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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 193-199

AntiMycobacterial activity of endophytic actinobacteria from selected medicinal plants


1 Department of Biotechnology, Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India
2 Centre for Drug Discovery and Development, Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India

Date of Submission21-May-2020
Date of Acceptance28-Jun-2020
Date of Web Publication12-Sep-2020

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


DOI: 10.4103/bbrj.bbrj_73_20

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  Abstract 


Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), poses a serious threat to human life. In addition, certain nontuberculous mycobacterial (NTM) species also cause infections in immune suppressed and even in immunocompetent individuals. With this view, the present study investigated the antimycobacterial properties of endophytic actinobacteria using Mycobacterium smegmatis as a surrogate model. Methods: Endophytic actinobacterial cultures were isolated from seven medicinal plants and their antimicrobial activity against two Gram-positive bacteria, three Gram-negative bacteria, fungi, and M. smegmatis was studied. Crude bioactive metabolites from one potential culture EAM4 were tested against Staphylococcus aureus, Escherichia coli, and M. smegmatis by disc-diffusion method, whereas activity against MTB H37Rv was tested by luciferase reporter phage (LRP) assay. The potential actinobacterial culture EAM4 was identified based on their 16S rRNA analysis. Results: Thirty actinobacterial cultures were isolated and were belonged to the genus Streptomyces. Maximum number of actinobacterial strains was isolated from Selaginella stellata (36.6%) and Pilea microphylla (36.6%). Gram-positive pathogens are inhibited by more number of actinobacterial cultures (>90%) than Gram-negative pathogens (50%). Twenty-eight of thirty actinobacterial cultures showed inhibitory activity against M. smegmatis. Bioactive metabolites from the potential culture EAM4 showed better inhibition against the S. aureus, E. coli, and M. smegmatis. In the LRP assay, 79% inhibition was exhibited against the MTB H37Rv. The potential actinobacterial strain EAM4 was identified as Streptomyces sp. based on their 16S rRNA analysis. Conclusion: Findings of the present study revealed that the endophytic cultures of Streptomyces investigated in this study are the promising source for isolating antimycobacterial metabolites effective against TB and nontuberculous mycobacterial pathogens.

Keywords: Antimycobacterial, endophytic, Mycobacterium smegmatis, Mycobacterium tuberculosis, Streptomyces


How to cite this article:
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;4:193-9

How to cite this URL:
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 [serial online] 2020 [cited 2020 Dec 5];4:193-9. Available from: https://www.bmbtrj.org/text.asp?2020/4/3/193/294864




  Introduction Top


Worldwide, tuberculosis (TB) remains among the top 10 causes of death. According to the World Health Organization Report (2019), 10 million peoples are estimated to fell sick with TB worldwide and 1.5 million people died.[1] Typically, after the 6–8 months of intensive antibiotic treatment, about 80%–90% of drug-sensitive TB patients are cured. However, the side effects due to combination anti-TB therapies result in the nonadherence of TB patients for treatment which, in turn, lead to the emergence of high levels of drug-resistant M. tuberculosis (MTB) strains. In addition, it takes up to 24 months of combination chemotherapy for the treatment of drug resistant TB.[2] Hence, there is a need to discover novel drugs to treat TB with lower toxicities and shorter duration of treatment.[3]

In addition, at the global level, the frequency and mortality due to diseases caused by the pathogenic nontuberculous mycobacteria (NTM) have been increasing constantly. In early days, NTMs are thought to be harmless environmental saprophyte bacterial members of mycobacterial genera and only very few species are risky to immune-suppressed individuals or those with imperfect lung structure. Nevertheless, now, some NTM species are infecting children and adults at a higher rate, even those who are immunocompetent.[4] Unfortunately, like TB, NTM infections are incredibly difficult to treat due to the inherent resistance of these bacteria to many common antibiotics.[5],[6] Hence, there is a timely need to discover new antibiotics effective against MTB and other NTM pathogens.

