Uncovering the mechanism of action of a historical antimicrobial against S. aureus and A. baumannii.

Natural products have provided most of our modern pharmacopeia, serving as active molecules or scaffolds for active molecules. Their use in drug development is often inspired by their traditional or historical medical use. For many decades, this discovery pipeline has focused on identifying a single molecule responsible for much of the biological activity of a "raw" natural product preparation (e.g., a whole-plant extract) and scoping this molecule for clinical potential. However, it is increasingly realized that historical/traditional remedies with significant biological activity may owe this activity to the combined action of multiple molecules. Concomitantly, microbiologists increasingly argue that effectively fighting antimicrobial-resistant infections will rely on combination therapies that combine multiple antimicrobials and/or adjuvant molecules. We previously reconstructed a complex historical remedy, Bald's eyesalve. Our reconstruction of this remedy had strong antibiofilm activity, which relied on the presence of multiple ingredients. Here, we report that Bald's eyesalve has multiple antibacterial effects on exemplar gram-positive (Staphylococcus aureus) and gram-negative (Acinetobacter baumannii) pathogens. Bald's eyesalve disrupts bacterial membrane integrity; inhibits the expression of genes associated with bacterial adhesins, virulence factors, and efflux pumps in both S. aureus and A. baumannii; inhibits quorum sensing in S. aureus; and causes the downregulation of genes involved in de novo nucleotide biosynthesis in S. aureus. Finally, we show that this multifaceted mechanism of action makes it difficult for S. aureus, A. baumannii, and Pseudomonas aeruginosa to evolve resistance against Bald's eyesalve. Bald's eyesalve could be used to identify a defined cocktail of natural products suitable for preclinical testing as a multi-target antibacterial preparation to which resistance may arise more slowly than current single-molecule antibiotics.IMPORTANCEThe increasing mortality and economic cost of antimicrobial resistance (AMR) have made it one of the biggest threats to global health. Available antibiotics are in short supply due to the slow progress in the discovery of new antibiotics. Hence, there is a need for alternative treatment options against difficult-to-treat infections. We have identified a natural product cocktail based on a historical remedy with broad-spectrum antibacterial activity and a multifaceted mechanism of action. We have shown that there is slower resistance evolution to this cocktail compared to mainline antibiotics, and it could be used as the foundation of an alternative treatment to antibiotics in clinical settings.