Comparative In Vitro
Antimicrobial Activity of Three different Macro Fungus Extracts Against
Clinical Isolates of Gram-Negative Pathogens
Abdul Aleem1, Noor Fathima Anjum2
and Kavitha A.N.3
Department of Pharmaceutics1,
Department of Pharmaceutical Chemistry2, Department of Pharmaceutics3
Krupanidhi College of Pharmacy, Bangalore
ABSTRACT:
The
three species of edible mushrooms fistulina hepatica,
cantharellus cibarius,
lepistanuda were collected from the Western Ghats and
malnad forest area of Karnataka. Methanolic extracts
of these three edible mushrooms were used in this study. Among the isolated
mushrooms fistulina hepatica and cantharellus cibarius were showed satisfactory
results. Determination of antimicrobial activity profile of all the isolates
tested against a panel of standard pathogenic bacteria indicated that the
concentrations of bioactive components directly influence the antimicrobial
capability of the isolates. Agar diffusion assay showed considerable activity
against all bacteria. These results are discussed in relation to therapeutic
value of the studied mushrooms using the cup plate and paper disc methods. Gram
negative microorganisms studied were Escherichia
coli, Solmonella typhi, Morexella, H.influenza. Neisseria gonorrhoeae.
For
examination of pathogenic microorganisms in the cup plate and disc diffusion
method, methanolic extracts of concentrations of 250 and 500 micrograms per cup
and disc were used. All the three extracts have demonstrated significant
antimicrobial activities in both methods.
KEYWORDS: Mushrooms; Antimicrobial activity; Gram-negative
pathogen; Agar diffusion.
INTRODUCTION:
The
treatment of infectious diseases with antimicrobial agents continues to present
problems in modern day medicine with many studies showing a significant
increase in the incidence of side effects and
resistance that pathogenic microorganisms build against several
antibiotics 1,2. However, attention has been paid to extracts of
biologically active compounds isolated from mushroom species used in herbal
medicine to treat infections recently 6.
Mushroom
is a macro fungus with a distinctive fruity body, it has a cap and a stem,
grows naturally from the month of April to July in India, there are 5000
species of mushroom were recognized in that about 1800 species are identified
for medicinal use, mushroom is relatively cheep
source of high quality food protein, rich in crude fiber, low fat and good
vitamins, As well as mushroom has shown hypotensive
effect, immunomodulatory and anti tumor activity8
etc.
There
are no previous comparative studies on the antimicrobial activity of 3 selected
mushroom, but there are some reports indicating the
antimicrobial activities of some mushrooms. In the present work, the
antimicrobial activities of all three separate extracts of selected mushroom
against clinical isolates of gram-negative pathogens are described10.
MATERIAL AND
METHODS:
Among
the picked adult mushrooms, three edible species were selected from non gilled
fungal group in the month of June. Identification was done by comparing their
morphological, anatomical and physiological characteristics and monographs with
description through electronic data on identification keys of mushrooms 8, the collected mushroom species were
cleaned air dried and finally grounded to a coarse powder before extraction
separately 6.
Preparation
of methanolic extracts:
Preparation
of methanolic extracts of three different mushrooms species was done separately
based on procedures with some modifications. A fine-dried mushroom powder
sample (250 g) was extracted by stirring with 250 ml of methanol at 25°C at 150
rpm for 24 hours and filtered through Whatman no. 4 paper. The residue was then
extracted with two additional 250 ml portions of methanol. The combined
methanolic extracts were evaporated at 40°C to dryness. The organic solvent in
the extracts was removed by a rotary evaporator. For the entire analysis,
compounds of extract were dissolved in dimethylsulfoxide
(DMSO), and filter-sterilization was done through a 0.22 μm
membrane filter. Extracts were kept in the dark at 4°C for not more than 1 week
prior to use 5.
Microorganisms:
The
microgranism strains of Escherichia coli, Solmonella typhi, Morexella, H.influenza, Neisseria gonorrhoeae specimens were collected from the
institutional microbiology lab. The
numbers of strains for each microorganism were fifty, except for N. gonorrhoeae,
which were thirty strains. To identify the microorganisms, specimens were
cultured on different general, enrichment, selective and/or differential media
and underwent biochemical tests 3,4
Microbiological assay:
The
antibacterial activities of extracts were determined by the agar diffusion
method, using different reservoirs [cup (11) and disc (12)].
