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International Journal of Antimicrobial Agents 33 (2009) 591–592 Contents lists available at ScienceDirect International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag Letter to the Editor Antimicrobial properties of shiitake mushrooms (Lentinula edodes) Sir, The shiitake mushroom, Lentinula edodes (Berk.) Sing, is a culi- nary delicacy and has traditionally been used as medicine in Asia dating back over 2000 years. Shiitake mushrooms contain pro- tein, lipids (primarily linoleic acid), carbohydrates, fibre, minerals, vitamins B1, B2 and C, and ergosterol, the D provitamin [1]. One of the major compounds isolated from the shiitake mushroom is lentinan [(C 6 H 10 O 5 ) n ], a polysaccharide with a mean molecular mass of 500 kDa which acts as an activator for macrophage T- lymphocytes and other immune effector cells that modulate the release of cytokines, which in turn may account for i ts indirect antitumour and antimicrobial properties. In this study, a known quantity of shiitake freeze-dried pow- der was suspended in an equal weight/volume of sterile 0.1% (w/v) peptone saline (PS) (CM0733; Oxoid Ltd., Basingstoke, UK) and filter-sterilised through a 0.22 ␮m syringe filter (Millipore (UK) Ltd., Watford, UK) before microbiological challenge. Sterile PS and antibiotic susceptibility discs containing 5 ␮g of ciprofloxacin (MAST Diagnostics Ltd., Bootle, UK) were employed as negative and positive controls, respectively. All test organisms (Table 1) were cul- tured on Columbia blood agar (CM0331; Oxoid Ltd.) supplemented with 5% (v/v) defibrinated horse blood and incubated for 24 h at 37 ◦ C. Under aseptic conditions, dilutions of each isolate were pre- Table 1 Organisms tested for positive inhibition by aqueous extracts of freeze-dried shiitake mushrooms (Lentinula edodes). Organism a Source Notes Paenibacillus lentimorbus, Bacillus licheniformis, Bacillus subtilis, Pseudomonas mevalonii, Klebsiella/Enterobacter sp., Sphingobacterium multivorum, Microbacterium sp., Stenotrophomonas sp. Mushroom composts Medically relevant opportunistic infections of patients Pseudomonas syringae subsp. savastanoi and P. syringae subsp. phaseolicola (4 UK races, strain nos.: NCPB-604, 605, 606, 607, 1320, 1321, 2571,1494, 1752, 3613, 4016); Pseudomonas pv. tolasii (2192, 1116, 3148, 387, 741, 1311, 2193, HB101) Plant and soil sources in crop residues (bacteria present in aerosolic dusts) (Central Science Laboratory, UK) Environmentally important, agrienvironment disseminated (soil/plant dust), can serve as a reservoir for Pseudomonas opportunistic infections in healthcare environments Pseudomonas aeruginosa Plant and soil sources Opportunistic plant and environmental pathogen Bacillus subtilis, Bacillus cereus, Serratia marcescens, Serratia spp./Rahnella, Cupriavidus Seed surfaces and composted fertiliser pellets, compost wastes Dormant pathogen of risk to animal and human health MRSA, Alcaligenes faecalis, Escherichia coli 0157 Environmental pathogens Associated with endogenous opportunistic infections in immune compromised patients Fungal isolates Aspergillus niger, Aspergillus flavus Mushroom composts Isolated at Applied Plant Science Division, Agri-Food & Biosciences Institute, Belfast, UK Fusarium graminearum Cereal seeds fungal pathogen Mycotoxins such as deoxynivalenol (Vomitoxin, ribotoxic) interrupt human and animal immune modulatory responses MRSA, meticillin-resistant Staphylococcus aureus. a All isolates listed above are from the culture collections of those held at Agri-Food & Biosciences Institute stock. pared individually in 0.1% PS, equating to a 0.5 McFarland standard (ca. 10 6 colony-forming units/mL), which was then inoculated onto Mueller–Hinton agar (CM0337; Oxoid Ltd.) with a sterile cotton swab, followed by fresh extracts (10 ␮L); the inoculum was allowed to dry prior to incubation. The plates were examined visually and any inhibition was noted and its diameter measured and recorded. Bioassay of the extracts showed that all the fractions exhibited qualitative inhibitory activity against all bacteria and fungi tested. Prompted in part by the response to opportunistic pathogens, a number of these metabolites or their aqueous extracts are currently being tested for their efficacy against a range of human clinical pathogens. As a result of the demonstration of its antibacterial and antifun- gal activity, we subsequently attempted to identify the chemical components of the shiitake from extracts and have provision- ally identified 34 compounds whose structure assignments were based largely on the interpretations surrounding mass spectral (MS) data deduced from previously reported metabolites. We have identified a number of new antimicrobial metabolites not previously identified in shiitake mushrooms. These include cyclo- heximide (an antibiotic substance that acts as a plant growth regulator, butcauses human liver toxicity and a reduction in protein synthesis), bostrycoidin (bioactive in vitro against Mycobacterium tuberculosis), anticarcinogenic alkaloids (muscarine, choline), tan- nins (epiafzelechin) and terpenoids (adiantone), cyclopiazonic acid (a natural food contaminant) and aspergillomarasmine. The lower 0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.10.018 592 Letter to the Editor / International Journal of Antimicrobial Agents 33 (2009) 591–592 level of activity of polar compounds in water-soluble extracts (Fraction 3) indicated that the antibacterial properties of shiitake mushrooms may have arisen largely from less polar organic com- pounds and the data also suggest that the higher activity may be due to the presence of disulphides, lenthionine compounds in the organic extracts. The aqueous extracts also confirmed the pres- ence of eritadenine and a metabolite with a molecular weight of 237 (C 15 H 11 NO 2 ), possibly a derivative of viridicatine, together with gallic acid (deduced from the ion peaks accompanying the MS data (M + H) + 171.2 with molecular weight 170). Gallic acid has been shown to have antibacterial properties and also exerts anticar- cinogenic activity on cancerous cells in animals. The presence of a tannin precursor provides the basis for epiafzelechin, with a molec- ular weight of 274, and the presence of a fungal metabolite with a molecular weight of 285, bostrycoidin (C 15 H 11 NO 5 ), was also estab- lished via GC–MS methods. Bostrycoidin is an antibiotic substance and has been shown to be active in vitro against M. tuberculosis. A number of other metabolites that were previously only obtained via organic solvent extractions are also reported in our freeze-dried aqueous extracts and include eritadenine, deoxyeritadenine, cyclo- heximide, 1,2,4,6-tetrathiepane, muscarine, choline and the sterol C 28 H 44 O 6 , and a fungal metabolite usually found in other mush- rooms’ cycloheximide (MW 281), found in the ethyl acetate-soluble portion of the methanol extract and in the methanol-soluble por- tion of the freeze-dried extract in our studies. It is beyond the scope of this current report to evaluate the quantitative yield for all the reported metabolites at present as well as how they com- pare with other mushrooms. It is expected that the occurrence of some of these metabolites may be in much smaller quantities in the shiitake mushroom than in others. However, this can be regarded as an advantage as they may not impose toxic side effects but none the less may be sufficient to express antibiosis, an ideal scenario for a natural medicinal product. Although other recent studies examining the presence of antimicrobial components in native plant varieties of the British Isles was unable to demonstrate any antibacterial or antifungal properties [2], with the exception of garlic (Allium sativum), the current study is encouraging and high- lights a potent antimicrobial fungus, which now requires further investigation and scientific follow-up. Acknowledgments The authors wish to acknowledge David Nelson and Graham McCollum for their help with this work. They also thank the Agri- Food & Biosciences Institute (AFBI), the University of Ulster and the Nuffield Foundation for their support and assistance in this research. Funding: JEM was partly supported by a Department of Health and Social Services and Public Safety (DHSSPS) Research Develop- ment Office grant (RRG 9.9), Belfast, UK. Competing interests: None declared. Ethical approval: Not required. References [1] Zuccato E, Calamari D, Natangelo M, Fanelli R. Presence of therapeutic drugs in the environment. Lancet 2000;335:1789–90. [2] Woods-Panzaru S, Nelson D, McCollum G, Ballard L, Millar BC, Maeda Y, et al. An examination of antibacterial and antifungal properties of constituents described in traditional Ulster cures and remedies. Ulster Med J 2009;78: 1–3. Juluri R. Rao a B. Cherie Millar b John E. Moore b,c,∗ a Environmental Pathology, Applied Plant Science Division, Agri-Food & Biosciences Institute, Newforge Lane, Belfast BT9 5PX, UK b Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Lisburn Road, Belfast BT9 7AD, UK c School of Biomedical Sciences, Centre for Molecular Biosciences, University of Ulster, Cromore Road, Coleraine, Co. Londonderry BT52 1SA, UK ∗ Corresponding author. Tel.: +44 28 9026 3554; fax: +44 28 9026 3991. E-mail address: jemoore@niphl.dnet.co.uk (J.E. Moore) 16 October 2008 . http://www.elsevier.com/locate/ijantimicag Letter to the Editor Antimicrobial properties of shiitake mushrooms (Lentinula edodes) Sir, The shiitake mushroom, Lentinula edodes (Berk.) Sing, is a culi- nary. aseptic conditions, dilutions of each isolate were pre- Table 1 Organisms tested for positive inhibition by aqueous extracts of freeze-dried shiitake mushrooms (Lentinula edodes). Organism a Source. cells that modulate the release of cytokines, which in turn may account for i ts indirect antitumour and antimicrobial properties. In this study, a known quantity of shiitake freeze-dried pow- der