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Studies on Bioactive Actinomycetes in a Niche Biotope, Nambul River in Manipur, India | OMICS International
ISSN: 1948-5948
Journal of Microbial & Biochemical Technology

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

Debananda S. Ningthoujam*, Suchitra Sanasam, Salam Nimaichand

Microbial Biotechnology Biotechnology Laboratory (MBRL), Department of Biochemistry, Manipur University, Canchipur, Imphal 795003, India

*Corresponding Author:
Debananda S. Ningthoujam
Microbial Biotechnology Biotechnology Laboratory (MBRL)
Department of Biochemistry, Manipur University
Canchipur, Imphal 795003, India
E-mail: [email protected]

Received date: May 05, 2011; Accepted date: November 21, 2011; Published date: December 07, 2011

Citation: Ningthoujam DS, Sanasam S, Nimaichand S (2011) Studies on Bioactive Actinomycetes in a Niche Biotope, Nambul River in Manipur, India. J Microbial Biochem Technol S6:001 doi: 10.4172/1948-5948.S6-001

Copyright: © 2011 Ningthoujam DS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

As part of our ongoing studies on actinomycete diversity in Manipur, an underexplored zone falling in the Indo- Burma 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.

Keywords

Nambul river; Bioactive; Antifungal; Novel species; Streptomyces

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].

Materials and Methods

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 CaCO3 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.

Antimicrobial assay

Test organisms: The test bacteria used were the Gram positive organisms Staphylococcus aureus (MTCC 96), Micrococcus luteus (MTCC 106), and Bacillus subtilis (MTCC 121), and the Gram negative bacteria Escherichia coli (MTCC 739) and Pseudomonas species (DN1); and the test fungi used were Candida albicans (MTCC 227) and Aspergillus niger (MTCC 1344). All the reference strains were procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India except for DN1 [16] which is a strain isolated in our laboratory.

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 [21-24]. 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].

16S rDNA amplification and sequencing were carried out for the bioactive isolates (having an inhibition zone of more than 17 mm diameter against the test organisms) using the primers (8F, 5’-AGAGTTTGATCCTGGCTCAG- 3’; 357F, 5’- CTCCTACGGGAGGCAGCAG-3’; 1100R, 5’-GGGTTGCGCTCGTTG-3’; 1492R, 5’-GGTTACCTTGTTACGACTT- 3’). The 16S rDNA sequences were submitted to EzTaxon server version 2.1 [25], which contain manually curated databases of type strains of prokaryotes, for sequence analysis. Related strains were selected for alignment by CLUSTAL W program and phylogenetic analyses were done according to the neighbour-joining method [26] using the MEGA version 4.1 [27,28]. To determine the support of each clade, bootstrap analysis was performed with 1000 replications [29].

Results and Discussion

Isolation of actinomycetes

A total of 156 actinomycetes were isolated from the Nambul River, of which 47 (NRB1-1 to NRB1-47) were from the bank, 69 (NRS1-1 to NRS1-69) from the river bed, and 40 (NRP1-1 to NRP1-40) from the aquatic rhizospheric samples.

Antimicrobial assay

Based on the results of primary screening, 23 strains (11.1%) showed an inhibition zone of more than 50%, against one or more of the test pathogens These isolates were then shortlisted for secondary screening (Table 1). Of 23 strains subjected to secondary screening, 9 (39.1%) isolates (NRB1-19, NRB1-44, NRP1-13, NRP1-14, NRP1-18, NRP1- 26, NRP1-35, NRS1-11b and NRS1-18) showed good antimicrobial activities with inhibition zone diameters of 17 mm or more against one or more of the test organisms. Among these bioactive isolates, 2 (NRS1- 11b and NRS1-18) were found to be purely antibacterial and 6 (NRB1- 19, NRB1-44, NRP1-13, NRP1-18, NRP1-26 and NRP1-35) had broad antimicrobial activities. Interestingly, the strain NRP1-14 specifically showed potent antifungal activity against C. albicans and A. niger. None of the isolates was found to be bioactive against Pseudomonas aeruginosa. Reports of antimicrobial actinomycetes from freshwater habitats are rare. Elliah et al. [30] obtained 30 actinomycete isolates from sediments of Krishna River in Andhra Pradesh, India, of which 16 (53.3%) exhibited excellent antagonistic properties in cross streak method. On detailed submerged fermentation studies, it was found that 12 isolates (40.0%) had antibacterial and 9 (30%) had antifungal activities. Five (16.6%) isolates showed both antibacterial and antifungal activities. Singh et al. [31] isolated 37 actinomycetes from phoomdi (floating putrefying vegetation) in Loktak Lake in Manipur, India. Twentyone (56.7%) isolates showed antimicrobial activities against test microorganisms in primary screening. Of these, 12 (32.4%) were found to have broad spectrum (antibacterial and antifungal) activities.

