The antibacterial activity associated with garlic (Allium sativum
L.) was identified by Cavallito in 1944 as being due to diallylthiosulfinate which was given the trivial name allicin [1
]. In vivo allicin is formed by the catalytic action of alliin-lyase (E.C.220.127.116.11) on alliin (S
-cysteine sulfoxide) (Scheme 1
). Enzyme and substrate are compartmentalized separately in cells and become mixed after mechanical damage. Allicin is the first major sulfur-containing volatile to be produced and gives freshly crushed garlic its typical odor. Allicin has a broad spectrum of cellular targets and it is effective against bacteria, fungi, oomycetes, and protozoa [3
]. Allicin’s mode of action is still not fully understood [9
However, allicin is a reactive sulfur species and undergoes a thiol-disulfide type exchange reaction (Scheme 2
a) with available thiol groups, or more specifically, with thiolate ions [10
]. However, in contrast to a standard thiol-disulfide exchange reaction, a molecule of water is generated from the thiosulfinate oxygen atom. The mixed disulfide formed can enter into further standard exchange reactions with fresh thiols and a redox cycling cascade can ensue, which in cells is also driven by various catalytic enzymes, e.g., thioredoxins and glutaredoxins (Scheme 2
b). Thus, the effects of allicin on cellular thiol homeostasis of proteins and cellular redox buffers, such as glutathione, can be profound. For example, allicin reacts with accessible cysteines in proteins and can inactivate essential enzymes [13
]. Allicin reacts with glutathione, shifts the cell redox potential to a more oxidized state and causes disulfide stress [9
]. In this regard, allicin has been described as a cellular redox toxin [15
]. Allicin is taken up readily by cells and has a calculated logP = 1.29 ± 0.13 [16
]. The ability to pass easily through cell membranes contributes to allicin’s effectiveness as an antibiotic [17
]. Furthermore, independently of its chemical reactivity, allicin’s physical properties allow it to cause transient pore formation in biological membranes and artificial lipid bilayers [18
]. This effect of allicin on membranes might explain the synergistic effect of allicin with membrane-active antibiotics like amphotericin-B and polymixin-B [19
Importantly, allicin is a volatile substance and can be effective against microorganisms via the gas phase. Since there are no volatile antibiotics currently available for clinical use, this makes allicin a very interesting candidate molecule. It therefore makes sense to develop new strategies using allicin-based drugs, perhaps combined with other conventional antibiotics, for direct treatment of lung infections via the pulmonary rather than the oral route. In this regard, the successful treatment of tuberculosis patients by inhalation of garlic vapor has been reported in the ‘pre-antibiotic’ era [21
The antimicrobial effect of garlic or allicin was already documented for several human pathogenic bacteria in vitro and in animal trials [9
]. Generally, these reports have been restricted to a few particular bacterial species, rather than a systematic treatment of a pathogenic group, for example lung pathogenic bacteria, as we report here.
Currently, the emergence of bacteria pan-resistant to current clinical antibiotics is a threat to effective treatment of infectious disease. Resistance to penicillin, the first commercially available antibiotic, was already documented in 1940 before its release into clinical practice in 1943. The appearance of resistance, rapidly following discovery or introduction into clinical practice, is also the trend for later antibiotics [25
]. The majority of antibiotic classes and drugs were discovered before the 1970s and there have been few new discoveries reported since (Figure 1
Several MDR (multiple drug resistant) strains of human pathogenic bacteria e.g., Pseudomonas aeruginosa
, Streptococcus pneumoniae
, Staphylococcus aureus
, Acinetobacter baumannii
, and Mycobacterium tuberculosis
have been reported and there is desperate need for new strategies and new classes of antibiotics [29
]. In this regard, drugs like allicin, with multiple sites of action, are particularly desirable as this hinders the emergence of resistance. The seriousness of the situation is emphasized by the recent report of the death of a patient infected by a pan-resistant isolate of Klebsiella pneumoniae
against which all 26 antibiotics allowed for clinical use in the USA were ineffective [34
In the work reported here, we tested the antimicrobial effect of allicin as a vapor on several clinical isolates of lung pathogenic bacteria and used standardized EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines to determine minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). The toxicity of allicin towards human lung epithelial cells and rat precision-cut lung tissue slices (PCLS) was investigated and the protective effect of glutathione was shown. The feasibility of developing allicin as part of a treatment regime for lung infections is discussed.
