Antibacterial potential of antagonistic Streptomyces sp. isolated from marine sponge Dendrilla nigra

Abstract

The role of Streptomyces sp. (BTL7) in synthesis of antibacterial agents reported from the marine sponge Dendrilla nigra was evaluated. Selective isolation of actinomycetes was performed on the newly developed selective media, Sponge Agar (SA) 1 and SA 2. The growth rate and antibiotic production were increased on the media supplemented with sponge extract. The chosen isolate BTL7 showed inhibitory interaction with Micrococcus luteus and the extracellular products contained potent antibacterial agents. The minimum inhibitory concentration of BTL7 against M. luteus was 44 μg protein/ml and the minimum bactericidal concentration was 88 μg protein/ml. Peak antibacterial activity was observed at 72 h in batch culture. Based on the findings, it could be inferred that bacterial endosymbionts sponges could form a reliable source for bioprospecting of next generation pharmaceutical agents.

Marine sponge, Dendrilla nigra, Bacterial-endosymbionts, Strepotomyces, Antibacterial-activity

1 Introduction

The biology of the bacteria – sponge relationship has elicited considerable interest among researchers investigating marine organisms as sources of natural products [1]. Antimicrobial compounds of sponge-associated bacteria suggested that microbial symbionts play a critical role in the defense of their host sponge [2,3]. Marine sponges contain wide array of bioactive secondary metabolites [4–9]. In some instances, the origin of these compounds has shown association with bacterial endosymbionts [10]. For example, Vibrio sp. associated with the sponge Dysidea sp. was shown to synthesize cyototoxic and antibacterial tetrabromodiphenyl ethers [11].

Most of marine invertebrates harbour microorganisms that include bacteria, cyanobacteria and fungi, within their tissues where they reside in the extra- and intra-cellular space [12,13]. Secondary metabolite production can be ascribed to symbiotic microorganisms only when synthesis has been demonstrated in cultures isolated from the host species [14], and it remains possible that these compounds are simultaneously produced by the host. In many instances, the limited availability of sponge material may preclude the commercial production of bioactive compounds [15]. These limitations could be overcome if the need to harvest sponges from the natural environment was eliminated using large-scale laboratory culture that would provide a consistent yield and extraction of bioactive compounds from symbiotic bacteria. The present study evaluates antibacterial potential of Streptomyces sp. BLT7 isolated from a marine sponge, Dendrilla nigra.

2 Materials and methods

The host sponge D. nigra was collected as bycatch in the fishing nets from the southeast coast of India [9]. A portion of collected specimen was subjected to in situ inoculation in Emerson agar (Himedia, Mumbai, India) using a mini clean-air cabinet (Proline^TM). The remaining portion was stored under liquid nitrogen and transported to the laboratory for bacterial isolation. In addition to the standard media compositions (Emerson agar (EA), casein starch agar (CSA), Raffinose-Histidine (RH) medium and GS medium [16]), two new media formulations were prepared for the isolation of actinomycetes. Aqueous extract of sponge tissue was prepared in phosphate buffered saline (PBS) and was filter sterilized (0.2 μm, Millipore) prior to addition to media. Organic extract was prepared from the sponge tissue by consecutive extractions with hexane and dichloromethane and methanol (1:1). The combined extract was concentrated in a rotary vacuum evaporator (Buchi, Flawil, Switzerland) at 40 °C. Isolation was performed on two selective media, Sponge agar (SA) 1 and 2. The main composition of the SA was 10 g raffinose, 1 g l-histidine, 0.01 g FeSO₄·7H₂O, 1 g K₂HPO₄, 0.02 g CaCO₃, 0.5 g MgSO₄·7H₂O, 15 g agar and 20 g NaCl. The medium SA 1 contained 10% aqueous extract and medium SA 2 contained 10% organic extract. For the isolation, 1 cm² area of D. nigra tissue was excised using sterile scissors and homogenized in a tissue homogeniser (Omni, Marietta, USA) using PBS. The aliquot was serially diluted in PBS up to 10⁻⁶ dilution and inoculated on the SA plates. The plates were incubated face up for 7 days at 25 ± 2 °C and examined daily for colony patterns and growth. The most common morphotypes observed on the SA 1 and 2 were picked for the determination of antagonistic property. Burkholder agar diffusion assay was adopted to screen for inhibitory interactions [17]. Biochemical, morphological and physiological characteristics of the antagonistic isolate BTL7 were determined using standard methods [16,18,19]. Antibiotic sensitivity profile was determined by Kirby–Bauer disc diffusion method [20].

