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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 1  |  Page : 88-93

Antimycobacterial Properties and Metabolite Profiling of Fish Gut-Associated Streptomyces sp. MCA2


1 Department of Biotechnology, Sathyabama Institute of Science and Technology (Deemed to be University), Chennai, Tamil Nadu, India
2 Centre for Drug Discovery and Development, Sathyabama Institute of Science and Technology (Deemed to be University), Chennai, Tamil Nadu, India

Date of Submission18-Jan-2021
Date of Acceptance21-Feb-2021
Date of Web Publication13-Mar-2021

Correspondence Address:
Dr. Manikkam Radhakrishnan
Centre for Drug Discovery and Development, Sathyabama Institute of Science and Technology, Chennai - 600 119, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_9_21

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  Abstract 


Background: Infectious diseases such as tuberculosis and other opportunistic diseases affecting the lungs are major threats concerning public health. These pathogens have gained resistance to the currently available drugs which has prompted the discovery of novel antibiotics to fight against such pathogenic organisms. With unique pharmaceutical and biotechnological applications, Actinobacteria are considered possessing rich source of natural products. Biodiscovery of actinobacterial metabolites depends on the identification and recovery from unexplored environments and effective assessment of their metabolites. Literature states that among various ecosystems, fish are promising yet understudied source. Methods: Potential strain was screened from 13 fish gut-associated actinobacterial strains for their antimycobacterial and antitubercular properties. Ideal fermentation methodology has opted for bioactive metabolite production. Optimization procedures, minimum inhibitory concentration determination, characterization studies, and metabolite profiling through gas chromatography mass spectrometry were performed. Results: Potential strain MCA2 was selected for their antagonistic activity against Mycobacterium smegmatis. Bioactive metabolite of potential strain was produced through agar surface fermentation and extracted using ethyl acetate. Antitubercular activity was confirmed by >50% reduction in relative light unit against Mycobacterium tuberculosis H37Rv. The potential strain MCA2 was identified as Streptomyces sp. based on its phenotypic characteristics. Conclusion: The outcome of this study outlined the antimycobacterial potential of isolated fish gut-associated Streptomyces sp. strain confirming that fish guts are promising sources for isolating metabolites active against tuberculosis and other nontuberculous mycobacterial diseases.

Keywords: Actinobacteria, antimycobacterial, fish gut, Mycobacterium smegmatis, Mycobacterium tuberculosis, metabolite profiling, Streptomyces


How to cite this article:
Nekkanti DS, Sasikumar U, Baskaran A, Kaari M, Venugopal G, Radhakrishnan M. Antimycobacterial Properties and Metabolite Profiling of Fish Gut-Associated Streptomyces sp. MCA2. Biomed Biotechnol Res J 2021;5:88-93

How to cite this URL:
Nekkanti DS, Sasikumar U, Baskaran A, Kaari M, Venugopal G, Radhakrishnan M. Antimycobacterial Properties and Metabolite Profiling of Fish Gut-Associated Streptomyces sp. MCA2. Biomed Biotechnol Res J [serial online] 2021 [cited 2021 May 12];5:88-93. Available from: https://www.bmbtrj.org/text.asp?2021/5/1/88/311102




  Introduction Top


Actinobacteria are unicellular, aerobic, and Gram-positive bacteria that produce a broad range of secondary metabolites with pharmacological potentials. Researchers have been keenly interested in the production of bioactive secondary metabolites which leads to the discovery of antibiotics.[1] In search of Actinobacteria with new gene clusters, researchers have started mining the unexplored/understudied sources obtaining novel secondary metabolites that are of great interest in the current era.[2]

Investigations on microbes present in the gut of fish started in the late 1920s which stated that the bacterial level in the fish gut is low and derived from their environment. A recent discovery of fish gut microbes described their role in immune defense, development, nutrition, resistance against harmful pathogens, and metabolite hemostasis. Actinobacteria reside as endosymbionts inside the gut of fish. Literatures on fish gut Actinobacteria depict their excellent genomic diversity and biosynthetic potential being richest yet understudied source.[3]

Mycobacterial diseases including tuberculosis (TB) caused by Mycobacterium tuberculosis and other opportunistic diseases among immune-compromised patients by nontuberculous mycobacteria (NTM) like Mycobacterium cansasii are major threat to humans worldwide.[4],[5] Despite the commercial availability of antibiotics for tuberculosis, emergence of resistance to the currently available drugs has urged the situation to discover novel antibiotics against M. tuberculosis and M. cansasii. The increase in mortality due to pathogenic NTM at a global level has forced the researchers to search for novel antibiotics. Multidrug therapies available for such disease-causing pathogens have their own limitations especially their long regimen.[6],[7]

Anti-TB antibiotics have been produced by Actinobacteria, precisely the genera, Streptomyces. Diverse actinobacterial genera are also involved in producing anti-TB metabolites.[8] Despite these constant discoveries, the need for development of drugs against TB and NTM still engages the researchers as the pathogens develop multidrug resistance.[9] In the present study, bioprospecting of Actinobacteria was carried out from fish gut, previously isolated and stored in Actinobacteria culture collection, Sathyabama Institute of Science and Technology, and their antagonistic activity was studied against Mycobacterium smegmatis and M. tuberculosis.