We have previously shown in agar diffusion assays that allicin compared well on a mol-for-mol basis with ampicillin and kanamycin against E. coli
], and there have been numerous individual reports that allicin, often in garlic juice rather than the pure substance, was effective against human pathogens, including MDR strains and MRSE [9
]. However, because allicin reacts with thiols, it is rapidly titrated out by glutathione in bodily fluids and it is therefore not suitable for clinical application via the oral route [37
]. Subcutaneous application of garlic extract has been used in mice to treat lung infections with Pseudomonas aeruginosa
and led to the disappearance of the bacteria in the infected lungs [23
]. However, scaling up for application in humans would require approximately 50 garlic bulbs per day per person and the authors concluded that this was not feasible. In clinical trials on the effect of consuming garlic capsules on cystic fibrosis patients, although positive tendencies were seen, treatment didn’t produce significant improvements [37
]. Because of the problems of attaining effective concentrations of allicin where it is needed when it is consumed orally, direct inhalation in the case of lung infections seems an attractive alternative. The successful treatment of tuberculosis patients by inhalation of garlic vapor has been reported [21
Allicin vapor was inhibitory to the growth of the clinical isolates tested with the exception of the very resistant P. aeruginosa
DSM2659 strain (Figure 2
). These results confirm ‘proof of principle’ that it could be possible to administer allicin via the pulmonary route rather than the oral route to combat lung infections. Furthermore, it may be possible to use sublethal concentrations of allicin in conjunction with other antibiotics. A synergistic action of allicin with beta-lactams (cefazolin, oxacillin, and cefoperazone) was shown to decrease the MIC to P. aeruginosa
]. A synergistic action between allicin and the antifungals amphotericin-B and polymixin-B has also been reported [19
MIC for the bacteria tested ranged from 32 to 128 µg/mL allicin with the majority of strains being completely inhibited by 64 µg/mL allicin, except for P. aeruginosa
DSM2659 which had an MIC of 512 µg/mL allicin. A similarly high MIC was found for P. aeruginosa
DSM50071 which might suggest that allicin is likely to be less effective against P. aeruginosa
infections than against other pathogens [9
]. Encouragingly, the MDR strains of S. pneumoniae
were equally as susceptible to allicin as the non-MDR strains (Table 1
). The majority of clinical isolates tested had MBCs from 64 to 128 µg/mL allicin, whereas P. aeruginosa
DSM2659 again showed greater resistance to allicin than other pathogenic strains (MBC = 1024 µg/mL).
Despite the MDR strains being as susceptible to allicin as the non-MDR strains, the overall performance of allicin compared to clinical antibiotics was poor (Table 2
). Nevertheless, for those antibiotics against which the MDR strains were resistant, the quantitative comparison with allicin was in some cases favorable, for example with S. pneumoniae
-10, S. Africa19A
-13 and Poland23F
-16 and erythromycin and clindamycin MIC > 256 µg/mL and for allicin 64 µg/mL. On this basis, allicin might be considered as a treatment for MDR strains. Where there is allergy to a particular antibiotic, allicin might also be considered.
Mammalian cells proved highly susceptible to allicin with EC50
values for the lung epithelial cell lines near the 64 µg/mL value taken as the general MIC for most bacteria (Figure 3
and Table 1
). Caco-2 and NIH/3T3 cells were even more susceptible than lung epithelial cells with EC50
of 19.5 and 21 µg/mL, respectively, well below the MIC of 64 µg/mL allicin for bacteria. Incorporating 1 mM GSH into the growth media raised the apparent EC50
value in all cases, however, this is because the effective dose of allicin is reduced in a simple reaction with the GSH to make S
Rat PCLS were even more susceptible to allicin than cells in culture with EC50
values of ~14.5 µg/mL allicin in the LDH and WST tests. The EC50
values were raised more than seven-fold to 106.4 µg/mL allicin (LDH test) and three-fold 45.5 µg/mL allicin (WST test) when the rat slices were supplied with 1 mM GSH (Table 3
). Hess et al. [38
] recently showed that the toxicity of a range of industrial chemicals in PCLS correlated well with in vivo aerosol application in rats for compounds stable in water. Therefore, the results we report here are not encouraging for developing allicin for treating lung infections, however, these in vitro data must be considered in the context of the in vivo situation. By virtue of their function, lung epithelial cells have to be protected against oxidative insults and the cells are well and continually supplied with GSH (1 mM GSH in whole blood) and have high levels of GSH and other intrinsic antioxidant systems [39
]. We have shown that GSH levels in E. coli
drop significantly during allicin stress and we have shown that GSH levels are important for the resistance of yeast to allicin with mutants deficient in GSH synthesis and metabolism being particularly susceptible [9
]. This intrinsic protection against allicin due to endogenous GSH and other low molecular weight thiols in the bacteria themselves, must be overcome before cells will be inhibited by allicin. The GSH concentration in alveolar fluid (epithelial lining fluid, ELF) has been reported to be 0.4 mM, 140 times more than in plasma [44
] and in rat lungs 2 mM GSH has been reported [42
]. This is completely different to the in vitro situation for cells in culture which are cut off from continual blood circulation. Mostly, lung-pathogenic bacteria colonize the gel layer of the mucus, which is separated from the epithelial cells by an additional sol layer [45
]. Therefore, it is very likely that bacteria colonizing the bronchial and alveolar air spaces have a less GSH-rich environment than the epithelial cells lining the lungs. High levels of GSH were found in ELF, which mainly consists of mucus and cells. As the pathogens (bacteria) would be on the surface of the mucus they would be more exposed to the inhaled allicin, whereas the epithelial cells would be more protected. This increased exposure of lung cells, but not the invading bacteria, to GSH in the bathing fluids may help with differential susceptibility of bacteria vs. lung cells in clinical treatment of infection. This question can only be clarified by experiments in animal models, which at present are beyond the scope of this investigation. Nevertheless, there are historical precedents for the successful treatment of lung infections by garlic preparations. Thus, Minchin [21
] used a specially designed mask to treat tuberculosis patients twice daily by one-hour inhalation periods with crushed garlic preparations containing ‘oleum allii’. Minchin also reported using oleum allii inhalation prophylactically “in the homes of, and by the members of, families notably affected by pulmonary tuberculosis”. These pioneering studies dropped out of focus as streptomycin, without the unpleasant smell of allicin, was introduced in 1944 and used as a treatment for tuberculosis.
