Parmotrema nilgherrense: potential antimicrobial activity against drug resistant pathogens




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Parmotrema nilgherrense: potential antimicrobial activity against drug resistant pathogens
Shaily Javeria1, Sushil Kumar Shahi1*, Mamta Patra Shahi2, and DK Uperti3

1Bio-resource Tech Laboratory, Microbiology Department, CCS University, Meerut-250005, India

2Microbiology Department, Meerut Institute of Engineering and Technology, Meerut--250005, India

3Lichen Laboratory, National Botanical Research Institute, Lucknow-226001, India.
*Corresponding author: shahi.sk@rediffmail.com

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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.

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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 micro-organisms (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

    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.

    1. 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.

    1. 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.

    1. 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 pre-sterilized 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.



    1. 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.

  1. 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.




  1. 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, gram-negative 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.



  1. 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

Thanks are due to Head, Department of Microbiology, Chaudhary Charan Singh University, Meerut for providing the facilities.



References

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Table 1: Antibacterial screening of P. nilgherrense extracts against drug Table 2: MIC of P. nilgherrense

resistant bacterial strains against bacterial pathogens


Drug resistant

bacterial strains

Bacterial Growth Inhibition % (BGI℅).

Ethyl acetate

Methanol

Benzene

Acetone

P. aeruginosa

100

70

60

80

P. fluorescens

100

46

40

100

Proteus vulgaris

100

60

100

100

Shegilla flexneri

100

100

70

70

Klebsiella pneumoniae

100

60

60

60

Salmonella typhi

70

60

60

60




Drug resistant

bacterial strains

MIC (µl)

P. fluorescens

3.125 × 10-5

Proteus vulgaris

3.125 × 10-5

Shegilla flexneri

6.25 × 10-4

K. pneumonia

12.5 × 10-3

P. aeruginosa

0.78125 × 10-7

Table 3: Resistant profile of 13 antibiotics against tested bacterial strains


Antibiotics

Inhibition zone of Bacteria (mm)

S. typhi

P. aeruginosa

P. fluorescens

P. vulgaris

S. flexneri

K. pneumoniae

Ca

8.3±0.48

1.0±0.40

0.5±0.25

0.3±0.25

01±0.70

5.5±0.65

Co

06±0.40

0.5±0.50

1.0±0.40

0.5±0.29

0.8±0.75

7.3±0.41

G

7.5±0.65

9.8±0.86

7.2±0.48

5.5±1.60

7.8±0.75

06±0.41

Ci

15±1.22

7.5±0.48

0.3±0.25

1.3±0.75

1.5±0.86

6.8±1.11

Cf

16.7±0.48

06±0.41

09±0.41

16.8±0.30

07±0.71

13.8±1.25

Mr

15±1.10

10.5±0.65

6.3±2.10

10.8±0.48

12.5±1.04

12.5±0.87

Tb

7±0.41

7±0.71

10.5±0.29

12±0.41

11.5±0.65

04±0.41

Mo

5.3±0.48

13±0.71

7.8±0.48

7.5±1.07

07±0.91

08±0.91

Of

1.3±0.33

4±0.71

7.3±0.48

8±1.35

11.7±0.63

8.5±0.65

Sc

8.5±0.65

2.3±0.47

11.5±0.29

12.3±1.40

7.5±1.19

7.5±0.65

Le

11±0.41

6.3±0.48

10.8±0.48

15.7±0.85

11.6±0.48

11.5±0.65

I

10.7±0.48

8±0.41

11.3±0.48

12.3±0.60

10.8±0.60

7.8±0.48

Ce

7.5±0.65

0.8±0.48

0.5±0.29

0.8±0.75

1.5±1.19

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







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