The extracellular products (ECPs) from the isolate BTL7 (which were determined to have antibacterial activity) were obtained using the modified cellophane plate technique [21]. Briefly, sterilised cellophane sheets were placed on the surface of SA1 plates and inoculated by spreading 0.5 ml of a 24 h broth culture with a sterile swab. After 72 h of incubation at 25 ± 2 °C, cells were washed off from the cellophane with PBS (pH 7.4). The cell suspensions were centrifuged at 10,000 ×g for 30 min at 4 °C (Plastocrafts^TM) and the resulting supernatant was filter-sterilized and used for bioscreening.

Ammonium sulfate precipitation and subsequent dialysis (desalting) were made for the isolation of protein fractions of ECPs. The total protein content was determined according to Bradford [22]. Antibacterial potentials of ECPs were determined using the bacterial type cultures such as Pseudomonas aeruginosa (MTCC741), M. luteus (MTCC106), Bacillus cereus (MTCC430), E. coli (MTCC40), Salmonella typhi (MTCC733), Staphylococcus aureus (MTCC87), Vibrio fisheri (MTCC1738), Klebsiella pneumoniae (MTCC530), B. subtilus (MTCC441) and Clostridium botulinum (MTCC1215) obtained from Microbial Type Culture Collections (MTCC), Chandigarh, India. For the assay, the inoculum was prepared by adding 5 ml of sterile nutrient broth (Himedia) onto the 18 h fresh slant culture of appropriate bacterium. The tube was shaken gently using a vortex mixer (Remi) and the resultant suspension was collected in a sterile test tube. The first base layer was prepared with 10 ml of 1.5% agar. Six numbers of sterile porcelain beads of 7 mm diameter was placed on the base layer. The overlaid seed layer was prepared by pouring 15 ml of molten nutrient agar (42 °C) containing 0.2 ml of prepared inoculum. After the seed layer solidified, the porcelain beads were removed carefully with a sterile forceps. The consequent wells were filled with the appropriate test compound and control. After 24 h of incubation, the inhibition zone diameter was measured. Modified tube dilution method [23] was used for determining minimum inhibitory concentration (MIC). Minimum bactericidal concentration (MBC) was determined by subculturing the MIC and higher concentration on solid media for bacterial growth. The concentration, which showed no growth, was considered as MBC.

Population growth rate and generation time were determined by analyzing the growth curve of batch a culture. The microbial numbers were measured at consecutive intervals using Petroff–Hausser chamber. Parallel plating was performed for determining the total viable count. The ECPs of culture collected at different time intervals were used for the determination of antibacterial activity. The generation time was extrapolated from the growth curve. The generation time (g) was calculated according to Cappuccino and Sherman [18].

3 Results

We performed in situ inoculation in order to ensure intact isolation process. However, the results indicated that the successful isolation of endosymbionts depended greatly on the media composition. Invariably no growth was observed on the EA plates inoculated in situ and in vitro. Similarly, when the CSA, RH medium and GS medium were used without sponge extract the level of growth was low (Fig. 1). The media composition was standardized during the preliminary experimentations. Based on the growth rate (in terms of colony numbers and size), new media formulations were developed and designated as SA 1 and SA 2. Both media showed similar growth rate in many instances. Most common and similar morphotypes (7 numbers) observed on the SA 1 and SA 2 were selected for antibacterial screening and biochemical characterization.

1

Growth pattern of endosymbionts on various media compositions.