  Methods Top


Cultivation of actinobacterial strain

Thirteen fish gut-associated actinobacterial strains were obtained from the Actinobacterial Culture Collection, Centre for Drug Discovery and Development, Sathyabama Institute of Science and Technology. All the cultures were grown on International Streptomyces Project 2 (ISP2) agar medium supplemented with 50% seawater at 28°C for 7–10 days. Cultural characteristics recorded include growth, colony consistency, aerial mass color, presence of reverse side pigment, and soluble pigment production.[10] This study was reviewed and granted a waiver of individual informed consent by the Ethical Review Committee of the Institute of Science and Technology (Deemed to be University), Chennai, Tamil Nadu, India.

In vitro screening for antimycobacterial activity

Antagonistic activity of fish gut-associated Actinobacteria was tested against M. smegmatis by adopting agar plug method.[11] Agar plugs of 5 mm diameter were cut from the actinobacterial cultures grown on ISP2 agar and were placed over 7H9 medium seeded with test pathogen, M. smegmatis. All the plates were incubated at 37°C for 24 h for observing the zone of inhibition.[12] Actinobacterial strain that showed maximum activity was selected as the potential strain for further studies.

Extraction and production of bioactive compounds

Effect of solid-state and submerged fermentation on the most potential strain was investigated. Spores of potential actinobacterial strain were inoculated onto ISP2 agar plates and 100 ml of ISP2 broth. ISP2 agar plates were incubated at 28°C for 12 days. ISP2 broth flasks were incubated in a rotary shaker with 95 rpm for 12 days. For every 24 h, agar plug from ISP2 agar plates was taken and tested against M. smegmatis.[12] Each 2 ml of ISP2 broth was taken and centrifuged at 10,000 rpm for 10 min. The cell-free supernatant was tested against M. smegmatis by adopting well diffusion method.[13] The optimal fermentation method was then chosen based on the activity of agar plugs/cell-free supernatant against the pathogen.

In vitro screening of crude extracts for antitubercular activity

Luciferase reporter phage (LRP) assay was applied for testing antitubercular activity of the crude extract against test pathogen M. tuberculosis H37Rv. The M. tuberculosis isolates were maintained on Lowenstein–Jensen medium. The working standard of crude extract was prepared using 10% dimethyl sulfoxide and filtered using 0.45 μm filters. Propagation of high titer mycobacteriophage phAETRC202 (obtained from the Department of Bacteriology, National Institute for Research in Tuberculosis, Chennai, India) was achieved using M. smegmatis in Middlebrook 7H9 complete medium. The LRP assay was performed by following the procedure detailed by Ashok et al., 2020.[14] A reduction of 50% or more in the relative light unit (RLU) denotes the activity of crude extracts against MTB.

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) of the actinobacterial crude extract was determined against M. smegmatis in 96 well microtiter plate. The initial concentration of the crude extract was prepared to 512 μg/ml, which was then diluted up to 2 μg/ml. 10 μl of 24 h grown inoculum was added and the final volume was made to 200 μl by 7H9 broth. Culture broth served as a positive control and inoculum along with broth served as a negative control. Plates were incubated for 24 h at 37°C. After the incubation, optical density was measured at 630 nm using ELIZA reader (Biotech-EL × 800) and the percentage of bacterial inhibition was calculated by using the formula:



Partial purification of crude extract

Partial purification of crude extract containing antimycobacterial compound was done by adopting thin layer chromatography analysis. The number of components present in the crude extract was analyzed by thin-layer chromatography using readymade silica gel coated plates (20 mm × 20 mm) (Merck). Crude extract was dissolved in a small volume of ethyl acetate and placed on the TLC sheet using a glass capillary tube. The chromatogram was run by using different combinations of solvent systems such as chloroform: methanol, chloroform: methanol: dichloromethane, butanol: acetic acid: water, hexane: acetone, hexane: methanol, chloroform: ethyl acetate, hexane: ethyl acetate. Rf values of the separated compounds were calculated by adopting the standard formula.[15]

