Investigation of Antimicrobial Activity of Different Trametes versicolor Extracts on Some Clinical Isolates

Abstract

Intensive drug uses against species which have clinical significance and cause severe diseases have triggered the development of resistance in these species. It is necessary to discover new potential drugs against clinical isolates because many antimicrobial drugs become ineffective in the coming years. In this research, we used different extracts from Trametes versicolor fruiting bodies for investigating antibacterial and antifungal activities on some clinical isolates. For this purpose, five bacterial species (Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa) and a fungal species (Candida albicans) were selected as clinically important isolates and four different crude extracts (chloroform, water, ethyl acetate and ethyl alcohol) of Trametes versicolor were tried on these species in terms of antimicrobial sensitivity with disc diffusion method. Obtained data were analyzed using SPSS 14 software. At the result, some of extracts were found to have antimicrobial activity on clinical isolates although all extracts generally show antimicrobial effect. Chloroform extract was found to have the highest antimicrobial activity on S. aureus and E. faecalis while ethanol extract was observed as more effective on P. aeruginosa, E. coli, B. subtilis and C. albicans isolates. It can therefore be suggested that, some of Trametes versicolor extracts are promising antimicrobial agents.

Full text

Investigation of Antimicrobial Activity of Different Trametes
versicolor Extracts on Some Clinical Isolates
Farklı Trametes versicolor Özütlerinin Bazı Klinik İzolatlar
Üzerindeki Antimikrobiyal Aktivitesinin İncelenmesi
Research Article
E. Özgör et al.
/ Hacettepe J. Biol. & Chem., 2016 , 43 (3), 267–272
Erkay Özgör*, Meltem Ulusoy, İrem Çelebier, Semiha Selda Yıldız, Nevin Keskin
Hacettepe University, Department of Biology, Microbiology and Parasitology Laboratory, Beytepe, Ankara, Turkey
ÖZET
Klinik önemi olan ve ciddi hastalıklara sebep olan türlere karşı yoğun ilaç kullanımı bu türlerde direnç
gelişimini tetiklemektedir. İlerleyen yıllarda birçok antimikrobiyal ilaç etkisiz hale geleceğinden bu türlere
karşı yeni potansiyel ilaçların keşfedilmesi gereklidir. Bu araştırmada, Trametes versicolor üreme yapılarından
elde edilen farklı özütler, bazı klinik izolatlar üzerinde antibakteriyel ve antifungal aktiviteleri araştırmak
için kullanılmıştır. Bu amaç doğrultusunda, klinik önemi olan izolatlar olarak 5 bakteri türü (Staphylococcus
aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa) ile bir fungus
türü (Candida albicans) seçildi ve Trametes versicolor’un 4 farklı ham özütünün (kloroform, su, etil asetat,
etil alkol), bu türler üzerinde antimikrobiyal duyarlılık açısından disk difüzyon yöntemi kullanılarak denendi.
Elde edilen veriler SPSS 14 programı kullanılarak analiz edildi. Çalışmanın sonunda, tüm özütler genel olarak
antimikrobiyal etki gösterse de özütlerden bazılarının klinik izolatlar üzerinde antimikrobiyal aktiviteye sahip
olduğu bulunmuştur. Kloroform özütünün S. aureus ve E. faecalis üzerinde en yüksek antimikrobiyal aktiviteye
sahip olduğu bulunurken etanol özütünün P. aeruginosa, E. coli, B. subtilis ve C. albicans izolatları üzerinde
daha etkili olduğu gözlenmiştir. Bu nedenle, bazı Trametes versicolor özütlerinin umut verici antimikrobiyal
ajanlar olabileceği söylenebilir.
Anahtar Kelimeler
Trametes versicolor, Antimikrobiyal aktivite, Antifungal aktivite, Klinik izolatlar.
ABSTRACT
Intensive drug uses against species which have clinical significance and cause severe diseases have triggered
the development of resistance in these species. It is necessary to discover new potential drugs against cli-
nical isolates because many antimicrobial drugs become ineffective in the coming years. In this research, we
used different extracts from Trametes versicolor fruiting bodies for investigating antibacterial and antifungal
activities on some clinical isolates. For this purpose, five bacterial species (Staphylococcus aureus, Entero-
coccus faecalis, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa) and a fungal species (Candida
albicans) were selected as clinically important isolates and four different crude extracts (chloroform, water,
ethyl acetate and ethyl alcohol) of Trametes versicolor were tried on these species in terms of antimicrobial
sensitivity with disc diffusion method. Obtained data were analyzed using SPSS 14 software. At the result,
some of extracts were found to have antimicrobial activity on clinical isolates although all extracts generally
show antimicrobial effect. Chloroform extract was found to have the highest antimicrobial activity on S. aureus
and E. faecalis while ethanol extract was observed as more effective on P. aeruginosa, E. coli, B . subtilis and
C. albicans isolates. It can therefore be suggested that, some of Trametes versicolor extracts are promising
antimicrobial agents.
Key Words
Trametes versicolor, Antimicrobial activity, Antifungal activity, Clinical isolates.
Article History: Re ceived: Jan 19, 201 6; Revised: May 16 , 2016; Accepted : Jun 20, 2016; Avai lable Online: Jul 31, 2016.
DOI: 10.15671/HJBC.20164420569
Correspondence to: E. Özgör, Ha cettepe Unive rsity, Depart ment of Biolog y, Microbiolog y and Parasitol ogy Laborato ry, Beytepe, An kara, Turkey
Tel : +90 312 297 801 1 Fax: +90 312 2 99 2028 E-Mail: er kay@hacettepe.edu .tr

