The in vitro susceptibility of Scedosporium prolificans to ajoene, allitridium and a raw extract of garlic (Allium sativum)

Stephen R. Davis1,*, Rosemary Perrie1 and Rafael Apitz-Castro2

1 Mycology Unit, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia; 2 Instituto Venezolano de Investigaciones Científicas (IVIC), Lab. Trombosis Experimental Centro de Biofísisca y Bioquímica, Caracas, Ap. 21827, 1020-A, Venezuela

Received 15 June 2002; returned 29 October 2002; revised 6 January 2003; accepted 10 January 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro susceptibility of 20 medical isolates of Scedosporium prolificans to ajoene, allitridium and raw garlic extract derived from cloves of garlic (Allium sativum) was tested using the NCCLS reference method (with minor modifications) for broth microdilution. The results demonstrate that both garlic derivatives and raw garlic extract appear to have in vitro activity against S. prolificans.

Keywords: antifungal, ajoene, allitridium, garlic


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The medical use of garlic (Allium sativum) has been part of folk medicine for thousands of years, and it is among the oldest of all cultivated plants. In 1944 at the Sterling–Winthrop chemical company in the USA, it was demonstrated that allicin, a constituent of garlic, had both antibacterial and antifungal properties.1 This compound was patented, but its clinical use was abandoned because of the substance’s odour.1 Since then, it has been determined that it is the breakdown and condensation products of allicin, and not allicin itself, that are responsible for most of the antifungal properties of garlic in vivo.2

The antifungal properties of one of these condensation products, ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide], were successfully demonstrated against Candida albicans and Aspergillus niger by Japanese researchers.3 In gel form, it is at present undergoing broad-scale trials in the treatment of tinea, with a demonstrated success rate equivalent to that obtained by using terbinafine cream in tinea treatment.4

Another breakdown product of allicin is diallyl trisulphide, and this product is the main component of a Chinese garlic extract called ‘allitridium’, which has been used successfully as an intravenous treatment for cryptococcosis in China.5 This product has also been shown to be active against other fungi such as Aspergillus spp. and Candida spp., as well as bacteria such as methicillin-resistant Staphylococcus aureus.6

Scedosporium prolificans (syn. Scedosporium inflatum) as a pathogen was first described in 1984.7 This fungus was found to be difficult to treat, and its inherent resistance to known antifungals both in vitro and in vivo has been documented.8 Apart from the innate resistance of this fungus to the available antifungal agents, this fungus has the ability to produce spores during growth in tissues, and these spores are able to disseminate from the site of inoculation to distant organs.9 A study performed in Spain on 16 patients with deep S. prolificans infections (disseminated infection in 14 patients, and fungal pneumonia and meningoencephalitis in the other two) showed that 14 (87.5%) of these patients died despite antifungal therapy.10

The most promising systemic antifungal treatment for this mould at present is combination therapy, specifically terbinafine with azoles, particularly itraconazole.11 The clinical outcome of treatment using this type of therapy is being evaluated at the time of writing this paper.

Because of the limited options for the treatment of infections caused by S. prolificans, we decided to investigate the in vitro antifungal susceptibility of S. prolificans to ajoene, allitridium and raw garlic extract.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Source and preparation of garlic derivatives

Garlic cloves (948 g; organically grown, obtained from a local grower at the Central Market in Adelaide, South Australia) were crushed using a food blender, and this mash was squeezed and filtered through cheesecloth; 150 mL of raw garlic extract was obtained. This particular garlic extract was assayed at 8.93 g/L protein [using Microprotein (M-TP) reagent; Beckman Synchron CX Systems, Diamond Diagnostics Inc., Holliston, MA, USA] by the Core laboratory at the Women’s and Children’s Hospital, Adelaide. The filtrate was stored at –20°C and was thawed before use. Unpublished data from the Mycology Unit at the Women’s and Children’s Hospital, Adelaide, suggest that the freezing of garlic extract helps preserve antifungal activity.

Allitridium (Harvest Pharmaceutical Company, Shanghai, China) was supplied in individual 2 mL vials, each containing 30 mg of active component suspended in distilled water containing Tween 80. The batch number was 010005, and the product had an expiry date of 30 June 2002. The preparation of this product is proprietary.