Microbes, especially the members of the phylum Actinobacteria, are the prolific producers of anti-TB antibiotics for several decades.[7],[8],[9] Two important antibiotics, streptomycin and rifamycin, in current anti-TB regimen are produced by the members of Actinobacteria. Several other anti-TB metabolites were also reported from different actinobacterial genera.[10],[11] However, there is a turndown in the newness of antimycobacterial metabolites from Actinobacteria due to the comprehensive searching of usual sources which results in the re-discovery of known metabolites. As an alternative, exploring Actinobacteria from underrepresented sources or ecosystems may find the lane for isolating novel antimycobacterial metabolites.

Medicinal plants are customarily explored for several pharmaceutical compounds including those with anti-TB properties. However, none of them reached the marketable level for TB treatment. In parallel, microbes associated with the healthy plant parts without causing any ill effects to their host plants, called endophytes, are documented as the richest source for novel bioactive metabolites with useful antibacterial, antifungal, and anticancer properties.[12],[13] There are few anti-TB antibiotics such as munumbicin B effective against multidrug-resistant (MDR) MTB that are reported from endophytic Streptomyces.[14] The present study investigated the endophytic actinobacteria from selected medicinal plants collected from Kerala, India, for their antimycobacterial properties against Mycobacterium smegmatis and MTB.


  Methods Top


Sample collection and pretreatment

A total of 7 medicinal plants as listed in [Table 1] were collected from the Vadakara region of Kerala state in India during November 2018. Specific plant parts, as mentioned in [Table 1], from each plant were collected and thoroughly washed for a minute using running tap water to take away the soil and other organic debris. The washed plant parts were dried, crumbled into small fragments, and processed with a four-step surface sterilization method. Briefly, plant parts are separately sterilized by immersing it in 70% ethanol for 1 min, sodium hypochlorite for 5 min, 90% ethanol for 30 s, and a final rinse with distilled water.[15]
Table 1: Endophytic actinobacteria isolation from medicinal plants

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Isolation of endophytic actinobacteria

Each sample was then ground using a clean motor and pestle with 5 ml of distilled water. The resultant homogenates were serially diluted 10−5 dilutions using sterile distilled water. Hundred microliter aliquots from the 10−3, 10−4, and 10−5 dilutions were plated on starch casein nitrate agar (SCA) media (supplemented with nalidixic acid (20 μg/ml and nystatin 50 μg/ml) and spread using flame sterilized L-rod. Colonies with actinobacterial morphology have been selected after 4 weeks of incubation at 28°C.[16] Morphologically, different colonies of Actinobacteria were retrieved from SCA with suspected actinobacterial morphology that have been retrieved from incubated plates and inoculated onto Yeast Extract Malt Extract (YEME) agar medium and incubated for 7 days at 28°C. The retrieved actinobacterial cultures were stored in glycerol broth (20%) and slants of YME agar.[17]

Characterization of endophytic actinobacteria

Cultural characteristics of actinobacterial cultures were recorded after 10 days of growth at 28°C on the YEME agar medium. Actinobacterial cultures were observed for their growth level, colony consistency, the aerial mycelia color, reverse side pigment, and soluble pigment production. Micromorphological features were examined by the method of slide culture using YEME agar. On the 10th day of incubation, slides were observed under a bright-field microscope with × 40. The noted micromorphological features include the vegetative mycelium, reproductive mycelium, and mycelia fragmentation if any.[18]

Screening for in vitro antimicrobial activity

Agar plug method[17] was followed to screen the actinobacterial cultures against a panel of test pathogens, namely Staphylococcus aureus ATCC 29213, Bacillus cereus,  Escherichia More Details coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13882, and Candida albicans. All the pathogenic cultures were grown on Mueller Hinton broth by incubating at 37°C for 18 h. Inoculum with 0.5 McFarland standard was spread using sterile cotton swan on Mueller Hinton Agar (MHA) plates. For the production of extracellular metabolites, all the endophytic actinobacterial cultures were cultivated for 10 days on YEME agar plates at 28°C. After incubation, the mycelial biomass was collected from the agar plates with the help of a sterile spatula. Then, 5-mm diameter agar plug containing the secreted actinobacterial metabolites was taken from the YEME agar medium and placed over MHA plates. The inhibition zone was measured after 24 h of incubation at 37°C and expressed in millimeter in diameter.

