INTRODUCTION:

Mangroves are salt-tolerant plant species inhabiting the intertidal region of the world. These forests have good adaptation with extreme conditions like high tide, salt concentration, temperature, and anaerobic soil condition¹. This productive ecosystem possesses diverse microorganisms that can produce different kinds of enzymes, antibiotics, secondary metabolites and natural products with immense agricultural, industrial and pharmaceutical applications².

During the past two decades, there is a dramatic increase in bacterial resistance due to the misuse of antibiotics and adecrease in the number of antibiotics discovered. Increasing antibiotic resistance becomes a significant threat to public health worldwide as it reduces the effectiveness of antibiotic treatment resulting in increased morbidity, mortality and health care costs³. In recent times new diseases are spreading very fast; hence the market demand for new drugs is very urgent. With the discovery of new technologies in chemical sciences, though it is now possible to develop engineered biosynthesis of antibacterial compounds, nature remains the richest and most versatile source for new antibiotics^4-5. Natural antimicrobial products are biologically active molecules used as a convenient source for finding novel drug compounds that are active against a wide range of targets^6-8. The main producers of antibiotics from natural sources are the microbes which are useful for novel secondary metabolites with a wide range of biological activities such as enzyme inhibitors, metal chelators, toxins, bioregulators, and immuno suppressants, insecticides, surfactants, and signalling molecules⁹. To date, most of the antimicrobial metabolites reported derive from Gram-positive actinobacteria^10-11. In comparison, report regarding antimicrobial compounds from mangrove actinobacteria has received relatively little attention. The metabolites produced by actinobacteria exhibit antimicrobial activity to several human and plant pathogen¹¹. In recent years, they have become increasingly recognized as a rich and unexplored source of novel antimicrobial compounds with therapeutic potential and have been the subject of extensive study.

Marine environments are the largest untapped source for the isolation of new actinobacteria with the potentiality to produce active secondary metabolites¹². Marine sediment is an inexhaustible resource that has not been properly explored. Since environmental conditions of the marine habitat are extremely different from terrestrial conditions, the microbial species present in this ecosystem are recognized as a source of novel antibiotics and anticancer agents with unusual structure and properties¹³.

Hence the present study aimed to isolate and identify actinobacteria isolates from the mangrove soil of Bhitarkanika and evaluate their antimicrobial potential against the selected pathogen.

MATERIALS AND METHODS:

Isolation of actinobacteria from the soil sample:

Soil samples were collected aseptically in a zip pouch bag below 1 cm of soil from different locations of Bhitarkanika mangrove forest, Odisha, India, and transported to the laboratory. Actinobacteria were isolated by the standard serial dilution method. One gram of soil was suspended in 9mL of sterile distilled water inside laminar flow. The dilution was carried out up to 10^-5 dilutions. A 0.1mL aliquots from the 10^-5 dilutions were spread on the ISP-2 (International Streptomyces project medium No. 2) (Himedia) medium and Actinomycetes isolation Agar (AIA) medium and incubated at 30ºC for 72 h. After 3 days of incubation, the morphologically distinct actinobacterial colonies that appeared were isolated.

Screening ofextracellular enzyme activity:

The isolated actinobacterial isolates were screened for different extracellular enzymes such as amylase¹⁴, protease¹⁵, lipase¹⁶, cellulase¹⁷, phosphate solubilization¹⁸and L-asparaginase¹⁹, following the standard method.

