Diallylthiosulfinate (Allicin), a Volatile Antimicrobial from Garlic (Allium sativum), Kills Human Lung Pathogenic Bacteria, Including MDR Strains, as a Vapor

Abstract

Garlic (Allium sativum) has potent antimicrobial activity due to allicin (diallylthiosulfinate) synthesized by enzyme catalysis in damaged garlic tissues. Allicin gives crushed garlic its characteristic odor and its volatility makes it potentially useful for combating lung infections. Allicin was synthesized (>98% pure) by oxidation of diallyl disulfide by H₂O₂ using formic acid as a catalyst and the growth inhibitory effect of allicin vapor and allicin in solution to clinical isolates of lung pathogenic bacteria from the genera Pseudomonas, Streptococcus, and Staphylococcus, including multi-drug resistant (MDR) strains, was demonstrated. Minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were determined and compared to clinical antibiotics using standard European Committee on Antimicrobial Susceptibility Testing (EUCAST) procedures. The cytotoxicity of allicin to human lung and colon epithelial and murine fibroblast cells was tested in vitro and shown to be ameliorated by glutathione (GSH). Similarly, the sensitivity of rat precision-cut lung slices (PCLS) to allicin was decreased by raising the [GSH] to the approximate blood plasma level of 1 mM. Because allicin inhibited bacterial growth as a vapor, it could be used to combat bacterial lung infections via direct inhalation. Since there are no volatile antibiotics available to treat pulmonary infections, allicin, particularly at sublethal doses in combination with oral antibiotics, could make a valuable addition to currently available treatments.

Keywords: Allium sativum; MDR strains; Pseudomonas aeruginosa; Streptococcus pneumoniae; allicin; antimicrobial; garlic; lung pathogenic bacteria; volatile antimicrobial agent.

Scheme 1

Enzyme-catalyzed biosynthesis of allicin.

Scheme 2

Allicin (1) is a reactive sulfur species (RSS) and undergoes a thiol-disulfide type exchange reaction in which two allicin molecules react with two thiols to produce water and two molecules of a mixed allyl disulfide (3). The reaction is shown overall in (a) and the formal oxidation states of the sulfur and the oxygen atoms are marked in red. In (b) mechanistic details are shown, breaking down the exchange reaction into component steps and showing further catalytic redox cycling of some of the components involving S-atoms with formal oxidation states of −2, −1, 0 and +1, respectively. Thus, initially, after reaction of allicin (1) with one thiol molecule (2), one allyl thial molecule (4) is formed and one molecule of a mixed allyl disulfide (3). The allyl thial is in tautomeric equilibrium with allyl sulfenic acid (5) which reacts readily with one further thiol molecule (2) to give a second mixed allyl disulfide molecule (3). These mixed allyl disulfides (3) are capable of reacting with any further thiol molecules in a standard thiol-disulfide exchange reaction shuttling interchangeably between the thiol and disulfide without producing water. If the thiols in question are cysteine residues in proteins, the protein disulfides formed (3) would be potential substrates for thioredoxins (TRX) and/or glutaredoxins (GRX) relying on reducing equivalents from NADPH-dependent thioredoxin reductases and glutathione to further cycle the reaction products. The thiols thus formed, including allyl mercaptan (6), could be catalytically re-oxidized to sulfenic acids (5) by a peroxiredoxin (PRDX) type of enzyme using H₂O₂, or oxidized directly but more slowly by H₂O₂, to yield sulfenic acids capable of further perpetuating catalytic cycling of the sulfur-containing intermediates.

Figure 1

Timeline of the introduction of novel antibiotic classes into clinical practice.

Figure 2

Antibacterial activity of allicin vapor. The Petri dish base with bacteria either spread on the agar surface (upper rows), or with bacteria-seeded agar (lower rows) were inverted over a 20 µL droplet of allicin solution in the center of a 9-cm diameter Petri dish lid. The absolute amount of allicin (µg) in the 20 µL droplet is stated. (a) The antimicrobial effect of allicin vapor on P. aeruginosa PAO1 SBUG8, PAO25, and the highly resistant DSM2659 strain, respectively; (b) The antimicrobial effect of allicin vapor on Streptococcus pyogenes, S. agalactiae, and S. dysgalactiae equisilimlis; respectively (c) The antimicrobial activity of allicin vapor on Streptococcus pneumoniae multi-drug resistant (MDR)-strains Spain^23F-1 and Poland^23F- and SNo 68665 and SNo 68668 and 16; (d) The antimicrobial effect of allicin vapor on Staphylococcus aureus; (e) example control plates placed over water droplets without allicin showed uniform bacterial growth.

Figure 3

Susceptibility of mammalian cell lines to allicin. Cell cultures were stressed for one hour by exposure to allicin at different concentrations, either in the absence or presence of 1 mM reduced glutathione (GSH). Cell viability was determined with the MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-test. The half maximal effective concentration (EC₅₀) for allicin was determined and is marked with a horizontal dashed line (- - - - -). The MIC value for the majority of tested bacteria (390 µM allicin, 64 µg/mL) is marked with a vertical dotted line (………). (a) Beas-2B (SV40-immortalized human bronchial epithelial cells); (b) A549 (human epithelial lung carcinoma); (c) Caco-2 (human epithelial colon tumor) and (d) NIH/3T3 (murine embryonic fibroblast), respectively.