A 1,000-Year-Old Antimicrobial Remedy with Antistaphylococcal Activity

Abstract

Plant-derived compounds and other natural substances are a rich potential source of compounds that kill or attenuate pathogens that are resistant to current antibiotics. Medieval societies used a range of these natural substances to treat conditions clearly recognizable to the modern eye as microbial infections, and there has been much debate over the likely efficacy of these treatments. Our interdisciplinary team, comprising researchers from both sciences and humanities, identified and reconstructed a potential remedy for Staphylococcus aureus infection from a 10th century Anglo-Saxon leechbook. The remedy repeatedly killed established S. aureus biofilms in an in vitro model of soft tissue infection and killed methicillin-resistant S. aureus (MRSA) in a mouse chronic wound model. While the remedy contained several ingredients that are individually known to have some antibacterial activity, full efficacy required the combined action of several ingredients, highlighting the scholarship of premodern doctors and the potential of ancient texts as a source of new antimicrobial agents.

Importance: While the antibiotic potential of some materials used in historical medicine has been demonstrated, empirical tests of entire remedies are scarce. This is an important omission, because the efficacy of "ancientbiotics" could rely on the combined activity of their various ingredients. This would lead us to underestimate their efficacy and, by extension, the scholarship of premodern doctors. It could also help us to understand why some natural compounds that show antibacterial promise in the laboratory fail to yield positive results in clinical trials. We have reconstructed a 1,000-year-old remedy which kills the bacteria it was designed to treat and have shown that this activity relies on the combined activity of several antimicrobial ingredients. Our results highlight (i) the scholarship and rational methodology of premodern medical professionals and (ii) the untapped potential of premodern remedies for yielding novel therapeutics at a time when new antibiotics are desperately needed.

FIG 1

Bald’s eyesalve. A facsimile of the recipe, taken from the manuscript known as Bald’s Leechbook (London, British Library, Royal 12, D xvii). The original text reads as follows. “Ƿyrc eaȝsealf ƿiþ ƿænne: ȝenim cropleac ⁊ ȝarleac beȝea emfela, ȝecnuƿe ƿel tosomne, ȝenim ƿin ⁊ fearres ȝeallen beȝean emfela ȝemenȝ ƿiþ þy leaces, do þonne on arfæt læt standan niȝon niht on þæm arfæt aƿrinȝ þurh claþ ⁊ hlyttre ƿel, do on horn ⁊ ymb niht do mid feþre on eaȝe; se betsta læcedom.” This is translated into modern English as follows. “Make an eyesalve against a wen: take equal amounts of cropleac [an Allium species] and garlic, pound well together, take equal amounts of wine and oxgall, mix with the alliums, put this in a brass vessel, let [the mixture] stand for nine nights in the brass vessel, wring through a cloth and clarify well, put in a horn and at night apply to the eye with a feather; the best medicine.” © The British Library Board, Royal 12 D Xll xvii. Reproduced with permission.

FIG 2

Bald’s eyesalve kills S. aureus in planktonic culture and in a synthetic wound biofilm model. (A) One hundred microliters of sterile distilled water (black line), the onion variant of the eyesalve (ES-O, red line), or the leek variant of the eyesalve (ES-L, blue line) (both from batch A) was added to a 200-µl mid-log-phase culture (10⁴ to 10⁵ cells) of S. aureus in synthetic wound fluid (SWF), and the optical density of the culture was measured during 18 h of incubation at 37°C. The mean results from four replica populations and the associated standard errors (shaded intervals; too small to see for ES-O and ES-L) are shown. (B) Two hundred microliters of ES-O or ES-L (batch A, filled circles, and batch B, open circles) or of each individual ingredient preparation was added to five 1-day-old cultures of S. aureus growing at 37°C in a synthetic wound (400-µl synthetic wound fluid rendered semisolid by adding 2 mg·ml⁻¹ collagen). After 24 h of further incubation, the collagen was dissolved to recover cells for agar plate counts. The control treatment was sterile distilled water left to stand for 9 days in the presence of brass, which was also present in all other preparations, to simulate the presence of a copper alloy vessel (see Materials and Methods). Asterisks denote treatments whose results were significantly different from those of the control.

FIG 3

The activity of Bald’s eyesalve against S. aureus biofilms requires several ingredients. Two hundred microliters of the onion variant of the eyesalve (ES-O), the leek variant of the eyesalve (ES-L) (batch C), or preparations missing a single ingredient were added to three 1-day-old cultures of S. aureus in synthetic wounds, and bactericidal activity quantified as described in Fig. 2. Red bars show the results for the full or reduced versions of ES-O, blue bars show the results for the full or reduced versions of ES-L, and the green bar shows the result for a variant of the eyesalve with neither onion nor leek (ES). The control treatment (grey bar) was sterile distilled water. Individual ingredients dropped out of the recipe (Minus) are coded as follows: O, onion; L, leek; X, oxgall; W, wine; G, garlic; B, brass. Reduced recipes were assessed as having full (F), partial (P), or no (N) activity in comparison with the results for the control treatment and the appropriate complete recipe.

FIG 4

The activity of Bald’s eyesalve against S. aureus biofilms requires the 9-day waiting period specified by the recipe. Immediately after preparation, 200 µl of the onion variant of the eyesalve (ES-O), the leek variant of the eyesalve (ES-L) (batch D), or sterile distilled water was added to five 1-day-old cultures of S. aureus growing at 37°C in synthetic wounds, and the bactericidal activity quantified as previously described (filled bars); the experiment was repeated using ES-O and ES-L (batch D) after the 9-day waiting period specified by Bald (open bars). Asterisks denote treatments whose results were significantly different from those of the control. While the control cultures for the 9-day experiment grew to slightly lower densities than the control for the fresh eyesalve (P = 0.005), this difference was small compared with the differences observed for ES-O and ES-L (1 to 2 log versus 4 to 5 log difference).

FIG 5

Bactericidal activity of Bald’s eyesalve in a mouse chronic wound model of MRSA infection. Six adult female Swiss-Webster mice were administered wounds and infected with ca. 10⁵ CFU S. aureus Mu50. Four days postinfection, mice were euthanized. Wound tissue was excised and cut into either three (n = 3 mice) or four (n = 3 mice) equal pieces, which were weighed, submerged in 300 µl sterile saline (one replicate from n = 6 mice), 100 µg·ml⁻¹ vancomycin (one replicate from n = 3 mice), the onion variant of the eyesalve (ES-O; one replicate from n = 6 mice), or the leek variant of the eyesalve (ES-L; one replicate from n = 6 mice) (both ES-O and ES-L were from batch B) for 4 h, and then rinsed in sterile saline and homogenized. Viable bacteria were enumerated, and the counts standardized per gram of tissue. Asterisks denote treatments whose results were significantly different from those of the control.