Identification of components by gas chromatography mass spectrometry

Analysis of the ethyl acetate extract was carried out by using gas chromatography mass spectrometry (GC-MS) instrument equipped with a capillary column (0.25 mm film thickness × 0.25 mm diameter × 30 m length). Column used is RXI 5 silicone MS. The instrument was operated in electron impact mode at ionization voltage, injector temperature (250°C) with split mode of injection, column temperature (90°C), column flow (0.99 ml/min), and pure flow (3.0 ml/min). The carrier gas used in helium (99.9%) purity at a flow rate of 1 ml/min and about 10 μl of sample was injected. It also has an oxygen trap which removes oxygen molecules and acts as a molecular sieve. The oven temperature was initially programmed at 90°C with 2 min hold time and then increased to 160°C and finally to 260°C with 5 min hold time.

Characterization of potential actinobacterial strain

Cultural characteristics were studied based on the growth of potential actinobacterial strain in different ISP media such as tryptone agar media (ISP 1), yeast extract malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salts starch agar (ISP 4), glycerol-asparagine agar (ISP 5), peptone-yeast extract-iron agar (ISP 6), and tryptone agar (ISP 7). All the media were prepared by following the guidelines described by Shirling and Gottlieb.[10] Inoculation of potential strain was done in all the different ISP media mentioned above and the plates were incubated for about 4 days at room temperature. Cultural characteristics recorded include the nature of growth, consistency, and aerial mass color if any. Physiological characteristics of potential strain were investigated through the utilization of carbon, nitrogen, and mineral sources.


  Results and Discussion Top


Sample retrieval

A total of 13 strains were collected and retrieved from SACC. Of 13 strains collected, only 8 showed good growth on ISP 2 media.

Morphological characteristics of fish gut actinobacterial strains

After retrieval, the strains were subcultured for observing their growth and characteristics. Majority of the cultures showed powdery, pasty, and leathery consistency, with aerial mass color ranging from white to yellow, as depicted in [Table 1].
Table 1: Morphological characteristics of fish gut-associated actinobacterial strains

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Antimycobacterial activity of fish gut-associated strains against Mycobacterium smegmatis

Of all the strains tested for antimycobacterial activity, 62.5% strains showed more than 12 mm zone of inhibition. Agar plug methods are in use for many decades for the preliminary detection of antagonistic activity exhibited by the extracellular metabolites of Actinobacteria.[16] The literature survey evidenced that there are no previous works on antimycobacterial activity of fish gut Actinobacteria. The strain which showed maximum zone of inhibition was considered for further analysis [Figure 1].
Figure 1: Antimycobacterial activity of fish gut-associated actinobacterial strains observed as zones of inhibition

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Among all the strains, the strain MCA2 has the highest zone of inhibition, and hence, it is selected for further studies. For statistical analysis, triplicate of strains MCA1 and MCA2 was performed and observed that MCA2 had more potential with a 30 mm zone of inhibition.

Selection of potential strain

The fish gut actinobacterial strain MCA2 [Figure 2] and [Figure 3] was selected as the potential strain as it possesses a broad range of antimycobacterial activity against M. smegmatis.
Figure 2: Potential actinobacterial strain MCA2

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Figure 3: Morphology of MCA2 under bright-field microscope

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Effect of fermentation method on bioactive compound production

The effect of fermentation method for potential strain MCA2 was checked for its antimycobacterial activity against M. smegmatis. The effect was studied by both agar plug and liquid well diffusion method for solid state/agar surface and submerged fermentation respectively. Since the strain showed activity only by agar plug method but not in well diffusion method till day 12 of fermentation, it was concluded that the actinobacterial strain produced bioactive compounds only in solid culture but not in liquid culture. Hence, for further bulk production of bioactive compounds from strain MCA2, fermentation was performed by adopting the agar surface fermentation method.

Production of bioactive metabolites by agar surface fermentation

Production of bioactive metabolites was done by adopting the agar surface method. Around 50 ISP2 plates were inoculated with strain MCA2 and incubated at room temperature for about 10 days. During the incubation period, extracellular metabolites secreted into the agar media were extracted. Most of the metabolites reported from actinobacteria were extracellular in nature and they were extracted using ethyl acetate.[11],[15]

Extraction of bioactive compounds

Bioactive metabolites were extracted using the solvent ethyl acetate in the ratio 1:2. Crude extract was obtained after performing evaporation in a rotary shaker evaporator and utilized for further studies.