268 E. Özgör et al.
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INTRODUCTION
Fungi have been a very good source of many
medical compounds for thousands of years.
Fungal substances have been as important source
of lead structures for new drug compounds. Due
to the discovery of penicillin that led to later
discoveries of potent antibiotics isolated from
microbial habitats [1,2]. In the last decades,
problem with antibiotic resistant bacteria has
emerged because of the wrong and intensive
drug uses. Bacterial and fungal pathogens have
evolved numerous defense mechanisms against
antimicrobial agents, and nowadays, the need to
discover new and more potent of these agents as
alternative to antibiotic therapy is stronger [3-5].
There are many different studies about
antimicrobial activity of different types of
fungal extracts from Slovakia, Nigeria, India and
China [3,6-9]. In these studies, fungal extracts
have been shown to be a potential drug against
bacterial and fungal disease agents. Studies with
macromycetes, especially Ganoderma lucidum
and Trametes versicolor, revealed that these fungi
have effective microbial inhibitory properties.
Trametes versicolor (L.) which commonly
known as turkey tail is a common polypore
mushroom found throughout the world. The
top surface of the cap shows typical concentric
zones of different colours. The fruiting body is 1–3
mm thick and has leathery texture. T. versicolor
contains biologically active compounds with
an enormous variety of chemical structures
including the protein-bound PSP, B-1,3-B-1,4
glucans, tetracyclic triterpenoids of lanostane,
fungisterol and B-sitosterol [10-12]. Furthermore,
Polysaccharide-K (PSK) which can be obtained
from mushrooms displays anticancer activity
in laboratory studies and preliminary human
researches [13,14]. Therefore, Trametes spp.
could be useful in the search of new potent
antimicrobial agents.
The aim of this study is to investigate
antibacterial and antifungal activities of different
extracts from Trametes versicolor fruiting bodies
against some clinical isolates including Gram-
positive, Gram-negative bacteria and Candida
albicans.
MATERIALS and METHODS
Preparation of Different Trametes versicolor
Extracts
Trametes versicolor fruiting bodies were collected
almost 500 gram from different forest areas
in Ankara and Zonguldak provinces, Turkey
during the spring season 2015 and macroscopic
identification of harvested fungi were done.
Fruiting bodies of T. versicolor were dried at
70°C and pulverized with laboratory blender. The
obtained powder was weighed and divided into
four groups. Each group was treated with 500
mL of four different solvent (chloroform, water,
ethyl acetate and ethyl alcohol) to find out which
solvent requires to obtain active compounds from
T. versicolor fruiting bodies. Powdered substances
in each extract were dissolved with the aid of
sonicator. After this process, each mixture was
filtered to remove solid residues and solvents
evaporated with rotary evaporator. The crude
residue of each extract was frozen at -80°C and
lyophilized [3]. These fungi extracts were stored
at + 4°C in refrigerator until use.
Test Microorganisms
Six clinically important strains of microorganisms
were tested in this research. Three Gram-
positive bacteria (Bacillus subtilis ATCC
6633, Staphylococcus aureus ATCC 25923,
Enterococcus faecalis ATCC 29212) and two Gram-
negative bacteria (Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC 27853) were
selected for antibacterial activity test. Candida
albicans ATCC 10231, a clinically important yeast
strain, was also selected for antifungal activity
test. Staphylococcus aureus and Enterococcus
faecalis were cultured in the blood agar (Sigma-
Aldrich, USA) at 37°C, Bacillus subtilis, Escherichia
coli and Pseudomonas aeruginosa were cultured
in the nutrient agar (Merck, Germany) at 37°C and
Candida albicans was cultured in the sabouraud
dextrose agar (Merck, Germany) at 30°C.
Antimicrobial Assay
Antibacterial and antifungal activity tests were
performed with disc diffusion method to find
antimicrobial sensitivities of selected clinical
isolates. Different Fungal extracts dissolved in

