Parmotrema nilgherrense: potential antimicrobial activity against drug resistant pathogens
Shaily Javeria1, Sushil Kumar Shahi1*, Mamta Patra Shahi2, and DK Uperti3
Bio-resource Tech Laboratory, Microbiology Department, CCS University, Meerut-250005, India
2
Microbiology Department, Meerut Institute of Engineering and Technology, Meerut--250005, India
3
Lichen Laboratory, National Botanical Research Institute, Lucknow-226001, India.
1
*Corresponding author: [email protected]
_____________________________________________________________________________________________________________________
Abstract
The antimicrobial properties of lichen Parmotrema nilgherrense was evaluated by using different solvents viz.
acetone, methanol, ethyl acetate and Benzene against 6 drug resistant bacterial strains viz., Klebsiella pneumoniae,
Salmonella typhi, Pseudomonas aeruginosa, Proteus valgaris, Shigella flexneri and Pseudomonas fluorescens. The
present study ethyl acetate extract exhibit maximum antibacterial activity against Pseudomonas aeruginosa (100℅),
Pseudomonas fluorescens (100℅), Proteus vulgaris (100℅), Shegilla flexneri (100℅), Klebsiella pneumoniae
(100℅) and Salmonella typhi (70%) and methanol extract exhibited minimum activity against Salmonella typhi
(46℅). The MIC of the ethyl acetate extract of lichen was found to be 0.78125×10 -7 µl against Pseudomonas
aeruginosa, while 3.125×10-5 µl against Pseudomonas fluorescens and Proteus vulgaris. The MIC of Shegilla
flexneri wa found to be 6.25×10-4 µl and the MIC of Klebsiella pneumoniae was found to be 12.5× 0-3 µl.
Key words: - Antibacterial activity, Parmotrema nilgherrense, different solvent extracts, drug resistant microbes.
_____________________________________________________________________________________________
1. Introduction
The challenge for today’s pharmaceutical industry
lies in the discovery and development of new
pharmacological active molecules (Behera et al.,
2005). According to a report issued by the World
Health Organization (WHO), plant species that are
currently used for medicinal purposes are about
20,000. Lichens have been used for medicinal
purposes throughout the ages, such as Cetraria
islandica, (Iceland moss), Lobaria pulmonaria and
Cladonia species were reported to be effective in the
treatment of pulmonary tuberculosis (Vartia, 1973).
Lichens are the symbiotic association of fungi
(mycobiont) and algae (phycobiont). They are an
important food for many animals, including man
(Richardson, 1988). They play very important role in
many pharmaceutical industries. Lichen forming
fungi produce antibiotic secondary metabolites that
protect many animals from pathogenic microorganisms (Lawrey, 1989). Plant product drugs and
herbal remedies have been employed since
prehistoric times to treat human and animal diseases
and several countries still rely on plants and herbs as
the main sources of drugs (Ogbonnia et.al 2008). A
number of investigators have studied the antibacterial
and antifungal activity of lichens. The first study of
the antibiotic properties of lichens was carried out by
Burkholder et al. (2008); Burkholder (1944). Vartia
(1973) reported antibacterial activity for several
lichens and other researchers have since then studied
the antibacterial activity several lichens against gram
positive and gram negative bacteria. Various workers
reported antifungal activity as well as antipyretic
properties of lichen (Tolpysheva and Yu, 1984;
Halama and Haluwin, 2004; Turk et al., 2003; 2005).
Lichens synthesize numerous metabolites which
comprise amino acid derivatives, sugar alcohols,
aliphatic acids, macrocyclic lactones, mono-cyclic
aromatic
compounds,
quinones,
chromones,
xanthhones, dibenzofuranes, depsides, depsidones,
depsones, terpenoids, steroids, carotenoids and
diphenyl ethers (Clix et al., 1984; Fiedler et al.,
1986). The development and spread of microbial
resistance to available antibiotics has prompted
investigators to study antimicrobial substances from
other sources. Owing to pronounced antimicrobial
activity of some of their secondary metabolites,
lichens are attracting much attention among
researchers as significant new sources of bioactive
substance (Ingolfsdottir et al., 1997; Hostettmann et
al., 1997). In this context the purpose of the present
study was to conduct in vitro evaluation of the
antibacterial activity of various lichen extracts viz.
ethyl acetate, methanol, acetone, benzene and ethanol
of Parmotrema nilgherrense against drug resistant
bacteria.
