Evaluation of Antimicrobial Activity of Oudemansiella sp (Basidiomycetes)
Iranian Journal of
Pharmaceutical Research (2004) 2: 103-107
Received: August 2003
Accepted: June 2004 |
Copyright ? 2004 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services |
Original Article
Evaluation of Antimicrobial Activity of
Oudemansiella sp (Basidiomycetes)
Hossein Vahidi* and Forogh Namjoyan
Department of Pharmacognosy, School of Pharmacy, Shaheed
Beheshti University of Medical Sciences, Tehran, Iran.
Abstract
Antimicrobial
activity of different culture extract of
Oudemansiella sp
grown on liquid medium (MYGP) were tested. Different fungi
(C. albicans, C. lipolytica, Saccharomyces
cervisiea, Cladosporium herbarum, and Aspergillus niger) and bacteria (Microccocus
luteus, E. coli, Staphylococcus aureus, and Staphylococcus
epidermidis) were used as test
organisms.
Various
antimicrobial assay methods including paper disc agar
diffusion and microdilution method, were employed to
determine possible activity of the extracts. Relative MIC
values showed strong activity of the ethyl acetate extract
of the fungus against tested bacteria and fungi, especially
filamentous fungi.
Key word:
Oudemansiella sp;
Antimicrobial; Basidiomycetes.
Introduction
The need for less toxic, more potent and
non anti - infectives antibiotics, as well as the evolving
resistance of microorganisms are some of the medical areas that
have posed a challenge to therapeutics since
1990s. A corresponding situation exists in the
agricultural sectors. These factors have the combined effect of
injecting a sense of urgency into the search for new bioactive
compounds (1). The basidiomycetes (mushrooms) are valuable as
gene pool sources, which have not yet been the subject of
extensive screening for any possible antifungal and
antibacterial activity (2).
Oudemansiella
(Fam. Tricholomatacea) is a saprophytic fungus, found mostly growing
on wood. Different antifungal agents have been isolated from
the Oudemansiella genus. Oudemansin and mucidin were isolated from the
Oudemansiella radicata, showing an antifungal activity (3).
Antifungal activity of Oudemansiella
platyphylla has also been
reported. The fungus has shown activity against C. albicans, C. tropicalis and A. fumigatus (4).
Experimental
Fungus material
The fungus was collected in November 1997
from the northern parts of Iran. Next, the fungus was
identified as Oudemansiella sp and stored in the Department of
Pharmacognosy at the School of Pharmacy, Shaheed Beheshti
University of Medical Sciences (Voucher number 01- Ram 97).
Growth and preparation of extracts
Tissue culture was carried out,
using the cap of the fungus (5). Mycelium of the fungus
was cultured in a liquid medium containing 10 g/l glucose, 10
g/l malt extract, 3 g/l Yeast extract and mycological peptone.
After 10 days the culture was filtered and filterate was
separately extracted with different solvents including ethyl
acetate, chloroform and petroleum ether respectively. Each
extract was concentrated under reduced pressure. The condensed
products were weighed and kept at 4°C perior to testing.
Test microorganisms
Microorganisms including fungi and
bacteria were obtained from the Persian Type Culture Collection
(PTCC) as well as the American type culture collection (ATCC).
Candida albicans (PTCC 5027), Candida
lipolytica (ATCC 825), Saccharomyces cervisiea (ATCC 9783), Cladosporium
herbarum (School of Health, Tehran
University), and Aspergillus niger (PTCC 5010), were used as the fungal tested
organisms and Micrococcus lutes (ATCC 9273), E.
coli (PTCC1330), Staphylococcus aureus (PTCC1112), and Staphylococcus
epidermidis (PTCC1114) used as the
bacterial tested organisms. For the bacterial suspension,
3× 107 cell /ml was prepared, compared to that of the Mc
Farland standard tube number 1. For the filamentous fungi and
yeast-like fungi, a suspension containing 1×106 spores or
cells per ml was prepared by using the Neubauer Counting
Chamber.