Screening for antimycobacterial activity using Mycobacterium smegmatis as surrogate

All the actinobacterial strains were tested for antimycobacterial activity against the NTM, M. smegmatis, by adopting the agar plug method as described above with some modifications. A loopful of M. smegmatis colonies were inoculated into 0.3 ml of Middlebrook 7H9 broth in a sterile Bijou bottle added with glass beads. The content was vortexed for 30 s and kept undisturbed for 5 min. Then, the suspension was made up to 5 ml using Middlebrook 7H9 broth. Then, 200 μl of M. smegmatis suspension was added to 5 ml of molten 7H11 agar and poured immediately onto Middlebrook 7H9 agar plate. Agar plug (5 mm in diameter) of actinobacterial cultures from the YEME agar was placed over M. smegmatis seeded MHA plates. The plates were sealed and incubated at 37°C for 48 h. The zone of inhibition was measured after incubation.

Bioactive metabolites production from EAM4

Endophytic actinobacterial culture EAM4, which showed maximum inhibition against M. smegmatis and other microbial pathogens, was selected for the bioactive metabolites production through agar surface fermentation.[17] Spores of the potential culture EAM4 were inoculated onto five YEME agar plates for the secretion of extracellular secondary metabolites. The mycelial growth from the culture plate was scrapped after 10 days of incubation with the help of a sterile spatula. The remaining agar medium which contains the secreted secondary metabolites was cut into pieces and extracted using ethyl acetate, methanol, chloroform, and hexane separately (1:2 ratio) for 24 h. The solvent portion was collected and concentrated using a rotary evaporator and quantified.

In vitro screening of extracts against Staphylococcus aureus, Escherichia coli, and Mycobacterium smegmatis

The solvent extracts of the actinobacterial culture EAM4 were tested by adopting the method of well diffusion against S. aureus, E. coli, and M. smegmatis. The main stock of the ethyl acetate extract at 10 mg/ml concentration was prepared using DMSO. The main stock was further filtered using a syringe filter (0.45 μ). One mg per ml of working stock solution was prepared by adding 100 μl of stock extract into 900 μl of solvent and mixed well. Inoculum of all three test pathogens was prepared and inoculated on to respective agar media by adopting the same procedure described in the preliminary screening. A well with 5 mm in diameter size was made on all the plates using sterile cork borer and loaded with 20 μl of different solvent extracts obtained from the culture EAM4. The inhibition zone was measured after 24–48 h of incubation at 37°C.

In vitro screening of extracts against Mycobacterium. tuberculosis H37Rv

The antitubercular activity of the ethyl acetate extract was tested by adopting luciferase reporter phage (LRP) assay[17] using MTB H37Rv as a test pathogen. Two mg per ml of working concentration of sample was prepared using 10% dimethyl sulfoxide (DMSO) and filtered using 0.45 μm filters. The viability of MTB H37Rv culture was maintained on Lowenstein–Jensen medium at 30°C ± 2°C.

High titer of mycobacteriophage phAETRC202 (obtained from the Department of Bacteriology, National Institute for Research in Tuberculosis, Chennai, India) was propagated using M. smegmatis in Middlebrook 7H9 complete medium. Approximately 350 μl of G7H9 broth supplemented with 10% albumin dextrose and 0.5% glycerol taken in cryovials and 50 μl of actinobacterial extract from main stock was added into it to achieve a final concentration of 200 μg/ml. Then, 100 μl cell suspension of MTB H37Rv was added into all the vials. The 10% DMSO was also included in the assay as the solvent control. After incubation for 72 h at 37°C, 50 μl of high-titer phage phAETRC202 and 40 μl of 0.1 M CaCl2 solution were added into the test and control vials. All the reaction vials were once again incubated for 4 h at 37°C. After incubation, 100 μl of the reaction mixture from each vial was transferred to luminometer cuvette and added with 100 μl of D-Luciferin solution. The relative light unit (RLU) was measured using the luminometer (Berthold) at 10 s interval time. Extracts showing a 50% reduction or more in RLU were considered as active against MTB.