Among the seven isolates (referred to as BTL1–7), BTL7 showed inhibitory interaction with M. luteus. Therefore, the antagonistic isolate BTL7 was used for evaluating the antibacterial potential and biochemical confirmation. Gram staining of the isolate BTL7 indicated that it was a gram-positive filamentous bacterium. The morphological studies prove that the colonies were white, opaque, rough, leathery and hard to remove due to branching filaments that have grown into the media. The bacterial strains BTL1–7 were non-motile, catalase positive and aerobic with an oxidative metabolism. Strain BTL7 produced extra-cellular enzymes like catalase, cellulase and gelatinase. Other properties are shown in Table 2. Since the biochemical characteristics were identical for all seven isolates, they were considered as same strains. However, the antibacterial activity was found to be associated with BTL7. Only strain BTL7 was thus utilized for further studies. The biochemical and morphological characterization indicated that the antibiotic-producing isolate was an actinomycete. Strain BTL7 showed high sensitivity towards chloramphenicol, tetracycline and neomycin and less sensitivity towards penicillin and ampicillin. Streptomycin resistance indicated its typical feature. Based on these, strain BTL7 was tentatively identified as Streptomyces sp.

2

Biochemical, morphological and physiological characteristics of the strain BTL7

Test/Characteristics	Strain BTL7
Gram staining	G+
Shape and growth	Long filamentous aerial mycelium
Motility stab	Non-motile
Growth on MacConkey plates	No growth
Tellurite reduction	+
Oxidase production	–
Catalase production	+
Indole production	–
O/F test	O
ONPG Hydrolysis	+
H2S production	–
Growth at 4 °C	–
Growth at 40 °C	+
Production of extra-cellular enzymes:
Amylase	–
Gelatinase	+
Cellulase	+
Urease	–
Caesinase	–
Acid from
Arabinose	–
Inositol	–
Lactose	–
Maltose	+
Rhamnose	–
Sorbitol	–
Glucose	+
Growth in NaCl:
0%	+
0.5%	+
1%	+
3%	+
6%	+
8%	+
10%	–
Antibiotic sensitivity pattern:
Chloramphenicol (25 mcg/disc)	+
Streptomycin (10 mcg/disc)	–
Tetracycline (10 mcg/disc)	+
Neomycin (30 mcg/disc)	+
Penicillin (5 mcg/disc)	+
Ampicillin (25 mcg/disc)	+

The ECPs of strain BTL7 successfully prevented the growth of the gram-positive bacteria to the extent of 60% whereas the inhibitory potential was decreased towards the gram-negative bacteria (40%). The inhibition zone produced by extracellular proteins of strain BTL 7 for P. aeruginosa was 21 mm and that for M. luteus was 19 mm (Fig. 2). The inhibition zone diameter formed for B. cereus, E. coli, S. typhi and S. aureus were 16 and 15 mm for V. fisheri and B. subtilus. The inhibition zone diameter for K. pneumoniae and C. botulinum was less than 10 mm.

2

Antibacterial activity of ECPs isolated from the isolate BTL7. (PA, P. aeruginosa; ML, M. luteus; EC, E. coli; VF, V. fisheri; SA, S. aureus; KP, K. pneumoniae; BC, B. cereus; BS, B. subtilus; ST, S. typhi; CB, C. botulinum.)

The MIC of BTL7-ECP for M. luteus was 44 μg protein/ml and the MBC was 88 μg protein/ml (Table 1). The MIC for V. fisheri was 176 μg protein/ml and the MBC was roughly 325 μg /ml. The MIC and the MBC of BTL7-ECP for P. aeruginosa was determined as 704 and 1408 μg protein/ml, respectively.

1

MIC and MBC of BTL7

Test organisms	MIC (g protein/ml)	MBC (μg protein/ml)
M. luteus	44	88
V. fisheri	176	352
P. aeruginosa	704	1408

The growth curve showed onset of lag phase immediately post inoculation and the exponential phase occurred 60–72 h post inoculation. The stationary phase occurred between 72 and 144 h and thereafter a decline in bacterial density was seen (Fig. 3). The generation time calculated to be 56 min. Peak antibiotic production occurred at 72 h in a batch culture (Fig. 3).

3

Growth curve and antibiotic production time of BTL7.