Antimicrobial assay
Paper disc agar diffusion method
A series of 90 mm Petri dishes containing
the Muller Hinton agar for the growth of bacteria and
the malt extract agar for the growth of
yeast-like fungi were prepared and each plate was
separately inoculated with different cultures of the
yeast like fungi and bacteria by swabbing aseptically on the
whole surface of the agar with cotton wool. A 6 mm diameter
filter paper disc was impregnated with 20 µl of each
extract in absolute ethanol (50 mg/ml). The discs were air
dried and placed aseptically at the center of the plates. The
plates were left undisturbed for 1 h to allow the extract to
diffuse into the agar. Chloramphenicol (0.050 mg) and Nystatin
(0.050 mg), dissolved in absolute ethanol, were also
impregnated onto the discs, air dried, and used as a positive
control. The plates were incubated at the desired temperature.
The growth inhibition, indicated by the areas of clear zone,
was measured. Evaluation of the inhibitory properties was
carried out in duplicates.
Microwell plate techniques
For the filamentous fungi, a 96 microwell
plate was used. 20 µl of the spore suspension and 100
µl liquid culture medium (malt extract broth) were added
to each well. 100 µl of the extracts in 10 % DMSO (100
mg/ml) was also added and plates were stored at 25°C for 48
h. Nystatin (100 mg/ml) was used as the positive control and
DMSO served as a negative control. After microscopic
examination, results were reported as the percentage of
inhibition of spore germination (100%, 80%, 50%, 20% and 0%)
(6, 7).
Minimum Inhibitory Concentration (MIC)
measurement
The MIC value of extracts showing a
strong activity was determined, using serial dilution method.
Serial dilutions of each extract were individually placed in
tubes labeled 1 to 6. Tube 1 was filled with 100 µl of
the extract stock solution ( 200 mg/ml, 10% DMSO) . Only 50
µl of the stock solution in tube 1 was transferred to
tube 2 and diluted with 50 µl of a 10% DMSO solution.
This procedure was repeated for solutions in tubes 2 to 6. Each
tube was then filled with 100 µl muller hinton broth
for bacteria and malt extract broth for the fungi and
also 100 µl spore or bacterial suspension to obtain a
serial dilution of the test materials ( 40, 20, 10, 5, 2.5 1.25
and 0.63 µg/µl). The resulting mixtures were then
stirred thoroughly and incubated at 25°C for the fungi and
37°C for the bacteria, overnight. Nystatin (200 mg/ml) and
chloramphenicol (200 mg/ml) were used as positive controls and
DMSO served as a negative control. Turbidity was taken as an
indication of growth and the lowest concentration which
remained clear after macroscopic evaluation was recorded as the
relative minimum inhibitory concentration. The MIC value was
recorded as the mean concentration of duplicates.
Results and discussion
Extraction of 500 ml fungus culture
filtrate gave the following yields: ethyl acetate extract
(0.05% W/V), chloroform (0.02% W/V) and petroleum ether
(0.01% W/V).
Results obtained from the controls
indicate that solvents had no effect on the fungal and
bacterial strains. Amongst the different extracts studied, only
the ethyl acetate extract showed a strong activity. The extract
showed high selectivity against M. luteus, exhibiting a
relatively high inhibition zone diameter (18.4 mm) (Table 1).
On the contrary, the efficacy of the extracts towards the
various yeast like fungi was non- selective, as the inhibition
zones showed little variation. The extracts also showed
strong activity against the filamentous fungi (Table 2), by
giving the lowest relative minimum inhibitory concentration
values of 10 µg/µl with C. herbarum (Table
3).
The chloroformed extract of the fungus
was particularly active against the tested microorganisms,
especially filamentous fungi. A relative minimum inhibitory
concentration values of 40 µg/µl with both Cladosporium herbarum and A. niger was observed. No strong activity was noted
when petroleum ether used as the solvent for extraction.
However, it is interesting to note that the extract showed
activity against the tested fungi. Therefore, the possibility
for presence of specificly active compounds within the
petroleum extract of the fungus is rather high. The ethyl
acetate extract has shown strong activity against spore
germination of C. herbarum and A. niger with 100 % and 80 % inhibition,
respectively. It is interesting to note that different extracts
showed strong activity against spore germination of the tested
microorganisms.
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