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.
Acclimatization Animals