Test isolates Test organisms
Gram positive bacteria Gram negative bacteria Yeast/fungi
MTCC
96
MTCC 106 MTCC 121 MTCC 739 DN1* MTCC
227
MTCC 1344
  Inhibition zone (in mm diameter)
Standard antibiotic  discs Erythromycin
16
Penicillin-G
18
Amikacin
18
Streptomycin
18
Rifampicin
12
Amphotericin-B
16
Nystatin
13
NRB1-1 - 13±0.29 16±0.76 - - - -
NRB1-9 - - 15±0.29 - - - 11±0.58
NRB1-19   17±0.29 18±0.76 19±1.0 - - 15±0.76
NRB1-20 - - 15±0.58 - - - -
NRB1-25 - - 16±1.0 - - - -
NRB1-29 - - 15±0.58 13±1.5 - - -
NRB1-33 - - 13±0.29 16±0.58 - - -
NRB1-44 16±1.0 - 18±0.76 - - - 17±1.0
NRP1-5 - 13±0.58 14±0.5 11±0.76 - - -
NRP1-13 - - 18±1.0 - - - 20±0.76
NRP1-14 - - - - - 20±1.0 21±0.58
NRP1-18 21±0.58 16±0.5 15±0.76 - - - 17±0.29
NRP1-20 - - 15±1.0 - - - -
NRP1-26 22±0.58 15±0.29 16±0.5 18±1.0 - 12±0.76 18±0.76
NRP1-28 - - 16±0.29 - - - -
NRP1-29 - - 15±0.76 - - - -
NRP1-35 - - 18±0.5 16±1.0 - - 18±0.76
NRP1-40 - - 13±1.0 - - - -
NRS1-1 - 12±1.5 15±0.29 - - - -
NRS1-11b 18±0.58 20±0.29 17±1.0 18±0.76 - - -
NRS1-18 13±0.58 - 15±0.58 17±0.29 - - -
NRS1-30 - - 14±1.0 - - - -
NRS1-39 - - 16±0.58 15±0.29 - - -

Table 1: Secondary Screening profile of the selected isolates exhibiting good antimicrobial activity in primary screening.

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 for plant growth promotion and biocontrol of pathogens (Table 2). These strains also had IAA producing abilities, with the exception of NRP1-14.

Test isolates P solubilization IAA Siderophore NH3 Production Chitinase activity
NRP1-14 + - + + +
NRP1-18 + + + + +
NRP1-26 + + + + +

Table 2: Plant growth promoting characteristics of the antagonistic actinomycete isolates.

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.

Name of the test NRB1-19 NRB1-44 NRP1-13 NRP1-14 NRP1-18 NRP1-26 NRP1-35 NRS1-11B NRS1-18
Gram’s staining + + + + + + + + +
Production of diffusible pigment - - - - - - - - -
Growth at
4°C
15°C
25°C
30°C
37°C
42°C
60°C
  -
-
+
+
+
-
-
  -
-
+
+
+
+
-
  -
-
+
+
+
+
-
  -
+
+
+
+
+
-
  -
-
+
+
+
+
-
  -
-
+
+
+
+
-
  -
-
+
+
+
+
-
  -
-
+
+
+
-
-
  -
-
+
+
+
-
-
Growth at pH range
5.2
7.0
8.0
9.0
10.0 
  +
+
+
+
+
  +
+
+
+
+
  +
+
+
+
+
  +
+
+
+
+
  +
+
+
+
+
  +
+
+
+
+
  +
+
+
W
-
  +
+
+
+
+
  -
+
+
+
+
Growth in the presence of
2% NaCl
5% NaCl
7% NaCl
10% NaCl
  +
+
W
-
  +
+
+
-
  +
+
+
-
  +
+
+
+
  +
+
+
-
  +
+
+
-
  +
+
+
W
  +
+
+
-
  -
-
-
-
Degradation of
Adenine 0.5%
Guanine 0.05%
Tyrosine 0.5%
Xanthine 0.4%
  +
-
-
-
  -
-
+
-
  -
-
-
-
  -
-
+
-
  -
-
+
-
  -
-
+
-
  -
-
-
-
  +
-
+
+
  +
-
-
-
Hydrolysis of
Casein
Starch
Urea
  +
+
-
  +
+
-
  +
+
+
  -
-
-
  +
+
+
  +
+
+
  -
+
-
  +
+
+
  +
+
-
Biochemical tests
Catalase activity
Oxidase activity
Methyl Red (MR)
Voges Proskauer (VP)
Citrate utilization
Indole production
Nitrate reduction
Gelatin liquefaction
H2S production
  +
+
+
-
-
-
+
-
-
  +
-
-
-
+
-
-
-
-
  +
-
+
-
-
-
+
-
-
  -
-
-
-
+
-
+
-
-
  -
-
-
+
-
-
+
-
-
  -
-
-
+
-
-
+
-
-
  -
-
+
-
-
-
+
-
-
  +
-
-
-
+
-
+
-
-
  -
-
-
-
-
-
-
-
-

Table 3: Biochemical and physiological tests of the bioactive actinomycete isolates.

