In vitro antimicrobial potential of organic solvent extracts of novel actinomycetes isolated from forest soil

Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, Riyadh 11451, Saudi Arabia. Department of Zoology, Thiru Vika Government Arts College, Thiruvarur, 610 003, Tamil Nadu, India. Department of Plant Biology and Biotechnology, Loyola College, Chennai, 600 034, India. Lifelong Education Center, Chonnam National University, Kwangju, 500-757, Republic of Korea Grassland and forage division, National Institute of Animal Science, RDA, Seonghwan-Eup, Cheonan-Si, Chungnam, 330-801, Republic of Korea.


INTRODUCTION
Actinomycetes are Gram-positive bacteria showing a filamentous growth.Actinomycetes are a group of organisms widespread in nature, and play a significant role in the future of biotechnology, because of their importance as producers of vitamins, enzymes, antitumour agents, immune modifying agents and, mainly, antibiotic compounds (Sofia and Boldi, 2006).According to Maya et al. (2011) number of novel molecules from actinomycetes were discovered.Streptomycetes, the Gram positive filamentous bacteria are widely distributed in a variety of natural and man-made environments, constituting a significant component of the microbial population in most soils.The results of extensive screenings have led to the discovery of about 4,000 antibiotic substances from bacteria and fungi, many of which have been applied in human medicine, veterinary medicine and agriculture (Adinarayana et al., 2007).Most of metabolites are produced from Streptomyces.Approximately 75% of metabolites originated from Streptomyces genus and at least 5000 documented bioactive compounds are known as being produced by Streptomyces genus (Vasanthabharathi et al., 2011).Most Streptomyces are used in the production of a diverse array of antibiotics including aminoglycosides, macrolides, β-lactams, peptides, polyenes, polyether, tetracyclines, etc (Augustine et al., 2005).In searching for new antibiotics, number of different bacteria, actinomycetes, Streptomycetes, fungi and algae have been investigated.To prevent exponential emergence of microorganisms from becoming resistant to the clinically available antibiotics already marketed, a periodic replacement of the existing antibiotics is necessary.In the present study, extraction of antimicrobial metabolites from Streptomyces and its antimicrobial effects on bacteria and fungi were studied.Previously, nutrients required for the antimicrobial compounds production were optimized under the shake flask condition for the identified Streptomyces strains (Arasu et al., 2012;2013).This study focused on the extraction of antimicrobial metabolites by using different solvents and evaluates minimum inhibitory concentration by broth micro dilution method.

Chemicals and solvents
Glucose and all other chemicals were obtained from Himedia (India).Organic solvents were procured from Himedia (India).

Liquid -liquid extraction
Actinomycetes were grown in modified nutrient glucose broth medium for six days.After incubation, the fermentation medium was collected and filtered through Whatman No.1 filter paper.The total culture filtrate 4000 ml was used for the series of solvent extraction by using hexane, chloroform, ethyl acetate and methanol.The low polar to high polar solvent was selected for the organic solvent extraction.Three folds volume of the solvent was mixed thoroughly with the broth by shaking them in 1000 ml capacity separating funnel and allowed to stand for 1 h.The solvents were removed using simple distillation and vacuum rotary evaporator at 40ºC.The extracts were stored at 4ºC until further use.Water extract was lyophilized and the concentrated metabolites were stored at 4ºC until further use.

Solid -liquid extraction
A metabolite which could not extract from fermented broth was extracted by this method.The spore suspensions of the culture were inoculated on Modified Nutrient Glucose Agar (MNGA) media and incubated at 28ºC for six days.Then, agar media with cultures were taken into a 500 ml flask containing 100 ml of methanol and kept in shaker for 2 h at 200 rpm.Then the suspension was centrifuged at 8000 rpm, 10 min to separate the organic phase and extracted twice.The methanol phase was concentrated by using vaccum at 35 0 C. The extracts were stored at 4ºC until further use.

Test organisms
The reference strains used in this study was procured from American type culture collection (ATCC) and Microbial type culture collection Chandigarh, India (MTCC).

Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) was performed according to the standard reference method (NCCLS, 1999).The extracts were dissolved in water + 2% dimethyl sulfoxide (DMSO).The initial concentration of extract was 10 to 0.156 mg/ml.The initial test concentration was serially diluted twofold.Each well was inoculated with 5 l of suspension containing 10 8 CFU/ml of bacteria.The antibacterial agent streptomycin was included in the assays as positive controls.The plates were incubated 24 h at 37°C.After incubation 5 μl of testing broth was placed on the sterile MHA plates for bacteria and incubated at respective temperature.The MIC for bacteria was determined as the lowest concentration of the extracts inhibiting the visual growth of the test cultures on the agar plate.Three replications were maintained.

Preparation of fungal spore
The filamentous fungi were grown on Sabouraud Dextrose Agar (SDA) slants at 28 o C for 10 days and the spores were collected using sterile doubled distilled water and homogenized.Yeast was grown on Sabouraud Dextrose Broth (SDB) at 28 o C for 48 h.