4. Materials and Methods
4.1. Allicin Synthese
Allicin was synthesized by oxidation of diallyl disulfide (DADS) with H2
as reported previously [46
The Pseudomonas aeruginosa
strains PAO1 SBUG8, PAO25, and DMS2659 were from the culture collection of the Institute for Applied Microbiology (IAM, RWTH Aachen). The Streptococcus pneumoniae strains
(MDR strains: Spain23F
-1 (PMEN-1), CSR14
-10 (PMEN-10), S. Africa19A
-13 (PMEN-13), Poland23F
-16 (PMEN-16) (http://www.pneumogen.net/pmen
), clinical isolates: SNo 67715, SNo 68668, SNo 68665), Streptococcus pyogenes
(clinical isolate SNo 67467), Streptococcus dysgalactiae equisilimlis
(clinical isolate SNo 67799, SNo 73742), Streptococcus agalactiae
(clinical isolate SNo 67764, SNo 69235), Staphylococcus aureus
(ATCC 43300 and clinical isolate SNo 68709), Klebsiella pneumoniae strains
(clinical isolates SNo 45412, SNo 45413), and Acinetobacter baumannii
(clinical isolates SNo 45541, SNo 45757, SNo 45760) were from the culture collection of the German National Reference Center for Streptococci (GNRCS), University Hospital, RWTH Aachen.
4.3. Antibiotic Activity of Allicin Vapor
Bacteria were grown over night at 37 °C on blood-CASO-agar plates. Bacterial colonies were resuspended in 5 mL MHB2 medium to an optical density at 600 nm (OD600
) = 0.2. Defibrinated sheep blood was added to 25 mL molten CASO-agar at 50 °C (5% v
), mixed with 300 µL of bacterial suspension and poured immediately into a Petri dish to make bacteria-seeded agar plates. Bacteria were either spread onto the agar surface. Different concentrated allicin solutions (20 µL) were pipetted onto the Petri dish lid and the solidified agar plate with bacteria was placed inverted over the lid. The amount of allicin in the droplet is indicated in Figure 2
. Bacterial growth was scored after incubation over night at 37 °C.
4.4. MIC and MBC Determination
Susceptibility testing was performed following the EUCAST guidelines together with the GNRCS using the broth dilution method in 96-well microtiter plate format [47
Bacteria were grown over night at 37 °C on blood-CASO-agar plates (CASO-Agar, Carl Roth, 5% defibrinated sheep blood Thermo Fischer Scientific GmbH, oxoid limited, Basingstoke, UK), P. aeruginosa without blood. Bacterial colonies were resuspended in 5 mL cation-adjusted Müller–Hinton Broth 2 (MHB2, Sigma-Aldrich, St. Louis, MO, USA) up to 1 McFarland. Double concentrated MHB2 (2.2 mL), 141 µL lysed horse blood (Thermo Fischer Scientific GmbH, oxoid limited, Basingstoke, UK) and 10 µL bacterial suspension were mixed and kept on ice until pipetting into microtitre plates.
A two-power dilution series of allicin in water (2–2048 µg/mL = 12 µM–12.6 mM) was prepared and 50 µL was mixed with 50 µL of bacteria suspended in blood-MHB2 medium (see above) in 96-well plates so that the final bacterial suspension in the test wells was 0.5 McFarland. Plates were covered with air-permeable, self-adhesive cling film (Carl Roth GmbH, Karlsruhe, Germany) and incubated at 37 °C for 20 h without shaking. The lowest allicin concentration without growth gave the MIC. For the determination of MBC 10 µL from each well were pipetted onto agar plates (see above) and incubated at 37 °C overnight. The lowest concentration without bacterial growth was the MBC.