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DMSO were prepared to a final concentration of
200 µg/mL. These solutions were regarded as
100% concentration and were diluted 1:1 ratio with
DMSO to obtain 50% concentrations . Suspensions
of all test microorganisms were prepared in
eppendorf tubes with using sterile distilled water.
Each tube was adjusted with microbial suspension
at the final density of 0.5 McF°. Antibiotics known
to be inhibition effect on isolates were used
as a positive control and DMSO was used as a
negative control during disc diffusion method.
Vancomycin was used for S. aureus and E. faecalis,
Erythromycin for B. subtilis, Norfloxacin for E. coli,
Piperacillin-tazobactam for P. aeruginosa and
Polymyxin B for C. albicans as an antibiotic. To
obtain meaningful results, microbial inoculations
were repeated three times for each parameter.
100 µl of microbial suspension was inoculated
into respective medium for each petri dish by
spread plate technique at disc diffusion method.
Discs were placed inoculated petri dishes and
100% and 50% concentrations of crude extracts
were added 15 µl on blank discs (5 mm). After
incubation at 37°C for bacteria and 30°C for yeast
for 36 hours, the inhibition of microbial growth
was evaluated by measuring zone diameters.
Statistical Analysis
After disc diffusion method, SPSS 14 software
was used for statistical evaluation of zone
diameters (in millimeters) measurement data.
The comparison of different extracts in each
isolate and between isolates was performed with
one-way ANOVA. These results were obtained
in 95% confidence intervals. The most effective
extract was also evaluated with Duncan test for
each clinical isolate.
RESULTS
The amounts of crude residues which are
lyophilisates of four different extracts made
from Trametes versicolor fruiting bodies are
shown in Table 1. Even though the products were
obtained with different amounts of each extract,
standardization is made using the residue in the
same proportions during disc diffusion method.
The antimicrobial activity (expressed as mm)
of four different fungi extracts from Trametes
versicolor against various strains of bacteria
and yeast are summarized in Table 2. Although
all extracts generally show antimicrobial effect,
some of them were found to have antimicrobial
activity on clinical isolates.
The comparison of zone diameters showed
that there is a significant difference between
the zones when extracts of 100% and 50%
concentrations compared with positive and
negative controls (p < 0.05). Antibiotics used as
positive control were found to be most effective
on all isolates. However, Different Trametes
versicolor extracts were found to have microbial
inhibition due to the negative control by Duncan
test (Table 3). Although it was not seen a
significant difference between the concentrations
of the extracts, it was found to be more effective
in 100% concentration of extracts (p > 0.05).
There was no significant difference between
the extracts when the antimicrobial activity of the
extracts on all isolates compared with one way
ANOVA (p > 0.05). However, there were significant
differences in the comparisons for each isolate (p
< 0.05). Chloroform extract was found to have the
highest antimicrobial activity on S. aureus and
E. faecalis (Figure 1) while ethanol extract was
observed to be more effective on P. aeruginosa,
E. coli, B. subtilis and C. albicans isolates (Figure
2). Furthermore, we did not determine significant
antimicrobial activity of tested water and ethyl
acetate extracts against selected clinical isolates.
DISCUSSION
Many researchers who tested different
macromycetes extracts against bacteria and
yeast observed their antimicrobial activities.
Macromycetes extracts contain lots of active
chemical compounds which show potential
antimicrobial activities. For example, terpenes,
organic acids, benzoic acid derivates and
quinolone were isolated from some species
such as Ganoderma spp., Lentinus edodes and
Leucopaxillus albissimus showed activity against
many different species of bacteria and yeast
[1,10,15-18].
Many different species of macrofungi
extracts were tested against different bacteria