Ajoene (R.A.C., Venezuela) was supplied as a solution containing 539 mg/mL ajoene in ethylacetate, and shown to be a pure 30:70 mixture of the E and Z isomers. The preparation of this solution has been described previously.12

Selection of isolates

A set of 20 clinical isolates of S. prolificans was selected from different patients. Ten isolates were from proven clinical infections (four from blood, four from tissue or bone, one from a wound and one from an ear swab). The other 10 isolates were from respiratory specimens, and the clinical significance of these isolates was not determined. A control isolate of Paecilomyces variotii ATCC 22319 was tested against raw garlic extract, allitridium and ajoene. All of the isolates of S. prolificans had previously been tested and had been shown to be multi-drug resistant, including to amphotericin B, during routine testing within this laboratory (see Table 1). P. variotii had been used in previous studies to determine MIC reference ranges for established and new antifungal agents, and so was chosen for this study.13


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Table 1.  Comparison of the MICs of garlic derivatives with the MICs of conventional antifungals for S. prolificans
 
Testing ethylacetate for antifungal activity

Five isolates of S. prolificans were chosen randomly from the set of 20 clinical isolates. Each isolate was used to make a suspension in water to a 0.5 McFarland standard turbidity. Each suspension was spread out onto an RPMI-1640 agar plate (auto-mod powder; Sigma, St Louis, MO, USA; lot 31k83031) using a swab. Unadulterated ethylacetate (25 µL; Merck; 99.5% pure) was placed onto each of five blank discs, and each disc was placed into the centre of the inoculated RPMI plates. The plates were incubated at 37°C for 48 h.

Broth microdilution method

Broth microdilution testing was performed according to the NCCLS M38-P method, with minor modifications, to determine MICs.14

For the garlic extract, a 1:2 serial dilution series was prepared using RPMI-1640 broth [a single lot of auto-mod powder (Sigma), lot 40k83091, supplemented with both 0.3 g/L L-glutamine and 0.165 M MOPS buffer (35.54 g/L) and without sodium bicarbonate], 0.01% DMSO (Merck, Australia; batch number 27329) and 0.03% gentamicin solution (Upjohn, Bentley, Australia; batch P880A, expiry date June 2002). The gentamicin solution was added to reduce the risk of bacterial contamination, and the DMSO to maintain the solubility of the garlic extract. Control wells used in this test demonstrated that the gentamicin had no antifungal activity at this concentration. Microtitre plates were prepared containing 100 µL of two-fold garlic dilutions with final concentrations ranging from 50% to 0.02% v/v.

For both allitridium and ajoene, drug dilutions were prepared following the additive two-fold drug dilution scheme for water-insoluble antifungal agents described in the NCCLS M38-P method.14 Stock solutions were diluted in 100% DMSO, and further diluted 1:50 in RPMI-1640 medium, and this resultant solution was used for a two-fold dilution series. Microtitre plates (plastic 96 well ‘u’ bottom trays; Sarstedt, Australia) were prepared containing 100 µL of the two-fold drug dilutions, with final concentrations ranging from 1024 to 4 mg/L for allitridium, and 64 to 0.025 mg/L for ajoene.

For each of the three tests, there was a set of sterility control wells containing RPMI only, and two sets of growth control wells, one containing RPMI broth alone and the other containing RPMI broth with DMSO. For the test involving garlic extract, the set of growth control wells containing DMSO also contained 0.03% gentamicin solution.

Inoculum preparation

The 20 isolates of S. prolificans and the P. variotii isolate were grown on potato dextrose agar slants at 35°C for 7 days to ensure maximum sporulation, and suspensions of these fungi were prepared in 0.85% saline. The resulting suspensions were standardized to an optical density of 0.15–0.17 absorbance for the Scedosporium isolates, and 0.09–0.11 for the Paecilomyces isolate using a spectrophotometer wavelength of 530 nm.13 These suspensions were then diluted 1:50 in RPMI-1640 broth, which corresponds to double the density needed of ~0.4 x 104 to 5 x 104 cfu/mL.