In vitro screening of antimycobacterial activity against Mycobacterium tuberculosis H37Rv strain

Antitubercular activity of ethyl acetate extract of the actinobacterial strain MCA2 showed 52.49% reduction in RLU against M. tuberculosis H37Rv at 500 μg/ml concentration, whereas at 100 μg/ml concentration, it only showed 40% of inhibition. From the results in [Table 2],[Table 3], it was concluded that the potential strain MCA2 is found to have antimycobacterial activity at 500 μg/ml concentration when tested by LRP Assay. Investigations of Radhakrishnan et al. 2014 reported that extracts from 39 actinomycetes showed activity against one or more M. tuberculosis and in particular 16 inhibited all the three M. tuberculosis strains.[11]
Table 2: Identification of compounds by gas chromatography-mass spectrometry

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Table 3: Physiological characteristics of strain MCA2

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Determination of minimum inhibitory concentration for crude extract

MIC was performed to determine the minimum concentration at which the drug can show its activity. It was performed against M. smegmatis using a 96-well plate. After incubation period, analysis was carried out in an ELISA reader at 630 nm. From the results obtained, MIC was found at 32 μg/ml, with 52.41% of inhibition.

Identification of compounds by gas chromatography mass spectrometry

The ethyl acetate extract collected from strain MCA2 was exposed to GC-MS analysis. GC-MS is done by injecting 1 μl of the sample with injection temperature (250°C) using a split mode of injection with a split ratio of 10. The spectrum has run for about 50 min. The spectrum is represented in [Figure 4]. Peak area, molecular formula, and molecular weight contributed to the identification of compounds. [Table 2] represents the compounds observed in the collected extract. The compounds tertrapentacontane and tetracosane showed a maximum retention time of 48.418 and 51.682, respectively, and have been proved to possess antibacterial, antifungal, and antioxidant properties.[17],[18],[19]
Figure 4: Gas chromatography mass spectrometry spectrum of the actinobacterial extract

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Characterisation and taxonomy of potential strain

Cultural characteristics

The cultural characteristics of strain MCA2 were observed under bright-field microscopy. The growth of MCA2 was studied in different ISP media. Good growth was observed in ISP 1 and ISP 2, while moderate growth was observed in ISP 4, ISP 5, and ISP 6. There was no growth observed in ISP 3 and ISP 7 media.

Physiological characteristics

Fish gut actinobacterial strain MCA2 was able to grow well when supplemented with various carbon sources. This strain was able to utilize various carbon sources such as inositol, lactose, rhamnose, raffinose, and starch [Table 3]. Of all the carbon sources supplemented, mannitol was less utilized. Among the various nitrogen sources supplemented such as peptone, soya bean, potassium nitrate, and soya bean source showed less growth. Various mineral salts were also supplied which included CaCl2, MnCl2, MgSO4, and FeSO4. Of all the four supplied, there was no growth observed in FeSO4. The effect of change in pH in the ISP 2 media was also studied. It was observed that aerial mass color changed by changing the pH from acidic to alkaline. At pH 9, the color change can be clearly identified. Based on the studied phenotypic characteristics, the potential strain MCA2 was identified as Streptomyces sp. Shirling and Gottlieb, 1966, had reported many methods for the characterization of Streptomyces.[10]


  Conclusion Top


The present study directed on bioprospecting of underexplored fish gut-associated Actinobacteria with special emphasis on isolation and characterization of antimycobacterial metabolites. Strain MCA2, a Streptomyces sp. isolated from the fish gut was found to exhibit promising antagonistic activity against M. smegmatis. Our study shows the immense potential of bioactive metabolites from Actinobacteria isolated from fish gut indicating assurance for prospective drug development programs.

Acknowledgment

We thank the management of Sathyabama Institute of Science and Technology (Deemed to be University), Chennai, for the research facilities provided.

Financial support and sponsorship

This work was supported by the management of Sathyabama Institute of Science and Technology, Chennai, by providing the research facilities.

Conflicts of interest

There are no conflicts of interest.



 
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Manigundan K, Revathy S, Sivarajan A, Anbarasu S, Jerrine J, Radhakrishnan M, et al. Bioactive potential of selected actinobacterial strains from Andaman marine ecosystems effective against Mycobacterium tuberculosis and other clinical pathogens. Indian J Geo Mar Sci 2019;48:1307-11.  Back to cited text no. 1
    
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Ashok A, Nandhini U, Sreenivasan A, Kaari M, Kalyanasundaram R, Manikkam R. Antimycobacterial activity of endophytic actinobacteria from selected medicinal plants. Biomed Biotechnol Res J 2020;4:193.  Back to cited text no. 14
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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