In vitro Antibacterial Activity of Himalayan Lichenized Fungi

The increase in the new strains of multi drug-resistant pathogens, the standard drugs have become less effective, thereby increasing the demand for the search of novel natural bioactive compounds from lichenized fungi. Despite rich diversity of lichenized fungi in Kumaon Himalaya, only a few of them have been screened for their biological activities. Present communication deals with antimicrobial activity of ethyl acetate extracts of Everniastrum cirrhatum, Usnea longissima, Flavoparmelia caperata and Ramalina conduplicans against five pathogenic bacteria. Among these, U. longissima and F. caperata extracts have revealed significant activity against all the bacteria while U. longissima was more active against Escherichia coli (13.0 mm; MIC=7.5 mg/mL) and Bacillus subtilis (11.6 mm; MIC=7.5 mg/mL). The F. caperata extract was active against E. coli (15.3 mm; MIC=15 mg/mL). Hence, there is an interest in the potential uses of antibiotics derived from lichenized fungi for pharmaceutical purposes. In vitro Antibacterial Activity of Himalayan Lichenized Fungi


Introduction
India is a rich centre of biodiversity contributing nearly 15% of the 13,500 species of lichenized fungi [1]. Of these, several lichenized fungi of the Himalayan region are said to effectively cure dyspepsia, bleeding piles, bronchitis, scabies, stomach disorders and many disorders of blood and heart [2][3][4]. Flavoparmelia caperata is part of Ayurvedic and Unani medicines under the name 'Chharila' as carminative and aphrodisiac and considered useful in treatment of intestinal worms and burns [5], Everniastrum cirrhatum relieves headache [6,7]. Ramalina conduplicans was put on wounds to stop bleeding, cure jaundice [8,9]. U. longissima has been used in the treatment of bone fractures and strains, and ulcers [10,11]. It is also used as a simple drug to stimulate menstruation or induce abortion. Reports on floristic, monographic, revisionary, pollution monitoring studies of lichenized fungi exist but little attention has been paid to the detailed chemical analysis and biological activity of lichenized fungi native to high altitude region of Himalaya.
The aim of present study is to investigate the antibacterial activity of extracts from Himalayan lichenized fungi viz. E. cirrhatum, U. longissima, F. caperata and R. conduplicans.

Plant materials
Lichenized fungi were collected from Kumaun (Uttarakhand) Himalaya during March 2015. The lichen specimens were identified with the help of published flora [17]. Voucher specimens have been deposited in Department of Botany, Kumaun University, and Almora.

Preparation of lichen extracts
Each lichen sample was washed to remove debris, dried, ground to powder and stored in a sterile glass bottle in the refrigerator. The powder (2-3 g) was added to 10 ml of ethyl acetate and left for 10 days at room temperature. The crude extract was filtered with Whatman No. 42 and solvent was evaporated to obtain dried extract. The extract was stored in refrigerator at ~4ºC.

Bacterial strains
The in vitro antibacterial activity was evaluated against pathogenic and clinically isolated 5 bacterial strains Pseudomonas aeruginosa (

Antibacterial activity by disc diffusion
Evaluation of antibacterial activity of lichenized fungi extracts was carried out by disc-diffusion method [18]. The samples were dissolved

Results and Discussion
The antibacterial activity data of lichenized fungal extract against five bacteria are presented in Tables 1 and 2. The maximum zone of inhibition was recorded against E. coli (19.0 mm), K. pneumoniae (16.3 mm) and B. subtilis (15.3 mm). Activity data revealed U. longissima and F. caperata to be more active against almost all the bacteria. U. longissima showed higher activity against Gram-negative bacteria E. coli (13.0 mm; MIC=7.5 mg/mL) and Gram-positive bacteria B. subtilis (11.6 mm; MIC=7.5 mg/mL), while F. caperata was more active against E. coli (15.3 mm; MIC=15 mg/mL) followed by K. pneumoniae (13.6 mm; MIC=15 mg/mL). The extract of E. cirrhatum was effective against E. coli (19.0 mm; MIC=15 mg/mL). The extract of R. conduplicans was found more active against B. subtilis (15.3 mm; MIC=15 mg/mL). It is interesting to note that the growth of S. typhimurium remained unaffected by any of the lichenized fungal extracts. The activity of extracts was noticed in the following descending order E. coli>K. pneumonia>B. subtilis>P. aeruginosa>S. typhimurium.
The inhibition data were subjected to PCA and HCA analysis (Figures 1 and 2). Group I, composed of the Gram-negative bacteria (E. coli and K. pneumonia), is characterized by high sensitivity to the extracts (13-19 mm). Group II is represented by Gram-positive bacteria B. subtilis distinguishable in the PCA as a distinct group (8.3-15.3 mm). Group III, which constituents Gram-negative bacteria P. aeruginosa and S. typhimurium was characterized by relatively resistant to all the extracts, especially S. typhimurium strain which showed high resistance to the extracts (<7 mm).
Secondary metabolites derived from natural products possess various benefits including antimicrobial properties against pathogenic and spoilage microbes. Major groups of compounds that are responsible for antimicrobial activity from plants include phenolics, phenolic in Dimethyl Sulfoxide (DMSO) to prepare desired concentrations. Inoculums of the microbial strains (1 × 10 6 CFU/mL) were plated using sterile swabs into petri dishes (90 mm) with 20 mL of Nutrient Agar, and then discs of Whatman paper-42 were soaked in sample solution (15 mg/mL) and placed onto inoculated petri dishes. Standard antibiotic streptomycin (15 mg/mL) was used as positive control and DMSO as negative control. The petri dishes were pre-incubated for 3 h at room temperature, allowing the complete diffusion of the samples and then, incubated at 37ºC ± 1ºC for 24 h [19]. The zones of inhibition were measured.

Antibacterial activity evaluation by agar dilution method
The evaluation of MICs was done using the agar dilution method with slight modifications described by the National Committee for Clinical Laboratory Standards [20]. Equal volume of each microbial strain culture, containing approximately 1 × 10 6 CFU/mL, was applied onto MHB supplemented with the extract at concentration ranging from (0.46-30 mg/mL) in tubes. These cultures were then incubated at 37°C for 24 h then cultures were finally inoculated on nutrient agar media to determine the growth of bacteria. Controls of bacteria without the extract were also applied. The concentration at which no visible growth was observed is considered as MICs.

Statistical analysis
Data were subjected to one-way Analysis of Variance (ANOVA) and the means were compared by Duncan Multiple Range tests at a level of significance of p<0.05 using SPSS 16.0 statistical software. The Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were performed using PAST statistical computer software package for evaluating correlation between antibacterial activity and extract.

Conclusion
There has been recurrence on the natural product chemistry researches in the recent years for new bioactive molecules that can replace synthetic additives and their potential use in the food and pharmaceutical industries. The results of the present study showed that some of the lichenized fungi extracts possess significant antibacterial activity. Further investigations of antimicrobial potential of these particular lichenized fungi in relation to human pathogens can be of pharmacological importance. Hence, there is an interest in the potential uses of antibiotics derived from lichenized fungi for the pharmaceutical industry.