Primary screening of antimicrobial activity:

The antimicrobial activities of isolated actinobacterial isolates were performed by cross streak method²⁰. The Pathogenic organisms selected and used for this test were Staphylococcus aureus (ATCC-3114), Salmonella typhimurium (ATCC-4312), Bacillus subtilis (ATCC-7123), Klebsiella pneumoniae (ATCC700603), Vibrio alginolyticus (ATCC17749), Vibrio parahaemolyticus (ATCC17802), Pseudomonas aeruginosa (ATCC27853) Escherichia coli (ATCC25922), Salmonella typhimurium (ATCC14028), Bacillus subtillus (ATCC6051) and Streptococcus salivarius (ATCC19258). The isolated actinobacteria were streaked in the center of the Mueller Hinton (MH) agar plate (g/L: acid hydrolysate of casein 17.5; starch 1.5; agar 17, pH 7.0) and incubated at 30^◦C for 72h. After 72h, the above selected pathogens are streaked at 90° angles to the selected actinobacterial strain and incubated for 24h at 37^◦C. The antagonism against each test organism was recorded. All experiments were carried out in triplicate (n=3)

Production of secondary metabolites:

The actinobacterial isolate showed a broad spectrum of extracellular enzyme activity, and antimicrobial activity in the primary screening method was selected for the production of extracellular secondary metabolites production under submerged fermentation conditions²¹. The selected isolates were grown in nutrient broth media for the production of secondary metabolites on an orbital shaker (220rpm) at 37 ± 2ºC for 3 days. Along with the test experiment, a control experiment was carried out without inoculating the actinobacterial culture in nutrient broth media. Then the actinobacterial culture was centrifuged at 5000 rpm for 20 min, and the cell- free clear supernatant was extracted with an equal volume of ethyl acetate (1:1)²¹.

Secondary screening of antimicrobial activity:

Agar-Well Diffusion (AWD) method was performed to screen the antimicrobial activity of the selected actinobacterial isolates. The tested pathogens were spread aseptically over the previously prepared Muller Hinton agar plate through spread plate techniques. Using six mm sterile borer, four to five agar cup wells were created on the above agar plate. A 50 μL of extracted broth supernatant of the isolated bacterium was added to such well and kept in a refrigerator for 2 h to allow the diffusion of the bioactive metabolite into the agar plate. After 2 h, the plates were incubated at 37°C in an incubator for overnight. The zone of inhibition (mm) produced around the well was recorded by an antibiotic zone reader²².

Gas chromatography-mass spectrometry (GC-MS) analysis:

For GC-MS, the secondary metabolites produced after fermentation was shaken for about 1 h at 28ºC at 180 rpm, concentrated to 10 mL using a rotary evaporator. The solvent phase was collected and evaporated in desiccators. The completely dried residues were re-dissolved in dimethyl sulfoxide (DMSO) and lyophilized for identification studies. Identification of the secondary metabolites of the extracts was carried out by GC-MS analysis using the protocol of Sengupta et al. ²³ with slight modifications. Analyses were conducted on Thermo Fisher ITQ 900 instrument (EI) and TG-SQC capillary column using helium as carrier gas with a flow rate of 1mL/min. Aliquots of 1µL of each sample were injected into the GC-MS system using an auto sampler. All spectra were recorded on an ion-trap mass spectrometer connected to TRACE ULTRA GC fitted with a liquid autosampler (Al3000). The major constituents were identified by matching their minimum similarity to the NIST library²¹.

Identification of the actinobacterial strain:

Culture characteristics such as colony appearance on ISP-2 agar medium, spore formation, arrangement of spore, andaerial mycelium of the selected actinobacterial isolate were observed under abinocular microscope (Olympus model no. MLX-B and CXi-21) according to standard methods. Physiological characterization such as growth at different temperature (10-40ºC), pH (3-9) and different NaCl concentration (2-10%) (w/v) were checked. In addition, biochemical tests such asVoges–Proskauer reaction, catalase, indole production, oxidase, urease, H₂S production, citrate, nitrate reduction, starch hydrolysis, methyl red, casein hydrolysis, arginine test, and utilization of sugar such as glucose, galactose, arabinose, sucrose, adonitol, lactose, sorbitol, fructose, mannitol, raffinose, salicin, rhamnose, xylose, and meso-inositol were also carried out. The results were compared with Bergey’s Manual of Determinative Bacteriology²⁴.