To
inoculate the media for assay, 1 ml from 106 cfu/ml
bacterial suspension, already harvested from surface of soybean casein digest
agar (Merck), was added to 100 ml molten Muller-Hintone
agar (Oxoid), (except for N. gonorrhoeae,
in which the culture and assay media was supplemented with 5% sheep’s blood).
Thirty ml of this inoculated medium were poured into petri
dishes and left until hardened. For the cup plate method 6 holes were punched
within the medium in each plate and 100 µl of each extract solutions (250 and
500 mcg/cup) were pipetted in triplicate into holes.
For the disc plate method, blank filter papers were placed on the surface of
the medium and impregnated with 10 µl, containing either 250 or 500 mcg of the
extracts 5.
Two
antibiotic standard discs were used as the positive controls for both methods.
Sensitivity was deduced by comparing the inhibition zone diameter produced by
the clarithromycin disc (for E. coli, Solmonella typhi, Morexella) or cefixim disc
(for H.influenza
and N. gonorrhoeae)
to those produced by extracts in both methods. The petri
dishes were incubated at 35ºC for 24 h, except for N. gonorrhoeae plates which were
incubated 48 h in the presence of 5% CO2. The inhibition zones were
measured with a caliberator and recorded as the mean
diameter of 3 replications in mm 5.
Data analysis:
Fisher’s
exact test was used to analysis data obtained from the zone of inhibition
produced by different extracts and positive controls. A p-value less than 0.05
were taken as the critical criterion for statistical significance 10.
DISCUSSION:
In
the present experiment, 3 species of wild edible mushroom isolated from the
Western Ghat forests and malnad
forest of Karnataka were evaluated for their anti microbial property. Obtained
methanolic extracts were screened for their antimicrobial efficacy. This assay
was carried out using the DMSO solvent. In this study, only two species fistulina hepatica and cantharellus cibarius were showed significant and
satisfactory results when compared to the third lepistanuda
isolates.
RESULTS:
The
results of the antibacterial activities of different mushroom extracts on the
collected strains are presented in Tables 1 and 2. Comparison of the effect of
extracts and positive controls is shown in Figures 1-5 (cup plate and disc
diffusion methods) 4.
Table 1.
Antibacterial activities of methanolic extracts of different mushroom isolates on E.coli, Solmonella typhi, Morexella, H.influenza and N. gonorrhoeae using cup plate method 6,,9.
|
Extract
|
|
E.coli (a)
|
Solmonella
typhi (a),
|
Morexella
(a),
|
H.influenza
(a),
|
N.gonorrhoeae
(b)
|
|
Amount
in cup
|
(Mcg)
|
R
|
S
|
%S
|
R/S
|
R
|
S
|
%S
|
R/S
|
R
|
S
|
%S
|
R/S
|
R
|
S
|
%S
|
R/S
|
R
|
S
|
%S
|
R/S
|
|
F. hepatica
|
250
|
16
|
31
|
65
|
0.61
|
20
|
30
|
62
|
0.62
|
25
|
26
|
52
|
0.92
|
25
|
25
|
50
|
1
|
18
|
12
|
40
|
15
|
|
|
500
|
12
|
38
|
78
|
0.31
|
17
|
33
|
65
|
0.53
|
22
|
29
|
48
|
0.72
|
23
|
27
|
55
|
0.75
|
16
|
14
|
46
|
1.13
|
|
C. cibarius
|
250
|
27
|
23
|
38
|
1.27
|
32
|
18
|
35
|
1.72
|
33
|
14
|
38
|
2.57
|
16
|
34
|
64
|
0.47
|
17
|
13
|
43
|
1.33
|
|
|
500
|
21
|
31
|
61
|
0.51
|
29
|
21
|
40
|
1.33
|
39
|
19
|
28
|
1.63
|
13
|
37
|
72
|
0.35
|
13
|
17
|
56
|
0.72
|
|
Lepistanuda
|
250
|
36
|
14
|
26
|
2.57
|
36
|
14
|
22
|
2.51
|
41
|
3
|
51
|
5.66
|
47
|
3
|
6
|
15.55
|
30
|
0
|
0
|
-
|
|
|
500
|
33
|
17
|
35
|
1.94
|
35
|
15
|
31
|
2.39
|
43
|
7
|
14
|
6.14
|
47
|
3
|
6
|
15.67
|
29
|
1
|
3.33
|
23
|
Table
2. Antibacterial activities of
methanolic extracts of different mushrooms. in terms % sensitivity of microorganisms on the
strains of E.coli(c), Solmonella
typhi (c), Morexella (c), H.influenza (c)and N. gonorrhoeace
(d) in comparison with standard discs of antibiotics using disc diffusion
method 4,6.
