Studies on Bioactive Actinomycetes in a Niche Biotope, Nambul River in Manipur, India

As part of our ongoing studies on actinomycete diversity in Manipur, an underexplored zone falling in the IndoBurma biodiversity hotspot, this paper reports bioactivity screening and characterization of bioactive actinomycetes from Nambul River. Bioprospecting studies on actinobacteria have been largely focused on terrestrial and, more recently, on marine ecosystems but freshwater habitats have been largely neglected and studies on freshwater actinomycetes are very scanty in India. Hence we investigated the actinomycete diversity in one of the freshwater rivers of Manipur, Nambul River in Manipur, India. A total of 156 actinomycetes were isolated from three samples of Nambul River. Based on the results of primary screening, 23 isolates were selected for secondary screening. Nine strains showed significant antibacterial or broad spectrum antimicrobial (antibacterial and antifungal) activities in the secondary screening. Phylogenetic analyses indicated that a majority of them were Streptomyces species though some rare actinobacteria were also recovered. Seven strains were identified as Streptomyces spp. while one strain each was identified as Nocardia sp. and Micromonospora sp. Three strains showed promising antifungal activities against human and plant pathogens. This study highlights the potential for discovering bioactive actinomycetes in underexplored niche biotopes such as river sediments. Studies on Bioactive Actinomycetes in a Niche Biotope, Nambul River in Manipur, India


Introduction
Actinomycetes are a group of physiologically versatile, high GC, gram-positive, filamentous bacteria found in most environments including terrestrial and aquatic habitats [1]. Streptomyces has been reported as the dominant genus in freshwater habitats whereas Micromonospora and related genera are predominant in freshwater and marine sediments [2].
There is increasing realization of the potential for wetlands as sources of actinomycetes that produce useful bioactive compounds. Cross [3] reported freshwater habitats as promising sources of bioactive actinomycetes. Okami [4] reported that actinomycetes of freshwater origin produce novel bioactive substances. There is an urgent need for screening of novel bioactive compounds from underexplored biotopes such as freshwater habitats. This is also dictated by the rise of emerging diseases and antibiotic-resistant human pathogenic bacteria such as multidrug resistant (MDR) strains of M. tuberculosis, vancomycin resistant enterococci (VRE), methicilin resistant Staphylococus aureus (MRSA), Pseudomonas aeruginosa and Candida albicans [5] etc. The focus is increasing towards novel biotopes, niche ecosystems and extreme environments for isolating novel bioactive strains [6] especially actinobacteria which produce nearly 80% of all known antibiotics [7]. Additionally the microbial profiles also serve as an indicator of freshwater ecological health [8].

Sampling and pretreatment
Sampling was done from three different sites of the Nambul River, which is one of the major rivers in Manipur (62.7 km in length), originating from Kangchup Hill range in the western side at an elevation of 1830 m above mean sea level. The river flows through the thickly populated area of the city and ultimately discharges into the Loktak Lake. The potentially polluted stretch of the river is within the Imphal Municipality area for a length of about 1.45 km and its tributary Naga Nala for a length of about 1 km. Soils and sediment samples were collected from the Nambul river bank, river bed and the rhizospheric sediments of river water vegetation in polyethylene bags, closed tightly, and stored in a refrigerator before processing.
Pretreatment of the soil samples were carried out by air-drying them at room temperature for about four weeks [9,10].

Enrichment and isolation
To further enrich the actinomycete population, 1.0 g air-dried sediment was mixed with 0.1 g of CaCO 3 and kept at ambient temperature for a week to enrich actinomycetes which usually prefer alkaline conditions and also to reduce the contamination of molds and fungi [11]. 1.0 g air-dried sediment was suspended in 99.0 ml of sterile distilled water and incubated in an orbital shaker at room temperature at 150 rpm for 30 minutes. The soil suspension was then serially diluted and 0.1 ml of 10 -3 to 10 -7 dilutions were spread plated in duplicates on Starch Casein Nitrate Agar (SCNA, pH 7.2) plates [12] supplemented with 50 µg.mL -1 each of nystatin and cycloheximide [13] and finally incubated at 28°-30°C for up to 4 weeks.
Selected actinomycete colonies were further purified on SCNA plates and pure isolates were maintained on modified Bennett's agar [14] slants at 4°C and as spore suspensions on 20% (v/v) glycerol at -20-°C [15] for further studies.  [16] which is a strain isolated in our laboratory.