Antifungal assays
The antifungal activity was performed according to the standard reference method (NCCLS, 1999).The extracts were dissolved in water+2% dimethyl sulfoxide (DMSO).The initial concentration of extract was 10 mg/ml.The initial test concentration was serially diluted twofold.Each well was inoculated with 5 l of suspension containing 10 4 spore/ml of fungi.The antifungal agent Fluconazole was included in the assays as positive controls; the plates were incubated for 24 h up to 9 days at 27°C for dermatophytic strains.MIC was defined as the lowest extract concentration, showing no visible fungal growth after incubation time.Experiment was carried out in triplicates.

RESULTS
Five different extracts from eight strains of actinomycetes were screened against four Gram-positive and eight Gram-negative bacteria; among them three strains extracts exhibited good activity against tested microbes (Table 1).Some extract had a significant activity for Gram-positive bacteria but not on Gram-negative bacteria.Ethyl acetate extracts of all the strains showed activity against more than four different bacteria.

Activity against Gram-positive bacteria
All the extracts from eight strains showed activity against tested Gram positive bacteria.The hexane extracts of ERI-1 exhibited activity against B. subtilis, S. aureus and S. epidermidis at a concentration of 5 mg/ml.Chloroform and methanol extract did not show antibacterial activity.Lyophilized water extract showed MIC of 5 mg/ml for S. aureus, S. epidermidis and E. faecalis.B. subtilis exhibited MIC of 2.5 mg/ml.Ethyl acetate extract showed MIC of 5 mg/ml to B. subtilis, S. aureus, S. epidermidis and E. faecalis.Hexane extracts of ERI-3 showed MIC of 5 mg/ml to S. epidermidis and B. subtilis for 10 mg/ml.Chloroform extract did not exhibit any activity against Gram positive bacteria.Ethyl acetate extract showed MIC of 10 mg/ml for B. subtilis, S. aureus and E. faecalis.For S. epidermidis growth was inhibited at a concentration of 2.5 mg/ml.Lyophilized water extract exhibited MIC of 5 mg/ml to B. subtilis and S. aureus.E. faecalis and S. epidermidis revealed MIC of 10 and 2.5 mg/ml respectively in the lyophilized water extract.
Hexane and ethyl acetate of ERI-26 did not show any activity against both Gram positive and Gram negative bacteria.The chloroform extract showed MIC of 10 mg/ml to all the tested bacteria.Methanol extract exhibited MIC of 2.5 mg/ml to B. subtilis, S. epidermidis and E. faecalis.Lyophilized water extract exhibited MIC of 5 mg/ml to E. faecalis and B. subtilis.S. epidermidis and S. aureus showed MIC of 10 mg/ml.Ethyl acetate extract of ERI-4 showed MIC of 5 mg/ml to S. epidermidis and S. aureus.B. subtilis exhibited MIC of 2.5 mg/ml for ethyl acetate extract and S. aureus revealed 10 mg/ml.Hexane, methanol and lyophilized water extract did not show any activity.Chloroform extracts of ATSI-13 showed MIC of 10 mg/ml to all the tested bacteria.Ethyl acetate extract exhibited MIC of 5 mg/ml.Hexane, methanol and lyophilized water extract of ATSI-13 did not show any activity against bacteria (Table 1).

Activity against Gram-negative bacteria
Most of the actinomycetes extracts did not inhibit the growth of Gram negative bacteria.Ethyl acetate extract of ERI-1 inhibited the growth of P. aeruginosa.Hexane, ethyl acetate and lyophilized water extract showed MIC of 5 mg/ml to E. coli.P. aeruginosa, K. pneumoniae, Xanthomonas sp, Erwinia showed MIC of 10 mg/ml to ethyl acetate and lyophilized water extracts.Ethyl acetate extract showed MIC of 10 mg/ml to S.typhi and V. fischeri.Hexane, chloroform, ethyl acetate and lyophilized water extract did not show any activity against P. vulgaris.Lyophilized water extracts of ERI-3 showed MIC of 10 mg/ml to all the tested bacteria.Ethyl acetate extract also showed MIC of 10 mg/ml to all the tested bacteria except S. typhi, V. fischeri and P.vulgaris.Hexane and chloroform extract of ERI-3 did not show any antibacterial activity.The methanol extract of ERI-26 exhibited MIC of 5 mg/ml to E. coli, P. aeruginosa and K. pneumoniae.Xanthomonas sp., Erwinia, S. typhi, V. fischeri, P. vulgaris showed MIC of 10 mg/ml.Lyophilized water extract showed MIC of 5 mg/ml to E.coli.Hexane, chloroform and ethyl acetate extracts of ERI-26 did not reveal any activity against tested bacteria.

Antifungal activity
Results of antifungal activity were summarized in Table 2.All the 8 strains showed varying degrees of antifungal activity.Most of the extracts inhibited more than four fungal strains.From the evaluation we found that ethyl acetate extracts inhibited the growth of fungus.Hexane and methanol extracts also nearly showed the same level

Table 1 .
Minimum inhibitory concentration of crude extracts obtained from selected actinomycetes against bacteria.