|
|
E. coli (c)
|
Solmonella typhi (c)
|
Morexella .(c)
|
H. influenza (c)
|
N.gonorrhoeae (d)
|
|
Disc
|
R S %S
R/S
|
R S %S
R/S
|
R S %S R/S
|
R S %S
R/S
|
R S
%S R/S
|
|
Amp
|
46 4
8.0 10.50
|
43 3
6.0 14.66
|
47 2
4.0 23.0
|
49 1
2.0 48.0
|
23 7 23.33
3.238
|
|
Amo
|
46 4 8.0
11.50
|
----------------
|
----------------
|
----------------
|
----------------
|
|
cefixim
|
31 19 38.0
1.63
|
34 16 32.0
2.12
|
45 5 10.0
9.0
|
47 3 6.0 15.66
|
27 3
10.0 9.0
|
|
clar
|
17 33 66.0
0.51
|
26 24 48.0
1.08
|
38 12 24.0 3.16
|
43 7 14.0 6.14
|
25 5 16.67 5.0
|
|
cefixim
|
41 9 18.0 4.55
|
14 16 53.3
0.87
|
----------------
|
----------------
|
----------------
|
|
Fistulina hepatica (a)
|
22 28 56.0
0.78
|
22 28 56.0
0.78
|
33 17 34.0 1.94
|
21 29 58 0.72
|
32 8 26.67
2.75
|
|
Fistulina hepatica (b)
|
16 34 68.0
0.47
|
17 33 66.0
0.51
|
30 20 48.0 1.50
|
18 32 63.0 0.56
|
19 11 36.67 1.72
|
|
Cantharelluscibarius (a)
|
26 24 48.0
1.08
|
21 29 58.0 0.72
|
36 14 28.0
2.57
|
40 10 20.0
4.0
|
18 12 40.0 1.50
|
|
Cantharelluscibarius (b)
|
24 26
52.0 0.92
|
18 32 64.0
0.56
|
34 16 32.0
2.12
|
37 13 26.0
2.84
|
14 16 53.3
30.87
|
|
 Lepistanuda (a)
Lepistanuda (b)
|
32 18
36.0 1.77
29 21 42.0 1.38
|
32 18 36.0 1.77
29 21 42.0 1.38
|
48 2 3.0 24.0
47 3 6.8 14.66
|
50 0
0 -
50 0
0 -29
|
30 0 0 -
1 3. 33 29.0
|
Figure 1 shows that different concentrations of Fistulina
hepatica (250 and 500 mcg/cup or disc) and Cantharellus cibarius extracts (500 mcg/cup or disc) have a similar
effect on E. coli, which is comparable with the positive control (clarithromycin). However, a lower antibacterial effect was
observed in comparison with positive control for Cantharellus cibarius extracts (250 mcg cup or disc)
(P<0.001 and P<0.05 respectively) and Lepistanuda
extracts (250 and 500 mcg/cup or disc) (P<0.001) 4,5.
Figure 1. Antibacterial activity of methanol extracts of
different mushrooms samples on 50 isolates of E. coli. Sensitivity was deduced
by comparing the inhibition zone diameter produced by clarithromycin
disc and the extracts using cup plate and disk method:
(*:
p<0.05. ***: p<0.001. Fisher test) 4, 5.
Figure 2 shows that a higher concentration of Fistulina hepatica extract (500
mcg/cup or disc) exhibits a significantly greater antibacterial effect, than
the positive control on Solmonella typhi (P<0.05). On the other hand a lower concentration
(250 mcg/ cup or disc) showed no significant difference, compared with the
positive control (P>0.05). There were no significant differences between the
effect of different concentrations of Cantharellus cibarius extracts (250 and 500 mcg/cup or disc) and the
positive control (P>0.05). Different concentrations of Lepistanuda
extracts (250 and 500 mcg/cup) have a lower degree of antibacterial activity
than the positive control, and this difference was significant (P<0.01 and P<0.05
respectively). However, there is no significant difference between the effect
of Lepistanuda extract and the positive control
(P>0.05) in the disc method 4,6.
