Isolation of Fungi from Mangrove Ecosystem of Mumbai and Evaluation of their Antibacterial Potential

In the current study, fungi from the mangrove ecosystem of Mumbai were isolated and their metabolites were screened for antibacterial potential. Two weeks old broth and mycelium were extracted using chloroform and methanol. Antibacterial property of solvent extracts was evaluated at various concentrations (2 - 10 µg/ml) against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumonia and Escherichia coli, by well diffusion method. Fungi isolated were identified as Aspergillus niger, Aspergillus flavus, Trichoderma harzianum, Cylindrocladium scoparium and Colletotrichum wuxiense . Results revealed that broth solvent extracts of isolates inhibited the growth of all gram-positive test bacteria, chloroform broth extract of Cylindrocladium scoparium, Colletotrichum wuxiense and ethanolic broth extract of Aspergillus flavus, Trichoderma harzianum exhibited antibacterial potential against gram negative test organisms. Chloroform and ethanol mycelium extracts of Trichoderma harzianum and Aspergillus flavus, respectively, exhibited 100% growth inhibition potential against all test organisms. The current investigation endorses the potent secondary metabolism of the identified isolates and their potential to synthesise antibacterial compounds.


INTRODUCTION
As per the WHO, the rising resistance of microbes against available antibiotics has left majority of the existing drugs obsolete. 1Since last two decades, the surge in the mortality rate due to infectious diseases is attributed to the emerging drug resistance of the microbes against available antibiotics. 2 Since the emergence of drug resistance, the scientific community and the pharmaceutical firms have looked up to natural resources to find novel medications to address this lethal threat. 3The biotic community of the marine ecosystem is presumed to be a rich source of biochemicals with wide therapeutic applications.In earlier studies, many compounds like nigrospoxydons A -C, epoxydon, dehydroxychlorofusarielin B, penicipyrone, 7-oxobrefeldin A, spirodioxynaphthalene etc., have been isolated and characterised from marine fungi and bacteria with potential to treat various human ailments. 4,5Recent technological developments and methodologies have made it possible to prospect the marine environment more effectively and to quantify compounds derived from marine sources chemically and biologically. 6,7hus, it has led to the discovery of new ways as well as a focus on these topics, with several marine microorganisms currently being used in medicine. 8econdary metabolites from marine fungi are an extremely important source of anti-infective compounds. 9,10From the marine environment, mangrove is an important ecological, economic and social ecosystem.Mangrove fungi are also called manglicolous fungi, which include mostly marine fungi along with a few terrestrial species which can be found in mangrove areas. 11There is a great deal of biodiversity in mangrove forests and they constitute a transitional ecosystem which contains organisms that are either terrestrial or aquatic. 12ue to their adaptation to extreme environmental conditions, mangrove fungi have a unique source of genetic code and of great scientific interest because they account for the second largest proportion of fungi on the planet. 13,14Mangrove fungi are an important source of genetic data because they have distinctive metabolism. 15,16tudies are being conducted to better understand their genetic transformation and metabolism of the numerous unique antimicrobial metabolites that have been discovered from mangrove fungi in order to use them in the development of drugs. 17,18As a biodiversity hotspot, the mangrove ecosystem offers unique opportunities to uncover bioactive and chemical compounds of medicinal nature. 19,20In the recent past, a number of studies have been carried out on metabolites of mangrove-associated bacteria and fungi which has resulted in the discovery of some novel bioactive compounds. 21,22In the present study, fungi have been isolated and identified from the Mangrove ecosystem of the Mumbai coastal region and their metabolites have been evaluated for antibacterial potential.

Sample collection and Identification of Fungi
Water sample was collected from the Gorai Mangrove area, in Mumbai, India and stored in a sterile vial till further use.Within 24 hours of collection, the diluted sample was spread on Potato Dextrose agar medium (PDA) and incubated at room temperature for 3 -4 days.PDA was supplemented with streptomycin to prevent bacterial contamination. 23,24Freshly cultured purified colony isolates were identified as described in earlier studies. 25

Metabolite Extraction and Fermentation
Broth fermentation was carried out in a 250 ml Erlenmeyer Conical flask.7][28] .After two weeks of incubation, broth of each isolate was treated with 50 ml Ethanol and 50 ml chloroform separately.The flasks were left on a rotating shaker set at 250 rpm for a day to allow metabolites to dissolve in the organic solvent. 26fter 24 hrs, broth was filtered using double Whatmans filter paper (qualitative paper with grade 597). 29The filtrate was dried on a rotary evaporator and the left thick paste-like substance was stored at 4°C in glass vials for further studies.Further, mycelium collected was washed with warm water to ensure no residue of solvent and nutrient medium is left.The washed mycelium was oven-dried and coarse powdered using mortar  and pestle.The dry mycelium coarse powder was extracted for its metabolites using ethanol and chloroform separately. 30,31In 50ml of solvent 10g of mycelium powder were added and left on a rotary shaker at 150 rpm for 48 hrs. 32The extracts were filtered after 48 hrs and the solvent was allowed to evaporate thus leaving behind a thick paste which was stored in glass vials at 4°C until further use.