Test procedure

Except for the sterility control well, each well was inoculated with 100 µL of each of the fungal suspensions, and trays were then incubated at 35°C without agitation in a moist chamber for 72 h.

MIC determination

The MICs were determined with the aid of a reading mirror, and the growth in each MIC well was compared with that of the growth control well and given a numerical score of 0–4, as recommended by the NCCLS M38-P method.14

The endpoint was taken as the first well that showed a reduction in growth of at least 50% of that of the growth control well (i.e. numerical score = 2). Purity checks and colony counts were performed by plating out 100 µL of a 1:100 dilution of the test inoculum onto Sabouraud’s agar (Oxoid, Basingstoke, UK) to confirm inoculum density.

The above procedure was repeated a second time in an identical fashion to obtain a second set of values.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MIC of the raw garlic extract for S. prolificans ranged from 0.2 to 0.4% v/v (1:512 dilution to 1:256), with an MIC50 of 0.2% and an MIC90 of 0.4%.

The MIC of ajoene for S. prolificans ranged from 2 to 8 mg/L, with an MIC50 of 4 mg/L and an MIC90 of 8 mg/L, whereas the MIC of allitridium ranged from 4 to 16 mg/L, with an MIC50 of 8 mg/L and an MIC90 of 16 mg/L. These results are compared with MICs of amphotericin B and itraconazole obtained for the S. prolificans isolates using the NCCLS M38-P method of broth microdilution testing (see Table 1).

The MIC of the raw garlic extract for P. variotii ranged from 0.2 to 0.4% v/v (1:512 dilution to 1:256), with an MIC50 of 0.4% and an MIC90 of 0.4%.

The MIC of ajoene for P. variotii ranged from 2 to 4 mg/L, with an MIC50 of 4 mg/L and an MIC90 of 4 mg/L, whereas the MIC of allitridium was 32 mg/L, with an MIC50 of 32 mg/L and an MIC90 of 32 mg/L.

All isolates of S. prolificans were confirmed as pure cultures, and colony counts performed were within the expected range of 20–50 cfu for 100 µL of the 1:100 dilution of the 1:50 dilution of the 0.5 McFarland equivalent.

There was no evidence of antifungal activity from ethylacetate. In all cases, the fungal lawn created by S. prolificans grew up to the disc.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the absence of commercially prepared standards, the protein assay performed on the prepared concentrated garlic extract was used as a simple method of comparing different preparations using an independent variable. The testing procedure we adopted was robust, as the results were reproducible and the MICs for both runs were within a one-fold dilution of each other. The ATCC strain of P. variotii was used as an in-house control to monitor reproducibility of our modified method.

Two separate studies have been described that demonstrated in vitro activity of garlic extracts against Cryptococcus neoformans. The first group obtained an allitridium MIC of 2.5 mg/L for C. neoformans.5 The second group obtained an MIC range of 6–12 mg/L when they tested their own concentrated garlic extract (containing 34% allicin) against three different clinical isolates of C. neoformans.15 These results are comparable to the MICs of ajoene and allitridium that we obtained for S. prolificans, and lend support to the idea that further studies may be worthwhile to determine whether garlic extracts have a place in the treatment of infections caused by S. prolificans, and perhaps other intractable fungal infections.

Gas chromatography studies16 have shown that a group of compounds [called the S-alk(en)ylcysteine sulphoxides], of which alliin is the predominant member, makes up between 0.53% and 1.3% of the fresh weight of garlic. We used this reported range to perform simple arithmetic based on the volume of extract derived from the dry weight of fresh garlic used in our test. Accepting an earlier observation that it is the breakdown products of alliin that are the predominant antifungal compounds,2 we obtain an MIC range of our prepared garlic extract against S. prolificans of between 6 and 64 mg/L. This range would have to be revised downwards because the calculation is based upon the maximum amount of alliin derivatives that could be present within the garlic. It can be seen from this simple calculation that the results for the prepared garlic extract are consistent with our other results.