2. Materials and Methods
2.1. Collection and Identification
Lichen was collected from Nainital (Utrakhand) area
(near Kaichi temple) and dried at room temperature
for 48 hrs. Collected lichen was identified by the help
of Dr. D.K. Upreti, lichen laboratory, NBRI,
Lucknow.
2.2. Preparation of extracts
The air dried lichen powder (4 g) was dipped in 8 ml
of each solvent (acetone, methanol, ethyl acetate and
benzene) separately for 48 hrs at room temperature.
The extracts were than filtered using Whatman filter
paper (No.1) and evaporated to dryness under
reduced pressure. The obtained residues, 8 ml of
Dimethly sulfoxide (DMSO) were added in each
filtrate for final concentration. The obtained solutions
were kept at room temperature for further
investigation.
2.3. Collection of microorganisms
Disease causing bacteria viz. Klebsiella pneumoniae
(MTCC 109), Salmonella typhi (MTCC 32216),
Pseudomonas aeruginosa (MTCC 2581), Proteus
valgaris (MTCC 744), Shigella flexneri (MTCC
1457), Pseudomonas fluorescens (MTCC 1748) were
obtained from Microbial Type Culture Collection
(MTCC), IMTECH, Chandigarh. All cultures were
kept on NAM (Nutrient agar media) at 40C for further
investigation.
2.4. Antimicrobial screening of collected lichen
extracts against test pathogens
The antimicrobial screening of the pathogenic
bacterial strains were carried out following the
Poisoned food technique (Grover and Moore, 1962)
with slight modification (Shahi et al., 1999). Lichen
extracts of 0.2 ml were mixed in 4.5 ml of presterilized sabouraud dextrose broth (pH + 5.6) and
than 0.5 ml bacterial culture suspension were added.
In control 0.2 ml DMSO (in place of the lichen
extract) were used in the medium in appropriate
amount. Inoculated culture tubes were incubated for
24 hour at 370C. After incubation, sterile disc of 6
mm (Hi Media) were dipped in to the broth (treated
as well as control) separately and aseptically
inoculated on the agar surface of the SDA medium in
plates. Inoculated petriplates were incubated at 370C.
Observations were (which were mean value of five
replicates in each case) recorded after 24 hr of
incubation. Percentage of Bacterial Growth Inhibition
(BGI%) was calculated as per formula.
BGI (%) dc – dt /dc × 100
Where, dc: colony diameter in control; dt: colony
diameter in treatment
2.5 Determination of MIC of extracts by microtiter
plate assay
MIC (minimum inhibitory concentration) expressed
as the lowest dilution, which inhibited growth judged
by lack of turbidity in the tube. A broth micro
dilution assay was adopted using 96 well micro titer
plates with resazurin. It was carried out to assess the
microbial growth and determine the minimal
inhibitory concentration (Sarker et al., 2007). The
resazurin (oxydation-reduction indicator) solution
was prepared by dissolving a 270 mg tablet in 40 ml
of sterile distilled water. A sterile 96 well microtiter
plate was taken for the test. 50 μl of test extracts were
pipetted into the first row of the microtiter plate A1.
Wells from A2 to H2 till A12 to H12 were dispensed
with 50 μl of nutrient broth. 50 μl of test extract was
transferred from test solution (A1-H1) to next wells
(A2-H2) and so on to create serial dilutions. 30μl of
the test culture were mixed in serially descending
concentrations to each well, from A2 to H2 till A12
to H12. In last 20 μl of resazurin solution was added
in all tested as well as control set. A11, A12 and H11,
H12 served as controls, 50 μl of DMSO was used in
place of extracts. The plates were incubated at 37oC
for 24 hours. The colour change was then assessed
visually. Any colour changes from purple to pink or
colourless were recorded as positive. The lowest
concentration at which colour change occurred was
taken as the MIC value. The average of two values
was calculated.
2.5. Determination of the strains sensitivity to
antibiotics
The susceptibility of the microbial strains to different
antibiotics were tested using disc diffusion method
Aboaba and Efuwape, 2001; Reynolds and
Martindale, 1996). Antimicrobial agents from
different classes of antibiotics (Hi media) were used
for susceptibility test.
3.
Results
The antibacterial activity of acetone, methanol, ethyl
acetate and benzene extracts of the lichen against the
tested microorganisms was estimated on the basis of
the percentage of inhibition of pathogenic strains, the
results were presented in Table 1.
The ethyl acetate extract showed minimum
activity against Salmonella typhi (70℅). The
maximum
activity
was
observed
against
Pseudomonas fluorescence (100℅), Proteus vulgaris
(100℅), Pseudomonas aeruginosa (100℅), Shegilla
flexneri (100℅) and Klebsiella pneumonia (100℅).