RESULTS AND DISCUSSION:

Isolation of actinobacteria and screening of extracellular enzyme activity:

In the present study, nine actinobacteria were isolated from mangrove soil samples of Bhitarkanika, Odisha, India, and named as Bhitarkanika actinobacteria (BAB), i.e., BAB -1 to BAB -9. All the nine isolated strains were screened for amylase, protease, lipase, cellulase, L-asparaginase, and phosphate solubilisation activity to determine the biotechnological potential. Although some of the isolates showed positive towards protease, amylase, cellulase, phosphate solubilisation, and L-asparaginase test (Fig.1) but observed negative activity towards lipase. Out of the nine actinobacterial isolates, one actinobacterial isolate, BAB-7, showed hydrolysis activity towards most of the enzymes tested. Microorganisms from mangrove ecosystems are rich sources of industrially important enzymes and antibiotics². In the present study, nine actinobacteria were isolated from the mangrove soil of Bhitarkanika and showed amylase, protease, cellulase, and phosphate solubilization activities. The above hydrolytic enzymes activity by different actinobacterial sp. from mangrove environment were also reported earlier^2,25,26

Fig. 1 Screening of (a) Protease, (b) amylase, (c) phosphate solubilization, (d) L-asparaginase and (e) cellulase activity by actinomycetes isolates, isolated from mangroves of Bhitarakanika

Preliminary screening of antimicrobial activity:

The preliminary antimicrobial activities of the actinobacterial isolates against ten selected human pathogens by the cross streak method showed that two actinobacterial isolates BAB-5 and BAB-7 possess antimicrobial properties, as evidenced by their antagonistic activities against the selected pathogens (Table-1). On the basis of the extracellular enzymes activity and primary antimicrobial activity (Table-1) the actinobacterial isolate BAB-7 is found to be the most efficient microorganism and selected for further study.

Table 1: Primary screening of actinobacterial isolates against pathogen by cross streak method

Pathogenic Strain With ATCC No.	BAB-1	BAB-2	BAB-3	BAB-4	BAB-5	BAB-6	BAB-7	BAB-8	BAB-9
K. pneumonia (ATCC700603)	R	R	R	R	S	R	S	R	R
V.alginolyticus (ATCC17749)	R	R	R	R	R	R	S	R	R
V. parahaemolyticus (ATCC17802)	R	R	R	S	R	R	S	R	R
P. aeroginosa (ATCC27853)	R	R	R	R	R	R	S	R	R
S. aureus (ATCC25923)	R	R	R	R	S	R	R	R	R
S. typhimurium (ATCC14028)	R	R	R	R	S	R	R	R	R
E.coli (ATCC25922)	R	R	R	R	S	R	S	R	R
B.subtillus (ATCC6051)	R	R	R	R	S	R	R	R	R
S. salivarius (ATCC19258)	R	R	R	R	S	R	S	R	R

S= Sensitive, R= Resistant

Secondary screening of the actinobacterial isolates against pathogens:

The selected actinobacterial isolate, BAB-7, was further screened for antimicrobial activity through Agar-Well Diffusion (AWD) method (Fig.2). The actinobacterial isolate, BAB-7, showed antibacterial activities against amaximum number of the tested pathogenic bacteria, with zones of inhibition ranging from 8–18 mm (Table 2). From the preliminary and secondary screening of antimicrobial activity, it has been observed that out of the nine actinobacteria isolates, BAB-7able to produce antimicrobial secondary metabolites against the tested human pathogen. There are vast reports on antimicrobial activity by actinobacteria such as Streptomyces sp., Spirillospora rubra, Spirillospora albida, Micromonospora sp., Rhodococcus sp., Nocardia sp., Gordonia sp., Saccharomonospora, Actinocorallia sp., Streptosporangium sp., Verrucosispora sp., Micromonospora sp. etc. which support the present investigation²⁷.