A small study was performed in the Ren Ji Hospital in Shanghai, China on the antifungal activity of the plasma of five patients after intravenous administration of allitridium.5 The study demonstrated at least a two-fold increase of anti- C. neoformans titres in the plasma of all five patients over pre-infusion levels using a standard in vitro microbiological assay. Anti-C. neoformans activity was also detected in the cerebrospinal fluid of four of the five patients after intravenous therapy with allitridium. Because the researchers believed that there was more than one antifungal chemical group within the garlic derivative, they made no attempt at measuring the plasma levels of specific molecules.

Reports from the People’s Republic of China indicate that the intravenous use of commercial garlic extracts is safe and relatively free of negative side-effects.17 Surveys performed in Shanghai indicate that serious toxicity during intravenous administration of allitridium, such as anaphylaxis, has not been recognized even when the extract was given for a month. Unpleasant side-effects that have been reported include abdominal cramping, nausea and thrombophlebitis at the intravenous site when allitridium is administered too rapidly. This may be contrasted with the usage of conventional amphotericin B in the western world, where infusion-related toxicities can occur in up to 60% of patients and renal impairment in up to 80% of patients receiving a 2 week course.18

Other factors to consider include the potential for a cheap antifungal drug for countries too poor to afford the latest western drugs, as well as the possibility of being able to exploit the known synergic interaction of allitridium (and probably other similar garlic derivatives) with amphotericin B,17 hopefully lowering the rate of dose-related toxicities due to treatment with amphotericin B alone.

The oral ingestion of this herb seems to have limited application for serious systemic fungal infections. Although it is known that antifungal activity is present in human serum half an hour after oral ingestion of garlic, the amount of unadulterated garlic extract that can be consumed at one sitting appears to be limited and does not allow therapeutic levels to be reached.19

Ajoene has already been mentioned as a potent inhibitor of fungi when used topically, and its use intravenously in humans has not been explored, although this compound has been successfully used intravenously in dogs.12

Garlic is a biologically active herb, and there has to be care taken with the administration of the unadulterated extract. It is antithrombotic,20 and can cause gastrointestinal problems in individuals20 when taken orally and contact dermatitis in some individuals21 when used topically. The risk of adverse drug interactions when using garlic or its derivatives concomitantly with other therapies has to be taken into account, but we believe that further research into garlic and its derivatives as antifungal agents in cases of serious, systemic disease is warranted.


    Acknowledgements
 
We would like to thank the following people from the Women’s and Children’s Hospital in Adelaide: Jan Bell of the Antibiotic Research Laboratory for her advice on methodology; Dr Paul Goldwater, deputy director of the Microbiology Department, for his strong support; Mrs Helen Alexiou, technical officer in the Mycology Unit, for her kind and generous support; and Mr Peter Norman and Ms Adrianna Santini for their help in media preparation. We would also like to thank Rao Jing from the Department of Dermatology at the Chonqing University of Medical Sciences in the People’s Republic of China for her assistance in obtaining allitridium from that country.


    Footnotes
 
* Corresponding author. Tel: +61-8-81617365; Fax +61-8-81617589; E-mail: sdavis{at}esc.net.au Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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18 . Butler, W. T., Bennett, J. E., Alling, D. W., Wertlake, P. T., Utz, J. P. & Hill, G. J. (1964). Nephrotoxicity of amphotericin B: early and late effects in 81 patients. Annals of Internal Medicine 61, 175–87.[ISI]

19 . Caporaso, N., Smith, S. M. & Eng, R. H. (1983). Antifungal activity in human urine and serum after ingestion of garlic (Allium sativum). Antimicrobial Agents and Chemotherapy 23, 700–2.[ISI][Medline]

20 . Gadkari, J. V. & Joshi, V. D. (1991). Effect of ingestion of raw garlic on serum cholesterol level, clotting time and fibrinolytic activity in normal subjects. Journal of Postgraduate Medicine 37, 128–31.[Medline]

21 . Jappe, U., Bonnekoh, B., Hausen, B. M. & Gollnick, H. (1999). Garlic-related dermatoses: case report and review of the literature. American Journal of Contact Dermatitis 10, 37–9.[CrossRef][Medline]