Antibacterial Activity
5 Metabolites at various concentrations were evaluated for their bioactivity against test organisms.Extracts were dissolved in dimethyl sulfoxide (DMSO) and 100 µl from each concentration was added to each well (6 mm) in nutrient agar plates seeded with test bacteria.The plates were kept in the refrigerator for 20-30 minutes at 4°C for diffusion of in the medium followed by incubation of plates for 24 hrs at room temparature.Streptomycin and DMSO were used as positive and negative control, respectively.After incubation, inhibitory activity was assessed by measuring the zone of inhibition in mm around metabolite-loaded wells.

RESULTS AND DISCUSSION
pH of the water sample collected from the mangrove site was found to be 7.6.The spread plate followed by a microscopic and staining study revealed the presence of 5 fungi in a water sample.The identified fungi were Aspergillus niger, Aspergillus flavus, Trichoderma harzianum, Cylindrocladium scoparium and Colletotrichum wuxiense (Table 1 & 2).
The broth of identified fungi was extracted by ethanol and chloroform separately.Solvent extracts after drying were stored in vials.The yield (metabolites) of each broth and mycelium extract of individual solvent and fungi is given in Table 3 and 4 6).
M a n g r o v e s c o m p r i s e a l m o s t 181,000 Km 2 and dominate the World's Forth coastline. 6Considering the kind of continuous changing environment the mangrove ecosystem has, fungi associated with mangroves help plants to sustain under turbulent environmental conditions. 11As a result of such interactions, mangrove fungi are believed to be the source of novel and unique natural compounds. 36In the last few decades, mangrove fungi have attracted researchers across the globe to investigate their metabolism which may lead to the discovery of natural compounds with therapeutic applications like antitumor, antiviral, antidiabetic and antimicrobial compounds. 37,38In the current study, results revealed the strong antibacterial potential of fungal metabolites against all test organisms.The antimicrobial potential of mangrove fungi has been reported in earlier studies. 21,6,20The extraction of broth and the nature of the solvent used plays an important role in the metabolite extraction.wherein they used different organic solvents with varied polarity.However, our results are contradictory to the studies earlier carried out by Reshi et al., 24 wherein the authors concluded that in addition to polar and non-polar organic solvents, ethyl acetate, being the mid-polar solvent must be used for metabolite  extraction from plants, bacteria and fungi.Many novel antimicrobial metabolites have been isolated from mangrove fungi and more and more studies are taken up to unravel the biologically active compounds. 39With increasing drug resistance, most of the available antibiotics have become obsolete, therefore the current circumstances demand natural and novel compounds to cater for the increasing drug resistance menace. 40

CONCLUSION
The research validates that various gram-positive and gram-negative bacteria are susceptible to the antibacterial effects of fungal metabolites.Further characterisation of bioactive metabolites is needed to identify the active compounds which may strengthen the existing antibiotics and also may pave the way for the discovery of new compounds of medicinal nature.

Figure 1 .
Figure 1.Antibacterial activity of Broth Extracts , respectively.Antibacterial property of metabolites was assessed by measuring inhibitory zone around the 6 mm wells loaded with extracts of different concentrations.The results revealed the bacterial growth inhibition property of all the broth and mycelium extracts (Table5& 6; Figure1 & 2).Extracts obtained from broth exhibited the growth inhibition property against all gram-positive test bacteria.Further, chloroform broth extract of Cylindrocladium

Table 1 .
Cultural and Morphological Characteristics of Isolates Isolate Media Colony size Surface Test organism used in the study were Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC 441, Enterococcus faecalis MTCC 439, Enterococcus faecium MTCC 9728, Klebsiella pneumonia MTCC 432 and Escherichia coli MTCC 64.The bacterial cultures was collected from MTCC Chandigarh, Punjab.Cultures were further maintained in laboratory as described earlier by Patankar et al.

Table 2 .
Isolated Fungi and Microscopy Similar inferences have been drawn earlier by Prasannan et al. and Zhang et al.,

Table 3 .
Weight of extracted metabolites from Broth fermentation (250 ml)

Table 4 .
Weight of Extracted metabolites from dry mycelium biomass (per 10 gm)

Table 5 .
Antibacterial activity of Broth extracts

Table 6 .
Antibacterial activity of Mycelial Extracts