Table 2: Secondary antimicrobial activity of the actinobacterial isolates, BAB-7

Pathogens	Zone of inhibition (mm)
K. pneumoniae	18mm
V. alginolyticus	17mm
V. Parahaemolyticus	15mm
P. aeroginosa	8mm
S. aureus	No zone found
S. Typhimurium	No zone found
E.coli	18mm
B. subtillus	No zone found
S. salivarius	13mm

ND=not detected

Fig. 2 Secondary screening of antibacterial activity by BAB-7

Gaschromatography-mass spectrometry analysis (GC-MS):

Identification of the bioactive constituents was carried out by GC-MS analysis of the ethyl acetate extract of the actinobacterial isolate BAB-7 (Fig. 3). The GC-MS chromatogram of the crudeextract showed the RT value corresponding to the peak at MS (EI, 70eV): m/z (%) = 154.65. Using the NIST database (NIST11 MS Database and MS Search Program v.2.0g), the primary peak found was similar to the derivative of 2-cyclohex-3-en-1-yl-2-oxoacetic acid (ketomycin).

Production of several antimicrobial metabolites by Streptomyces sp. like actinorhodin²⁸, undecylprodigiosin²⁹, streptomycin, etc.,³⁰were also reported earlier.

Fig. 3 GC-MS study of secondary metabolites from the actinobacterial isolate BAB-7

Identification of the actinobacterial strain:

Based on the morphological and biochemical analysis (Tale- 3), the actinobacterium, BAB-7, was found to be grey-white, positive for Gram staining, spore, catalase, oxidase, starch hydrolysis, nitrate reduction, and casein hydrolysis whereas found negative for H₂S, MR-VP test, indole, citrate, urease, and arginine dihydrolasetest. Based on these morphological and biochemical tests, the isolates BAB-7 was tentatively identified as Streptomyces sp. In the present study, out of the nine actinobacterial isolates, one actinobacterial isolate, BAB-7, showed maximum enzyme activity and antimicrobial activity against selected pathogen and identified as Streptomyces sp. through biochemical techniques. Protease, amylase, cellulase, and L-asparaginase activity by Streptomyces sp. reported in the earlier investigation is well established again in the present investigation ³¹.

Table 3: Biochemical characterization of the actinobacterial isolate BAB-7

Test	Results
Growth characteristics on ISP-2 agar	Whitish
Gram staining	+
Spore	+
Citrate	-
Methyl red	-
Voge’sProskauer	-
Catalase	+
Oxidase	+
H₂S Production	-
Nitrate reduction	+
Indole production	-
Urease	-
Starch hydrolysis	+
Arginine	-
casein	+
Carbon utilization
Glucose	+
fructose	+
lactose	+
arabinose	+
Meso-inositol	-
galactose	+
sucrose	-
adonitol	+
sorbitol	+
manitol	+
raffinose	-
salicin,	-
rhamnose	+
xylose	-

+ (Positive), - (Negative)

CONCLUSION:

In the present research investigation, the production of a compound similar to 2-cyclohex-3-en-1-yl-2-oxoacetic acid derivative by Streptomyces sp. from the mangrove environment of Bhitarkanika could be helpful for human therapeutics purposes against tested bacterial infection. The multiple enzymatic application of the strain would also be of great industrial significance. The result of this present investigation suggests that the actinobacteria Streptomyces sp. isolated from mangrove environment possess a significant potentiality to produce compounds having unique antibacterial activity with a new dimension to microbial natural product research.

ACKNOWLEDGMENT:

The authors are grateful to the authority, faculties, and staff of MITS School of Biotechnology, Bhubaneswar, for providing laboratory facilities to carry out this study.

CONFLICT OF INTEREST:

Declared none.

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Received on 15.02.2021 Modified on 25.05.2021

Research J. Pharm.and Tech 2022; 15(4):1653-1658.

DOI: 10.52711/0974-360X.2022.00276