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by Suay et al., [12]. These researchers determined
and confirmed antimicrobial activity of crude
macrofungi extracts.
From the obtained results, it could be
observed that ethanol was the best solvent
for extracting antimicrobial compounds from
Trametes versicolor. This suggestion was based
on the number of organisms inhibited and
the diameter of inhibitory zones produced.
Furthermore, chloroform extract of Trametes
versicolor has effect against S. aureus and
E. faecalis known as infectious disease agents.
These results demonstrated that various active
compounds can be isolated from different fungal
extracts. Thus, fungal extraction with different
solvents should be performed in order to obtain
active compounds to inhibit each clinical isolate.
Comparison of the concentrations (100% and
50%) of extracts revealed that there is no
significant difference between the concentrations
(Table 3). According to this result, inhibition of
clinical isolates can be performed with the lesser
amount of active compounds.
In conclusion, this study has shown that
different extracts (ethyl alcohol, ethyl acetate,
chloroform and water) have been used in-vitro
Table 1. The amounts of crude residues obtained from four different Trametes versicolor extracts.
Extracts Weight (mg)
Ethyl Alcohol 98
Ethyl Acetate 83
Chloroform 124
Water 149
Tab l e 2. Antimicrobial activity of different Trametes versicolor extracts against clinical isolates tested by disc diffusion
method.
Ethanol Ethyl acetate Chloroform Water
100% 50% 100% 50% 100% 50% 100% 50%
P.aeruginosa 10.33 9.33 6.00 6.67 7. 3 3 7.33 7.0 0 7. 3 3
S.aureus 5.33 5.00 7. 33 5.67 10.00 12.33 5.00 5.00
E.faecalis 6.33 6.33 5.67 6.33 6.33 7. 3 3 5.33 5.33
B.subtilis 9.33 8.67 6.00 5.00 6.33 8.33 8.67 7.0 0
E.coli 9.67 9.33 6.33 6.33 7.0 0 8.67 9.00 8.67
C.albicans 9.00 6.67 7. 00 5.67 6.33 6.33 7. 3 3 5.00
Values are mean inhibition zone of three replicates (mm).
Table 3. The comparison of 100% (A) and 50% (B) concentration of extracts with positive and negative controls.
CONS NSubset for alpha = 0.05
1 2 3
Duncana N 72 5.01
50% 72 7.0 7
100% 72 7.21
P 72 19.83
Sig. 1.000 0.79 1.000
Means for groups in homogeneous subsets are displayed. a. Uses Harmonic Mean Sample Size = 72.000.

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to inhibit the growth of some disease causing
bacteria and yeast. It can therefore be suggested
that, some of extracts are promising antimicrobial
agents.
ACKNOWLEDGEMENTS
We would like to express our thanks to Öznur İrem Yayalar
and Nermin Nazlı Ayan for help the collection of fungi
samples and Öyküm Öztürk for attribution to detect
Trametes versicolor macroscopic identification.
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Figure 1. Evaluation of antimicrobial effect of chloroform extracts of Trametes versicolor on Staphylococcus aureus by disc
diffusion method.
Figure 2. Evaluation of antimicrobial effect of ethanol extracts of Trametes versicolor on Bacillus subtilis by disc diffusion
method.

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