4.5. Effect of allicin on Mammalian Cells
Lung epithelial cell lines Beas-2B (SV40-immortalized human bronchial epithelial cells) and A549 (human epithelial lung carcinoma), Caco-2 (human epithelial colon tumor) and fibroblast NIH/3T3 (murine embryonic fibroblast) cells were tested for their sensitivity to allicin. Beas-2B, A549 and Caco-2 were cultivated in DMEM medium with penicillin/streptomycin 1% (v/v) (each 10,000 U/mL, Lonza, Verviers, Belgium) and fetal bovine serum (FBS) 10% (v/v) (Sigma-Aldrich, St. Louis, USA). NIH/3T3 cells were cultivated in Roti®-CELL RPMI-1640 (Carl Roth GmbH, Karlsruhe, Germany) media with penicillin/streptomycin 1% (v/v) and FBS 10% (v/v). Cells were cultivated in TC (tissue culture) dishes (60 mm, Sarstedt, Nümbrecht, Germany) at 37 °C and 5% atmospheric CO2 and sub-cultured weekly. For experiments, the cell culture was grown directly in 96-well plates (100 µL per well).
Cells were exposed to allicin (diluted in medium) for 1 h (controls with medium only). Allicin concentrations between 78 and 2500 µM (12.7–406 µg/mL) (two-power dilution series) were tested. In a further experiment, cells were incubated with GSH 1 mM (Sigma-Aldrich, St. Louis, MO, USA). After incubation, unreacted allicin was titrated out by the addition of 100 µL cysteine solution 5 mM (AppliChem GmbH, Darmstadt, Germany, dissolved in media). Cells were post-incubated for 23 h at 37 °C and 5% CO2. Cell viability was tested with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Carl Roth GmbH, Karlsruhe, Germany). 50 µL of MTT 0.5% (w/v) dissolved in phosphate-buffered saline (PBS) was added to each well and the plate incubated for 3 h at 37 °C and 5% CO2. Cells were lysed by adding 100 µL isopropanol and the A570 subtracted from A630 automatically in the plate reader (TriStar2 LB942, Berthold Technologies, Bad Wildbad, Germany). The results are presented as half maximal effective concentration (EC50) showing a 50% reduction in MTT response.
4.6. Effect of Allicin on Rat PCLS
Female Wistar rats (Rattus norvegicus) (270 ± 10 g) were purchased from Charles River (Sulzfeld, Germany). Animal studies were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (ID: 8.87-51.05.20.10.245, 16 April 2013) and performed following the Directive 2010/63/EU of the European Parliament. Animals were euthanized with a lethal dose of pentobarbital (60 mg/kg). After confirming that the animals did not show any reflexes, the abdomen was opened and the lung was removed. The lung was filled via the trachea with 37 °C 1.5% (w/v) low-melting agarose and the lung was transferred to ice-cold PBS. After the agarose solidified, the lung was sliced first into 10 mm thick cylinders and afterwards into 250 µm thin slices with a Krumdieck tissue slicer (Alabama Research and Development, Munford, TN, USA). The slices were placed in a 24-well plate (two slices per well). One well per treatment and per animal was used. Each experiment was performed with three animals.
PCLS were incubated at 37 °C and 100% humidity in MEM (minimal essential medium) supplemented with CaCl2
1.8 mM, MgSO4
0.8 mM, KCl 5.4 mM, NaCl 116.4 mM, glucose 16.7 mM, NaHCO3
26.1 mM, Hepes 25.17 mM, sodium pyruvate 1 mM, glutamine 2mM, MEM amino acids and vitamin mix. Agarose was removed from the slices by replacing the medium every 30 min for two hours and then every 60 min for the next 2 h before being used for experiments [48
The slices were incubated for 1 h with allicin (0.1 µM, 1 µM, 10 µM, 33 µM, 100 µM, 333 µM, and 1 mM) (16.2 ng/mL, 162.3 ng/mL, 1.6 µg/mL, 5.3 µg/mL, 16.3 µg/mL, 54.0 µg/mL, 162.3 µg/mL). Allicin was removed after incubation by washing the slices 3 times with PBS and post-incubating the tissue slices for 23 h.
Cell viability was determined using the lactate dehydrogenase activity test (LDH cytotoxicity detection kit, Roche) and via measurement of the mitochondrial metabolic activity with WST-1 (water soluble tetrazolium) kit (Roche Diagnostics GmbH, Mannheim, Germany).