The Acetone extract showed minimum activity
against Salmonella typhi (60℅), Klebsiella
pneumoniae (60℅) Shegilla flexneri (70℅),
Pseudomonas aeruginosa (70℅) and Pseudomonas
fluorescens (86.6℅). The maximum activity was
observed against Proteus vulgaris (100℅). The
Benzene extract was showed the minimum activity
against Salmonella typhi (60℅), Klebsiella
pneumoniae (60℅), Shegilla flexneri (70℅),
Pseudomonas aeruginosa (60℅) and Pseudomonas
fluorescens (40℅). The maximum activity was
observed against Proteus vulgaris (100℅). The
Methanol extract was showed the minimum activity
against Salmonella typhi (60℅), Klebsiella
pneumoniae (60℅), Pseudomonas aeroginosa (70℅),
Proteus vulgaris (60℅) and Pseudomonas
fluorescens (46℅). The maximum activity was
observed against Shegilla flexneri (100℅).
The MIC is the lowest concentration at
which the bacterial growth is inhibited by the lichen
extracts and could be judged by pink well showing
reduction of resazurin in the dilution series. The
results obtained after MIC determination are
presented in Table 2. The MIC of the ethyl acetate
extract of lichen was found to be 0.78125×10 -7 µl
against Pseudomonas aeruginosa, while 3.125×10-5
µl against Pseudomonas fluorescence and Proteus
vulgaris. The MIC of Shegilla flexneri was found to
be 6.25 × 10-4 µl and the MIC of Klebsiella
pneumoniae was found to be 12.5×10-3 µl.
The sensitivity of tested bacterial strains
against
Impenem
(I),
Meropenem
(Mr),
Ciprofloxacin (Cf), Tobramycin (Tb), Moxifloxacin
(Mo),
Ofloxacin
(Of),
Sparfloxacin
(Sc),
Levofloxacin (Le), Ceftazidime (Ca), Cephotaxime
(Ce), Gentamicin (G), Co-Trimoxazole (Co),
Ceftriaxone (Ci) and Gatifloxacin (Gt) (Himedia
Labs, Mumbai, India) are shown in Table 3.
4.
Discussion
The present study confirmed the presence and
absence of antibacterial substance in the extracts of
Parmotrema nilgherrense against 6 pathogenic
bacteria viz. Pseudomonas aeruginosa, Pseudomonas
fluorescens, Proteus vulgaris , Shegilla flexneri,
Klebsiella pneumoniae and Salmonella typhi. The
differences of antibacterial activity between lichen
extracts were dependent upon the solvent used for
extraction. Earlier studies did not find any
antibacterial activity of lichens extract in water
(Yilmaz et al., 2004; Tay et al., 2004). The probable
reason for this is that majority of active substances
present in the thalli of lichens are either insoluble are
poorly soluble in water. But in the present study ethyl
acetate extraction for great inhibition of test microbes
compared to other extracts. Rowe et al. (1989)
reported that the Turkey lichens, Evernia prunastri,
Pseudovernia furfuracea and Alectoria capillaris
were active against gram-positive bacteria and the
Candida albicans. All these studies indicate that the
lichens inhibit mostly gram-positive bacteria. Even
though most of the lichens have been reported to be
active against gram-positive bacteria, the actual
factors that affect the selective antibiotic activity
have not been identified. However, this may be
attributed to the biochemical and physiological
variations between gram-positive and gram-negative
bacteria. If so, it is of great interest to note that
Parmotrema nilgherrense inhibited the growth of
both gram positive and gram negative bacteria.
Studies of Burkholder et al. (1994) on 100
species of American lichens showed that 52% of
lichens were active only against Gram positive
bacteria. Vartia (1949; 1950) were reported that 75 of
149 Finnish lichen species inhibited the growth of
gram positive and gram negative bacteria. Silva et al.
(1986) also observed that most of the Brazilian
lichens were active against gram positive bacteria.
Rankovic et al. (2007) screened the
antimicrobial properties of acetone, methanol and
aqueous the lichens Lasallia pustulata, Parmelia
sulcata, Umbilicaria crustulosa and extracts of
Umbilicaria cylindrica. Antimicrobial activities of
the extracts of different lichens were estimated by the
disc diffusion test for gram-positive bacteria, gramnegative bacteria and fungal organisms, as well as by
determining
the
MIC
(minimal
inhibitory
concentration). The obtained results showed that the
acetone and methanol extracts of Lasallia pustulata,
Parmelia sulcata and Umbilicaria crustulosa
manifested antibacterial activity against the majority
of bacterial strains tested, in addition to selective
antifungal activity. The MIC of lichen extracts was
lowest (0.78 mg/ml) for the acetone extract of
Lasallia pustulata against Bacillus mycoides.
