Antibacterial Activity of Certain Ferns Against Multi Drug Resistant Organisms

Kathakali Nath1, Anupam Das Talukdar2, Mrinal Kanti Bhattacharya3*, Deeshikha Bhowmik1, Shiela Chetri1, Debarati Choudhury2, Abhijit Mitra2 and Amitabha Bhattacharjee1 1Department of Microbiology, Assam University, Silchar – 788011, Assam, India 2Department of Life Science and Bio informatics, Assam University, Silchar – 788011, Assam, India 3Department of Botany and Biotechnology, Karimganj College, Karimganj – 788710, Assam, India


Introduction
The effectiveness of conventional and current drugs is getting limited due to the rapid development of resistance by the pathogenic bacteria which often leads to treatment failure and subsequently leaving the clinicians with fewer antibiotics 1 . This increasing resistance to antibiotics has thus necessitated the need to search for new antibacterial agents.
Staphylococcus aureus responsible for various post-operative infections, endocarditis etc. which was once easily treatable, but in the recent years emerged out to be resistant against methicillin drug and still emerging resistance against vancomycin.
Gram negative bacterium such as Escherichia coli is a normal flora in human intestine but can cause lower urinary tract infection, coleocystis or septicaemia 2

Extract Preparation
The leaf samples were washed thoroughly 2 3 times with running tap water and once with sterile water, air dried, powdered and used for extraction. Fifty grams of each of the air dried and coarsely powdered plant material was extracted successively with 500 ml each of ethyl acetate, methanol and water in the increasing order of their polarity using a soxhlet evaporator 13 . Water extract was prepared by boiling (3-4 hours) in a hot water bath.

Antibacterial Activity of Fern Extract
Antibacterial activity of aqueous and solvent extracts was determined by agar well diffusion method. Inoculum containing 105cfu/ml (0.5Mc

Minimum Bactericidal Concentration of Fern Extract
Samples from the tubes used in the MIC assays, which did not show any visible growth after a period of incubation were sub-cultured onto a freshly prepared nutrient medium 15 . The minimum bactericidal concentration was taken as the lowest concentration of the extract that did not yield a single colony on the nutrient agar plate after 24 hours incubation period.

Synergism of Crude Extract with Conventional Antibiotics
A bacterial suspension was prepared and was standardized to give a resultant concentration of 1x10 8 cfu/ml. Bacteria was then seeded onto Mueller Hinton agar plates by swabbing with a sterile swab.
To the seeded plates antibiotic disc were placed and 6µl of the plant extract was added on the antibiotic disc. The same method was repeated for antibiotic disc alone and the plant extract alone. Then the plates were allowed to stand for 30 minutes and

Minimum Inhibitory Concentration
The minimum inhibitory concentration of Cyathea brunoniana and Pseudomonas aeruginosa was found to be 400mg/ml (Table 2).

Minimum Bactericidal Concentration
The minimum bactericidal concentration (Table  3) of the two ferns viz. Cyathea brunoniana and Pronephrium nudatum was found to be 200mg/ml.

Synergistic Activity
The in vitro synergism of ethyl acetate extract of Cyathea brunoniana and Pronephrium nudatum      21  Pronephrium nudatum an ethno-medicinally imperative fern, used for the treatment of pyorrhoea 10,23 . It has been seen in the present experiment that of the three extracts i.e., ethyl acetate, methanol and water, crude ethyl acetate frond of the fern was active against methicillin resistant Staphylococcus aureus (Table 1) with minimum inhibitory concentration of 400mg/ml ( Table 2). The present result may be validated by the antifungal activity of the plant against Aspergillus niger 24 . Increased activities of the antibiotics, which are used in Staphylococcal infection, were observed resulting from the synergistic effect of antibiotic and crude extract (Figure 1).

Conclusion
From the present study it is evident that the crude