Molecular characterization of potential actinobacterial culture EAM4 by 16S rRNA analysis

The genomic DNA of the potential culture EAM4 was isolated using solute ready genomic DNA kit and further analyzed by gel electrophoresis and quantified using a spectrophotometer (NanoDrop ND-1000, Thermo Scientific, Gloucester, UK). The 16S rRNA gene sequence of strain EAM4 was amplified using the primers: 27F 5´ AGAGTTTGATCMTGGCTCAG3´ (forward) and 1492R 5´ TACGGYTACCTTGTTACGACTT3´ (reverse). The polymerase chain reaction (PCR)-amplified product was sequenced and analyzed at Eurofins Genomics, Bangalore, India. The obtained 16S rRNA gene sequence was aligned with similar sequences available in GenBank using the MEGA 7 program. The aligned sequences were used to construct the phylogenetic tree by following the neighbor-joining algorithm in the MEGA 7 program.[19] The bootstrap method was used to determine the confidence of the branches (phylogenetic tree branches).[20] The partial 16S rRNA nucleotide sequence of strain EAM4 has been deposited into the GenBank database.


  Results and Discussion Top


Isolation and characterization of endophytic actinobacterial strains

In this present study, a total of 30 endophytic actinobacterial cultures (EAM 1–EAM 30) were isolated from seven medicinal plants collected from Kerala, India. The number of actinobacterial cultures isolated from different plants is given in [Table 1]. In some earlier studies, endophytic actinobacteria are reported from Aloe vera,[21]Ocimum tenuiflorum,[22],[23] and Piper nigrum[24] but not from Sida cordifolia, Sauropus androgynus, Selaginella stellate, and Pilea microphylla. In the present study, the maximum number of actinobacterial cultures was isolated from S. stellate (36.6%) and P. microphylla (36.6%) [Table 1]. Morphological characteristics of endophytic actinobacterial cultures are given in [Table 2]. On the YEME agar, the maximum of the cultures (90%) showed good growth with powdery colonies (80%) with grey aerial mycelium (50%). About 46.6% of the cultures produced brown reverse side pigment and 33.3% of the cultures produced brown or pale brown soluble pigments. All the cultures showed the presence of both vegetative (substrate) and reproductive (aerial) mycelium under bright filed microscopic observation. Based on the phenotypic characteristics, the isolated cultures were tentatively identified as Streptomyces. Similarly, in previous studies also, Streptomyces species are the predominant endophytic actinobacterial genera reported from different medicinal plants.[25],[26]
Table 2: Morphological characteristics of endophytic actinobacterial strains

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In vitro antimicrobial activity

In the preliminary screening for antimicrobial activity, among the 30 actinobacterial cultures, 27 cultures were active against S. aureus and 19 strains were active against B. cereus. Among the Gram-negative bacterial pathogens tested, E. coli was inhibited by a maximum of 16 actinobacterial cultures, followed by K. pneumoniae being inhibited by 15 strains, whereas P. aeruginosa was inhibited by 11 actinobacterial cultures [Figure 1]a. Sixteen actinobacterial strains were found to be active against C. albicans. Interestingly, actinobacterial cultures such as EAM3, EAM4, EAM6, EAM12, and EAM17 showed broad-spectrum activity against both the Gram-positive and Gram-negative bacterial pathogens and the fungi C. albicans [Table 3]. Similarly, Streptomyces sp., isolated from A. vera was exhibited maximum inhibition against Gram-positive bacteria, S. aureus and Micrococcus luteus followed by Bacillus subtilis and fungal pathogens Aspergillus niger, followed by Candida tropicalis, C. albicans, and Microsporum canis.[21] According to the early reports, the maximum number of actinobacteria inhibits Gram-positive bacteria than Gram-negative bacteria.[27] However, in this study, about 50% of the endophytic actinobacterial cultures were found to be active against Gram-negative bacteria with or without showing activity against Gram-positive bacterial pathogens. It also noted that in a previous study by Manikkam et al,[17] 50% of the actinobacterial cultures isolated from rare ecosystems in India showed activity against Gram-negative bacterial pathogens.
Figure 1: Antimicrobial activity of actinobacterial strains. (a). No. of actinobacteria strains showing the inhibition against various pathogens. (b). Zone of inhibition against Mycobacterium smegmatis