Antimicrobial assay
Initial antimicrobial assay of the putative actinomycete isolates was carried out using the cross-streak technique [17,18]. Actinomycete isolates which showed inhibition of >50% against the test organisms in the primary screening were further subjected to secondary screening by Kirby Bauer method [19] against the above test organisms. The actinomycete strains showing positive antimicrobial activities were subjected to phenotypic and genotypic characterization.

Biocontrol assay of the bioactive strains
Fungal pathogens were procured from MTCC, Chandigarh except for LSMU1(procured from Life Science Department, Manipur University). The bioactive strains were tested for biocontrol activity by dual culture method [20] against the rice pathogens, Fusarium oxysporum MTCC 287, Pyricularia oryzae MTCC 1477, Curvularia oryzae MTCC 2605 and Bipolaris oryzae LSMU1.

Phenotypic and genotypic characterization
The various morphological, physiological and biochemical characterization tests were carried out using the standard procedures [21][22][23][24]. The micromorphologies of the spore chains and the spore surfaces of 14 days old culture grown on Streptomyces agar were determined using Carl Zeiss microscope (AxioScope A.1, Germany, magnification 600X). The cultural properties of the strains were evaluated according to the guidelines of the International Streptomyces Project (ISP) as described by Shirling & Gottlieb [24].

Biocontrol assay of the bioactive strains
Three of the bioactive isolates, i.e NRP1-14, NRP1-18 and NRP1-26, showed antagonistic activity against one or more rice fungal pathogens [32]. These strains also exhibited phosphate solubilizing, siderophore, ammonia production and chitinase activities, showing their potential Degradation of Adenine 0.5% Guanine 0.05% Tyrosine 0.5% Xanthine 0.4%   Table 4: Growth morphology on different ISP and other actinomycete specific media.  (Table 2). These strains also had IAA producing abilities, with the exception of NRP1-14.

Phenotypic and genotypic characterization
Phenotypic characteristics of the bioactive strains and their growth morphologies on different ISP and other actinomycete specific media are shown in (Tables 3, 4.) The gross morphologies of the bioactive strains grown on SCNA media and their micromorphologies are shown in Figure 1. except for NRP1-35, which was negative for casein hydrolysis, and NRP1-14, which was negative for both casein and starch hydrolysis.
Rifaat [33] reported the predominance of Streptomyces in water sample and that of Micromonospora in sediments of the Nile

NRS1-11b
Streptomyces subrutilus DSM 40445 T (X80825) River. These Streptomyces strains were reported to have significant antimycotic activity. Elliah et al. [30] observed that Streptomyces strains, from Krishna river sediments in India, had significant antibacterial and antifungal activities. Our group had earlier showed potential for obtaining bioactive actinomycetes from niche habitats in Manipur including Nambul River [34]. The present study reemphasizes the promise of Nambul as source of antimicrobial actinomycetes. Although, freshwater habitats have been long ignored for actinomycete exploration, several recent reports corroborate the importance of such ecosystems for the search of antibiotic producing actinomycetes. A Streptomyces sp. AZ-NIOFD1, with broad-spectrum antimicrobial activity, was isolated from water sample of the Nile River in Egypt by Atta et al. [35]. Cwala et al. [36] reported Actinopolyspora sp. TR008, from Tyume River in South Africa which was active against both Gram positive and Gram negative bacteria. Sibanda et al. [37] recently stressed the significance of freshwater habitats as source of bioactive actinomycetes. They obtained actinomycete species belonging to Sachharopolyspora and Actinosynemma from Tyume River, South Africa. Crude extracts of these strains were found to exhibit potent antibacterial activity against both Gram positive and Gram negative bacteria.

Bifidobacterium bifidum YIT 4039 T (AB437356)
Our preliminary findings showed promise of obtaining bioactive (antibacterial and antifungal) actinomycetes in an underexplored habitat, Nambul River in Manipur, India. Further studies on actinomycete population in the plethora of wetlands in Manipur-lakes, rivers, ponds, and marshes etc.-hold promise for obtaining novel strains, or even species, of bioactive actinomycetes.