Characterization of the antibacterial activity of Bald’s eyesalve against drug resistant Staphylococcus aureus and Pseudomonas aeruginosa

Abstract

Bald’s eyesalve is an Anglo-Saxon medicinal remedy that has been used through ancient times to treat eye sty infections and may represent a source of ancientbiotics. This study assessed the efficacy of Bald’s eyesalve against several strains of Staphylococcus aureus and Pseudomonas aeruginosa, including a multi-drug resistant phenotype, and identified the principal compound conveying antibacterial activity. Bald’s eyesalve formulations were produced by combining garlic, onion or leek, wine, bovine bile, and brass, with specific ingredient omissions in several formulations, followed by incubation at 4 °C for 9 days. Bald’s eyesalve formulation ES-GBBr exhibited the greatest antibacterial activity against S. aureus and P. aeruginosa. Fractionation of ES-GBBr using molecular size exclusion and organic solvent partitioning isolated its antibacterial activity to the small molecule nonpolar fraction, and 1D 1H NMR revealed the identity of the antibacterial agent to be allicin. Depletion of allicin from this fraction by addition of exogenous cysteine established that all observable growth inhibition originated from allicin. Quantification of allicin demonstrated that its concentration was significantly greater in ES-GBBr compared to the ES-O formulation; however, this was not due to greater yield. The antibacterial activity of allicin against S. aureus was antagonized by other ingredients within Bald’s eyesalve, whereas they were additive or synergistic against P. aeruginosa. These results suggest that neither leek nor onion is necessary for the antibacterial efficacy of Bald’s eyesalve against S. aureus or P. aeruginosa, and while allicin was identified as the principal antibacterial agent present, its activity is influenced differentially in the presence of additional Bald’s eyesalve ingredients when used against S. aureus compared to P. aeruginosa. Ancientbiotics may provide a source of promising antibacterials; however, identifying the source of activity and assessing distinct formulations for cooperative effects are essential to using ancient remedies, such as Bald’s eyesalve, effectively against drug resistant pathogens.

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

“…Our results against planktonic cultures of S. aureus and P. aeruginosa align with a report by Fuchs et al 2018, that garlic alone, and specifically allicin, accounts for the majority of the planktonic activity of Bald’s remedy. Allicin is a defensive compound that is converted from alliin by the enzyme alliinase, upon damage to the plant tissue.…”

“…Our initial work indicated that all four ingredients in ESO were required to kill biofilms of S. aureus Newman in synthetic wound biofilms (20). A recent publication by Fuchs et al (2018; (21)) concluded that the bactericidal activity of ESO was due to the presence of allicin from garlic, however, the Fuchs et al study only investigated planktonic killing in standard Mueller-Hinton Broth (MHB). It is well known that planktonic cultures of bacteria can be up to 1,000 times more sensitive to antibiotics than the same isolates grown as biofilms (7), and that antibiotic sensitivity is highly dependent on growth medium (31).…”

“…Previous papers using HPLC have detected allicin concentrations ranging from 1 μg·ml −1 to 2 mg·ml −1 (33,52), a range that the results in this paper lie within. Fuchs et al 2018 used quantitative NMR (qNMR), which too has advantages and is being increasingly used in quantification of concentrations (54). It is more likely that our Bald’s eyesalve preparations really do have different allicin concentrations.…”

“…In this paper, we investigate the importance of the combination of ingredients for the anti-biofilm activity of a 10th century remedy used for eye infection from the manuscript known as Bald’s Leechbook (London, British Library, Royal 12, D xvii). This remedy has previously been shown to kill S. aureus biofilms by Harrison et al (2015) (20), Pseudomonas aeruginosa planktonic cultures (21) and recently Neisseria gonorrhoeae in a disk diffusion assay (22). The recipe, known as ‘Bald’s eyesalve’, requires equal volumes of garlic ( Allium sativum ) and another Allium species (referred to as cropleac in the original Old English) to be crushed together and mixed with equal volumes of wine and ox gall (bovine bile).…”

The safety profile of Bald’s eyesalve for the treatment of bacterial infections

“…These include oils from Cupressus sempervirens (Cypress) and Commiphora species (myrrh), being used to treat colds, coughs, and inflammation (8), and Artemisia annua (Artemisin) used to treat malaria (9). Bald’s eyesalve has previously been shown to possess antibacterial activity against Staphylococcus aureus in planktonic cultures and biofilms (10), and against Pseudomonas aeruginosa in planktonic cultures (11). A recent study from our laboratory found that Bald’s eyesalve had antimicrobial activity against a range of Gram-positive and Gram-negative organisms in planktonic cultures and biofilms (12).…”