4 Discussion

Culture-based studies indicated that the supplementation of host sponge extract in the culture media drastically increased the number of morphotypes. Earlier reports indicated that the expression of the antibiotic producing capabilities only be restored in sponge extract supplemented medium [10,24]. The secondary metabolites of host sponge D. nigra have demonstrated broad-spectrum antibacterial activity and inhibited the growth of all tested bacteria [9]. The present findings suggested that secondary metabolites of D. nigra might have been synthesized by the associated bacterial endosymbionts. Most of the available reports on antibacterial properties of sponges reveal their activity on gram-positive bacteria. Samples of 28 demosponges collected along the French coast indicated higher antibacterial activity against gram-positive bacteria (77%) than against gram-negative bacteria (53%) [25]. Literature indicates that sponges contain potent antibacterial secondary metabolites [26–28]. Many bacteria and cyanobacteria associated with sponges were found to be the sources of antibiotics and other bioactive compounds in marine environment. It was reported that wider biosynthetic capabilities of sponges were associated with the symbiotic microorganisms [29]. The marine bacteria, Pseudomonas species isolated from its host sponge Suberea creba collected from the Coral sea of New Caledonia, produced strong antibiotic quinines [30].

Enrichment and isolation of Streptomyces is of particular interest because of the widespread applications of its derived products. In the present study, antibacterial activity was observed in ECPs of strain BTL7. The ECPs of actinomycete strain (LL-31F508), isolated from an intertidal sediment collected in Key West, Florida, showed potent antimicrobial activity against Staphylococcus and Enterococcus spp. [31]. Literature shows that marine Streptomyces are potential source of antimicrobial agents. Two novel antimycin antibiotics, urauchimycins A and B, were isolated from a fermentation broth of a Streptomyces sp. Ni-80 [32]. The strain was isolated from an non-identified sponge. A marine actinomycete strain TP-A0597 produced two novel antibiotics, watasemycins A and B [33]. The marine Streptomyces sp. 173 showed insecticidal activity in brine shrimp bioassay [34]. The host sponge (D. nigra) of strain BTL7 showed wider range of bioactivity including brine shrimp cytotoxicity [9]. Systemic bioactivity profiling and chemical elucidation may therefore give forth potential novel antibiotics and bioactive agents.

Actinomycetes are useful biological tools producing antimicrobials against bacteria [35]. In general, Streptomyces are primarily saprophytic and are best known from soils where they contribute significantly to the turnover of complex biopolymers and antibiotics [35–38]. In the past two decades, however, there has been a decline in the discovery of new lead compounds from common soil-derived actinomycetes as culture extracts yield unacceptably many metabolites described previously. For this reason, the cultivation of marine actinomycete taxa has become a major focus in the search for the next generation of pharmaceutical agents [39].

The present study explored the role of endosymbionts in secondary metabolite synthesis. In some cases, the associated microorganisms may constitute up to 40% of the biomass of sponges [12]. Sponges are filter feeders and consume microorganisms from the inhaled seawater by phagocytosis. The relationships of marine invertebrates and microorganisms that may serve as food or which live either permanently or temporarily inside marine macroorganisms are highly complex and not yet understood [13,40]. Therefore these endosymbionts might have significant role in the protection of host sponges from predators and other infectious agents. The toxic metabolites (secondary metabolites) secreted by endosymbionts were presumed to be accumulated in sponge tissue. Literature shows that continuous supply of source organisms is a serious obstacle for the compounds, which have taken for preclinical/clinical trails [9]. The pharmaceutical industry is interested in the true source of secondary metabolites in sponges. In this regard, the present study implies that systemic drug development could be sustained using the associated endosymbionts instead of host sponges because the recollection of the host sponge is not necessary. The endosymbiont microorganisms could be stored in the laboratory for a long period and reactivated and/or scaled up in a bioreactor for further studies.

Acknowledgements

Authors are thankful to Rev. Fr. Premkumar, Correspondent and Secretary, Prof. A. Ubald Raj, Principal, Malankara Catholic College, Mariagiri, Kaliakavilai 629153. Help rendered by renowned taxonomist Dr. P.A. Thomas, Scientist Emeritus, CMFRI, Vizhinjam for the identification of sponge is acknowledged.