Media Isolate → NRB1-19 NRB1-44 NRP1-13 NRP1-14 NRP1-18 NRP1-26 NRP1-35 NRS1-11b NRS1-18
ISP1 AM Cream Grey Magenta Off White Cream Cream Grey Sandal
wood
Reddish
SM Cream Light Grey Magenta Cream Cream Cream Grey Cream Reddish
ISP2 AM Pale cream Cream Magenta Cream Pale Cream Pale Cream Grey Sandal
wood
Reddish brown
SM Cream Light Yellow Magenta Light Yellow Cream Cream Brown Light Yellow Reddish Brown
ISP3 AM Cream Cream P.G. Pale Cream Grey Grey Grey Cream Reddish
SM Cream Brown P.G. Cream Grey Grey Brown Cream Reddish Brown
ISP4 AM Cream Grey P.G. Off White Grey Grey Grey Sandalwood P.G.
SM Cream Light Grey P.G. Light Grey Grey Grey Brown Cream P.G.
ISP5 AM White Off White Light range Off White White White Grey Cream P.G.
SM White White Light Orange White Cream Cream Grey Cream P.G.
ISP6 AM White Pale Cream Magenta Cream Pale Cream Pale Cream Grey Cream Reddish Brown
SM Pale Cream Pale Cream Cream Brown Pale Cream Pale Cream Dark
Brown
Cream Reddish Brown
ISP7 AM Off White Cream P.G. Light Grey Grey Grey Grey Sandal wood P.G
SM Off White Light Grey P.G. Grey Grey Grey Light grey Cream P.G
SCNA AM Off White Grey Light Orange Grey Grey Grey Grey Off White Orange
SM Grey Brown Orange White Yellow Yellow Brown Yellow Brown
SA AM Cream Cream Magenta Pale Cream Cream Cream Grey Sandal
Wood
Reddish Brown
SM Cream Light Brown Cream Cream Light Brown Light Yellow Black Cream Brown
TSA AM Pale Cream Pale Cream Pale Cream Pale Cream Pale Cream Pale Cream Off White Cream P.G.
SM Pale Cream Pale Cream Pale Cream Pale Cream Pale Cream Pale Cream Light Grey Pale Cream P.G

Table 4: Growth morphology on different ISP and other actinomycete specific media.

microbial-biochemical-technology-actinomycetes

Figure 1: Morphological feature of the bioactive Nambul actinomycetes (NRB1-19, NRB1-44, NRP1-13, NRP1-14, NRP1-18, NRP1-26, NRP1-35, NRS1-11b, NRS1- 18) and their micromorphologies.

NRP1-13 grew at 25-42°C, pH 5.2-10, and tolerated up to 7% NaCl while NRS1-18 grew at 25-37°C, pH 7-10 and could tolerate < 2% NaCl. NRB1-19 and NRS1-11B grew well at 15-37°C, pH 5.2-10 and tolerated up to 7% NaCl. Four isolates (NRB1-44, NRP1-18, NRP1-26 and NRP1-35) grew at 25-42°C, pH 5.2-10 (though NRP1-35 grew poorly at pH 10) and tolerated 2-7% NaCl (NRP1-35 could grow even at 10% NaCl). NRP1-14 grew well at 15-42°C, pH 5.2-10 and tolerated 2-10% NaCl. Most isolates were positive for casein as well as starch hydrolysis, except for NRP1-35, which was negative for casein hydrolysis, and NRP1-14, which was negative for both casein and starch hydrolysis.

Results of phylogenetic analyses (Figure 2) of the bioactive actinomycetes revealed that Streptomyces was the predominant actinomycete genus among the Nambul river strains, though Micromonospora and Nocardia were also recovered. Seven strains were identified as Streptomyces species. NRB1-19 was most closely related to Streptomyces parvus (similarity index 100%), NRB1-44 to Streptomyces thinghirensis (similarity index 100%), NRP1-14 to Streptomyces mutabilis (similarity index 99.805 %), NRP1-18 to Streptomyces subrutilis (similarity index 100%), NRP1-26 to Streptomyces enissocaesilis (similarity index 99.728%), NRP1-35 to Streptomyces drozdowiczii (similarity index 99.428%) and NRS1-11b Streptomyces fragilis (similarity index 100%). NRP1-13 was found to be most closely related to Nocardia asiatica (similarity index 99.780%) and NRS1-18 to Micromonospora chalcea (similarity index 99.659%).

microbial-biochemical-technology-actinobacteria

Figure 2: Phylogenetic relationship of the bioactive Nambul actinobacteria with its closest homolog.

Rifaat [33] reported the predominance of Streptomyces in water sample and that of Micromonospora in sediments of the Nile 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.

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.

Acknowledgements

The authors acknowledge the kind help of Dr. S. Shivaji, Scientist (Deputy Director), CCMB, Hyderabad and Dr. S. Mayilraj, Scientist, MTCC, IMTECH, Chandigarh for providing the DNA sequencing facility.

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