs readily evolves for an individual AMP as well as a naturally occurring combination in vitro and provides broad protection against AMPs. Such non-specific resistance could result in strong selection on host immune systems that rely on cocktails of AMPs.
抗菌肽（AMP）是生命之树中高度保守的免疫效应物，并且以组合形式存在。在甲虫黄粉虫中，金黄色葡萄球菌接种后体内防御素和鞘脂素高度表达。防御素有很强的体外活性，但体内没有生存获益。鞘脂素提供体内生存获益，但体外没有活性。这表明体内存在增效作用，在这里，我们想用一种从仅两种丰富的 AMP 组合开始的体外底部方法来研究这种组合对耐药性进化的影响。我们在防御素和防御素与鞘脂素的组合存在下，用实验方法使金黄色葡萄球菌进化。基因组重测序表明，耐药性与 pmt 或 nsa 操纵子中的突变有关。具有这些突变的菌株表现出更长的迟滞期、更慢的 Vmax，而 nsa 突变体达到更低的最终种群大小。rpo 操纵子中的突变导致 nsa 突变体的迟滞期进一步增加，但 pmt 突变体没有。相比之下，最终的 MIC（最小抑菌浓度）不根据突变而有所不同。所有耐药株都显示出 AMP，但没有抗生素交叉耐药性。针对个体 AMP 以及体内自然发生的组合，昂贵的 AMP 耐药性很容易进化，并提供对 AMP 的广泛保护。这种非特异性耐药性可能导致宿主免疫系统受到强烈选择，宿主免疫系统依赖于 AMP 鸡尾酒。