Figure 2. Antibacterial activity of methanol extracts of
different mushrooms samples on 50 isolates of moraxella.Sensitivity
was deduced by comparing the inhibition zone diameter produced by clarithromycin disc and the extracts using cup plate and
disk method: (*: p<0.05. **: p<0.001. Fisher test) 4, 5.
Figure 3 shows that there are significant differences between the effect of Fistulina
hepatica (250, 500 mcg/cup) and Cantharellus cibarius extracts (500, mcg/cup) with the positive
control (P<0.001 and P<0.001, P<0.05 respectively) on Morexella. However, the effect of Lepistanuda
extract (250 mcg/cup) is significantly lower than the positive control
(P<0.001). The antibacterial effect of Fistulina
hepatica extract (500 mcg/disc) on Morexella is
significantly higher than the positive control (P<0.05). Fistulina
hepatica (250 mcg/disc) and Cantharellus cibarius (250, 500 mcg/disc) extracts have a similar
effect in comparison with the positive control.
Lepistanuda extract (250 and 500 mcg/disc)
have lower effects in comparison with the positive control (P< 0.001,
P<0.05 respectively) 4,7.
Figure 3. Antibacterial activity of methanol extracts of
different mushrooms samples on 50 isolates of salmonella typhi.
Sensitivity was deduced by comparing the inhibition zone diameter produced by clarithromycin disc and the extracts using cup plate and
disk method: (*: p<0.05. ***: p<0.001. Fisher test) 4, 5.
Figure 4 shows the effects of extracts and positive control on P.aeruginosa, Fistulina hepatica
(250 and 500 mcg/cup or disc) and Cantharellus cibarius (250 and 500 mcg/cup) extracts have a
significantly higher effect than the positive control (P<0.001). The effect
of Cantharellus cibarius (250
and 500 mcg/cup) and Lepistanuda extract (250 and 500
mcg/disc) extracts is similar to the positive control. Lepistanuda
extracts in the disc method do not have any effect on P.aerogenosa
(p<0.001) 4,8.
Figure 4.
Antibacterial activity of methanol extracts of different mushrooms samples on
50 isolates of salmonella typhi. Sensitivity was deduced
by comparing the inhibition zone diameter produced by cefixim
disc and the extracts using cup plate and disk method:
(*:
p<0.05. ***: p<0.001. Fisher test)4,5.
Figures 5 shows nearly the same effect of Fistulina
hepatica (250 and 500 mcg/cup) and Cantharellus cibarius (250 and 500 mcg/cup) extracts on N. gonorrhoeae, comparable with the positive control. The
antibacterial effect of Fistula hepatica extracts (250 and 500 mcg/disc) is
lower than the positive control (P<0.001 and P<0.05 respectively), while the Cantharellus cibarius extracts had almost no effect on N. gonorrhea
(P<0.001) 4,9.
5. Figure Antibacterial activity of methanol extracts of
different mushrooms samples on 30 isolates of N.gonorrhoeae.
Sensitivity was deduced by comparing the inhibition zone diameter produced by cefixim disc and the extracts using cup plate and disk
method:
(*:
p<0.05. ***: p<0.001. Fisher test) 4
ABBREVEATIONS:
|
A1
|
Fistulina hepatica 250 mcg/cup
|
|
A1
|
Fistulina hepatica 250 mcg/disc
|
|
A2
|
Fistulina hepatica 500 mcg/cup
|
|
A2
|
Fistulina hepatica 500 mcg/disc
|
|
B1
|
Cantharellus Cibarius 250 mcg/cup
|
|
B1
|
Cantharellus Cibarius 250 mcg/disc
|
|
B2
|
Cantharellus Cibarius 500 mcg/cup
|
|
B2
|
Cantharellus Cibarius 500 mcg/disc
|
|
C1
|
Lepista nuda 250 mcg/cup
|
|
C1
|
Lepista nuda 250 mcg/disc
|
|
C2
|
Lepista nuda 500 mcg/cup
|
|
C2
|
Lepista nuda 500 mcg/disc
|
|
Clarithromycin
|
10
mcg/ disc
|
|
Cefixim
|
30
mcg/disc
|
ACKNOWLEDGEMENT:
This
work is dedicated to my beloved parents
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