Genomics of experimental adaptation of Staphylococcus aureus to a natural combination of insect antimicrobial peptides

Olga Makarova,Alexandro Rodriguez-Rojas,Baydaa El Shazely,Paul Johnston,Javier Moreno Morales,Jens Rolff

doi:10.1038/s41598-018-33593-7

Abstract

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.

Highlights

In antibiotic treatments, often single drugs are successfully used to clear infections
We explore if this combination of two highly abundant Antimicrobial peptides (AMP) influences the evolution of bacterial resistance
In a recent experimental evolution study investigating S. aureus resistance evolution against three different AMPs from different organisms[12], we found a range of mutations associated with resistance[13]

Summary

Introduction

Often single drugs are successfully used to clear infections. An interesting observation is that during an infection antimicrobial peptides are expressed that have no known activity against the agent of infection This is surprising, given that insects for example have different receptors that can distinguish between classes of infectious microbes such as fungi or bacteria[8] and energetic costs of protein synthesis are considered to be high[3]. We want to use the simplest approach of understanding combinations of AMPs as observed in hosts, by using in vitro experimental evolution of bacteria to start with the minimum number in a combination: two We explore if this combination of two highly abundant AMPs influences the evolution of bacterial resistance. These results are in line with a growing number of studies that show a high probability of cross-resistance against AMPs14,15

Methods

Results

Conclusion