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Table 3: Antimicrobial activity of endophytic actinobacteria by agar plug method

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Screening for antimycobacterial activity using Mycobacterium smegmatis

In this study, M. smegmatis culture was inhibited by 28 actinobacterial cultures. The zone size was ranged between 10 and 25 mm in diameter [Figure 1]. Notably, 16 actinobacterial strains showed zone of inhibition between 20 and 25 mm in diameter [Figure 2]. It is difficult to use slow-growing mycobacterium like MTB for screening a huge number of compounds or extracts on a high throughput basis. Therefore, in previous studies, M. smegmatis is the surrogate of choice for antimycobacterial screening due to its nonpathogenic nature, faster growth rate than MTB. The bioassay using M. smegmatis as a surrogate usually serves to prioritize the candidates in anti-TB screening.[28] The active extracts or compounds further can be tested in more specificin vitro assays on pathogenic drug-sensitive, MDR-, and XDR-MTB strains. In some previous studies, extracts of endophytic actinobacterial strains are reported to be active against M. smegmatis,[29] whereas in the present study, the endophytic actinobacterial cultures were screened by the simple agar plug method in which 93% of actinobacterial strains were found to be active against M. smegmatis. Hence, this agar plug method is a simple and rapid approach to study the antimycobacterial activity of Actinobacteria using M. smegmatis as a substitute host.
Figure 2: Antimycobacterial activity of endophytic actinobacteria

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Production, extraction, and activity testing

In agar surface fermentation, the actinobacterial culture EAM4 showed good growth on YEME agar medium. Among the four different solvents tested, crude extracts were obtained only in the ethyl acetate (21 mg) and methanol (29 mg) but not in the chloroform and n-hexane. In the agar well diffusion method, only the ethyl acetate extract showed inhibition against S. aureus (16 mm), E. coli (10 mm), and M. smegmatis (23 mm). The activity exhibited by the methanol extract was not considered since it showed less than a 10 mm zone of inhibition. The previous study also reported that the ethyl acetate extract of actinobacterial strains was isolated from various sources which showed the maximum level of antimycobacterial activity.[29],[30]

In vitro screening of strain EAM4 ethyl acetate extracts against M. tuberculosis

In the antitubercular screening by the LRP assay, the ethyl acetate extract of strain EAM4 showed 79.93% inhibition against the standard laboratory strain MTB H37Rv. The present investigation is exactly correlated with other studies, showing that ethyl acetate extract of endophytic actinobacteria isolated from medicinal plant showing anti-TB activity against MTB H37Rv.[29],[31]

Characterization and identification of strain EAM4 by 16S rRNA analysis

Strain EAM4 produced good colonial growth with powdery consistency with brown soluble and reverse side pigment on the YEME agar medium. Both the aerial and substrate mycelium were observed under the microscopic observation. PCR amplification of the 16S rRNA gene from the strain EAM4 yielded 1491 base pair sequence size. The BLAST analysis revealed that the EAM4 16S rRNA gene sequence showed 99.87% similarity with the genus Streptomyces. Phylogenetic tree constructed based on the neighbor-joining method also indicated its closest similarity with Streptomyces maritimus and Streptomyces rochei (99%) and other Streptomyces species [Figure 3]. The 16S rRNA gene sequence of strain EMA4 was published in GenBank with the accession number KJ413010. There are several antitubercular antibiotics that are reported from different Streptomyces sp. but not from S. maritimus and S. rochei. However, in the present study, antitubercular activity was exhibited by the Streptomyces EAM4 which is close to S. maritimus and S. rochei. Moreover, the production of secondary metabolites is a strain-specific process rather than a genus and species specific.
Figure 3: Neighborjoining phylogenetic tree based on 16SrRNA gene sequences comparing strain EAM4 to Streptomyces species. The number on the branches indicates the percentage bootstrap values of 1000 replicates; only values >50% are indicated. Nocardia farcinica DSM43665 used as outgroup

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  Conclusion Top


Findings of the present study revealed that the endophytic cultures of Streptomyces investigated in this study are the promising source for isolating antimycobacterial metabolites effective against tuberculous and NTM pathogens.

Acknowledgement

The authors would like to thank the authorities of Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India, 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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