Antimicrobial peptides 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. To investigate this paradox we experimentally evolved S. aureus to increased resistance

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... t the defensin and a combination of the defensin and coleoptericin. Genome re-sequencing showed that resistance was associated with mutations in either the ytr or nsa operons, in both AMP treatments. Strains with these mutations show longer lag phases, slower Vmax and nsa mutants reach lower final population sizes. Mutations in rpoB were showed a further increase in the lag phase in nsa mutants but not in ytr mutants. In contrast, final MICs do not segregate by 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.