Aqueous extracts of all of the tested lichens were
inactive. Extracts of the lichen Umbilicaria
cylindrica manifested the weakest activity, inhibiting
only three of the tested organisms.
Thus, further study is necessary to
characterize the chemical constituents of the extracts
from lichen samples. In addition, the data may also
suggest that the extracts of lichen species tested
possess compounds with antibacterial properties,
which require further studies to determine
antimicrobial agents for therapy of infectious
diseases in human and plant diseases.
5.
Conclusions
Out of four extracts of lichen Parmotrema
nilgherrense, ethyl acetate extract showed maximum
antimicrobial activity against 6 drug resistant bacteria
viz. Pseudomonas aeruginosa, P. fluorescens,
Proteus vulgaris, Shegilla flexneri, Klebsiella
pneumoniae and Salmonella typhi. In future it can be
used for the development of antibacterial drug against
multidrug resistance bacteria.
Acknowledgment
11.
Thanks are due to Head, Department of
Microbiology, Chaudhary Charan Singh University,
Meerut for providing the facilities.
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Table 1: Antibacterial screening of P. nilgherrense extracts against drug
resistant bacterial strains
Drug resistant
bacterial strains
P. aeruginosa
P. fluorescens
Proteus vulgaris
Shegilla flexneri
Klebsiella pneumoniae
Salmonella typhi
Table 2: MIC of P. nilgherrense
against
bacterial
pathogens
Bacterial Growth Inhibition % (BGI℅).
Ethyl acetate Methanol Benzene Acetone
100
70
60
80
100
46
40
100
100
60
100
100
100
100
70
70
100
60
60
60
70
60
60
60
Drug resistant
bacterial strains
P. fluorescens
Proteus vulgaris
Shegilla flexneri
K. pneumonia
P. aeruginosa
MIC (µl)
3.125 × 10-5
3.125 × 10-5
6.25 × 10-4
12.5 × 10-3
0.78125 × 10-7
Table 3: Resistant profile of 13 antibiotics against tested bacterial strains
Antibi
otics
Ca
Co
G
Ci
Cf
Mr
Tb
Mo
Of
Sc
Le
I
Ce
S. typhi
8.3±0.48
06±0.40
7.5±0.65
15±1.22
16.7±0.48
15±1.10
7±0.41
5.3±0.48
1.3±0.33
8.5±0.65
11±0.41
10.7±0.48
7.5±0.65
P. aeruginosa
1.0±0.40
0.5±0.50
9.8±0.86
7.5±0.48
06±0.41
10.5±0.65
7±0.71
13±0.71
4±0.71
2.3±0.47
6.3±0.48
8±0.41
0.8±0.48
Inhibition zone of Bacteria (mm)
P. fluorescens
P. vulgaris
0.5±0.25
0.3±0.25
1.0±0.40
0.5±0.29
7.2±0.48
5.5±1.60
0.3±0.25
1.3±0.75
09±0.41
16.8±0.30
6.3±2.10
10.8±0.48
10.5±0.29
12±0.41
7.8±0.48
7.5±1.07
7.3±0.48
8±1.35
11.5±0.29
12.3±1.40
10.8±0.48
15.7±0.85
11.3±0.48
12.3±0.60
0.5±0.29
0.8±0.75
S. flexneri
01±0.70
0.8±0.75
7.8±0.75
1.5±0.86
07±0.71
12.5±1.04
11.5±0.65
07±0.91
11.7±0.63
7.5±1.19
11.6±0.48
10.8±0.60
1.5±1.19
K. pneumoniae
5.5±0.65
7.3±0.41
06±0.41
6.8±1.11
13.8±1.25
12.5±0.87
04±0.41
08±0.91
8.5±0.65
7.5±0.65
11.5±0.65
7.8±0.48
6.5±0.65
(I) Impenem, (Mr) Meropenem, (Cf) Ciprofloxacin, (Tb) Tobramycin, (Mo) Moxifloxacin, (Of) Ofloxacin, (Sc) Sparfloxacin, (Le)
Levofloxacin, (Ca) Ceftazidime, (Ce) Cephotaxime, (G) Gentamicin, (Co) Co-Trimoxazole and (Ci) Ceftriaxone