Anti-biofilm efficacy of a medieval treatment for bacterial infection requires the combination of multiple ingredients

Novel antimicrobials are urgently needed to combat the increasing occurrence of drug-resistant bacteria and to overcome the inherent difficulties in treating biofilm-associated infections. Research into natural antimicrobials could provide candidates to fill the antibiotic discovery gap, and the study of plants and other natural materials used in historical infection remedies may enable further discoveries of natural products with useful antimicrobial activity. We previously reconstructed a 1,000-year-old remedy containing onion, garlic, wine, and bile salts, which is known as ‘Bald’s eyesalve’, and showed it to have promising antibacterial activity. In this paper, we have found this remedy has bactericidal activity against a range of Gram-negative and Gram-positive wound pathogens in planktonic culture and, crucially, that this activity is maintained against Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes in a model of soft-tissue wound biofilm. While the presence of garlic in the mixture is sufficient to explain activity against planktonic cultures, garlic alone has no activity against biofilms. We have found the potent anti-biofilm activity of Bald’s eyesalve cannot be attributed to a single ingredient and requires the combination of all ingredients to achieve full activity. Our work highlights the need to explore not only single compounds but also mixtures of natural products for treating biofilm infections. These results also underline the importance of working with biofilm models when exploring natural products for the anti-biofilm pipeline.ImportanceBacteria can live in two ways, as individual planktonic cells or as a multicellular biofilm. Biofilm helps protect bacteria from antibiotics and makes them much harder to treat. Both the biofilm lifestyle and the evolution of antibiotic resistance mean we urgently need new drugs to treat infections. Here, we show that a medieval remedy made from onion, garlic, wine, and bile can kill a range of problematic bacteria grown both planktonically and as biofilms. A single component of the remedy – allicin, derived from garlic – is sufficient to kill planktonic bacteria. However, garlic or allicin alone do nothing against bacteria when they form a biofilm. All four ingredients are needed to fully kill bacterial biofilm communities, hinting that these ingredients work together to kill the bacteria. This suggests that future discovery of antibiotics from natural products could be enhanced by studying combinations of ingredients, rather than single plants or compounds.