Investigation on the bioactivity of culturable endophytic and epiphytic bacteria associated with ethnomedicinal plants

Introduction: The growing need for the bioactive compounds of ethnomedicinal plants for the treatment of diseases has resulted in exploitation of medicinal plants. The present investigation aimed to study the diversity of endophytic and epiphytic bacteria associated with ethnomedicinal plants and to explore their potential as source of bioactive compounds. Methodology: Characterization of culturable endophytic and epiphytic bacteria associated with 11 ethnomedicinal plants and their potential as natural antioxidants was assessed through free radical scavenging activity, total phenolics, total flavonoids, metal ion chelation, and antagonistic activity. Genetic screening to assess the potential of endophytes and epiphytes to synthesize bioactive compounds was achieved by screening for the presence of the non-ribosomal peptide synthetase (NRPS) gene. Result: The frequently isolated endophytic bacterium recovered was Bacillus sp. Antioxidative property of the bacterial extracts revealed endophytes with potent antioxidant activity and better antagonistic activity as compared to epiphytes. Genetic screening revealed the presence of the NRPS gene in seven plant-associated bacteria, indicating the production of natural products. Conclusions: The study indicated the extracts of bacterial endophytes associated with ethnomedicinal plants as good sources of natural products with potential application in oxidative stress. The isolates could be used as new bioactive agents.


Introduction
There is an emerging need for compounds derived from natural sources for the development of drugs to treat diseases, drug-resistant bacteria, and fungal infections [1].The present study carries significance because the tribal people of the northeastern region of India are known to use plants as folk remedies for treating a variety of ailments [2], and this has resulted in the exploitation of ethnomedicinal plants.In recent years, research on medicinal plants have attracted considerable attention globally [3][4][5], but very little is known about the bioactive properties of bacteria living in association with plants.Compounds derived from plants are largely products of plant metabolism.However, plant-associated microorganisms also produce bioactive compounds; taxol and camptothecin are examples of anti-cancer compounds synthesized by both plants and endophytes [6][7].Exploring bioactive compounds from associated microbes may not only reduce the unwanted destruction of ethnomedicinal plants [8], but may also provide an alternative source for the extraction of potential metabolites on a large scale.Endophytes are a specific group of microorganisms that can be found in internal, healthy plant tissue without causing visible damage to their hosts [9].Microorganisms that colonize plant surfaces are referred to as epiphytes.
The present study was an attempt to assess the diversity of predominant and culturable endophytic and epiphytic bacteria associated with ethnomedicinal plants and to explore their potential as natural antioxidants through assessment of free-radical scavenging activity, total phenolics, total flavonoids, metal ion chelation, and antagonistic activity.The biosynthetic potential of endophytes and epiphytes was considered via the detection of non-ribosomal peptide synthetase (NRPS) biosynthesis genes [10].NRPSs are involved in the biosynthesis of antibiotics and anti-inflammatory and immunosuppressive compounds.NRPSs are composed of a series of modules, and the adenylation (A) domain is targeted for the detection of NRPS biosynthesis genes [11][12].Though there are other methods for bioprospection of microbes, isolation in culture can be an important process for the study of microbes and discovery of novel biological activity.

Plant sample collection
Healthy plants used by different traditional medicinal practitioners (TMPs) were collected from different parts of northeast India based on their ethnomedicinal uses.The taxonomic identity of the plants was confirmed with the help of the herbarium curator of the parent university.All samples were collected in sterile polythene bags, brought to the laboratory, and used for isolation within 24 hours of collection.

Isolation and molecular characterization of endophytic and epiphytic bacteria
Endophytic and epiphytic bacteria were isolated from healthy plant samples devoid of any apparent infections per the method previously reported [13,14].Total genomic DNA was extracted using HiPurA bacterial and yeast genomic DNA Isolation Kits (HiMedia Labs, Mumbai, India).Polymerase chain reaction (PCR) amplification and sequencing of 16S rRNA gene was carried out in a 25 µL reaction mixture using general primers 27F 5'-AGAGTTTGATCCTGGCTGAG-3' and 1541R 5'-AAGGAGGTGATCCAGCCGCA-3'(Applied Biosystems, Foster City, USA) as described previously [15].The V6-V8 hypervariable region of bacteriaspecific 16S rRNA gene was amplified with the primer pair 984GCF 5'-AACGCGAAGAACCTTAC-3' and 1378R 5'-CGGTGTGTACAAGGCCCGGGAACG-3' [16,17].The DNA template replaced with sterile water was used as a negative control.The amplified 16S rRNA gene (approximately 1,400 base pairs) and V6-V8 region (approximately 400 base pairs) was then purified using QIAquick Gel Extraction Spin Kit (QIAGEN, Hilden, Germany).The purified PCR products were bi-directionally sequenced using both forward and reverse primers in a sequencer Genetic Analyzer (ABI 3130 Applied Biosystems) with Big Dye (3.1) terminator protocol as described previously [15].

Phylogenetic analyses
Sequence similarities were determined by the BLAST program against the database of type strains with validly published prokaryotic names in the EzTaxon 2.1 server [18].Molecular Evolutionary Genetics Analysis software (MEGA, version 4.0) [19] was used for phylogenetic analysis as described previously [15].

Bacterial extract preparation
All selected isolates were grown in 100 mL nutrient broth incubated in a shaker incubator at 120 rpm at 32°C for three days.The bacterial culture broth was centrifuged at 10,000 rpm for 15 minutes, and the supernatant was filtered using autoclaved 0.22 μm membrane filter paper.The filtrate was concentrated using a rotary evaporator (Stuart RE300P, Stuart Science Equipment, Stone, UK) under reduced pressure at 45°C.The concentrated aqueous extract of each selected isolate was then diluted to perform various assays.

Total phenolics
Total phenolic content was determined colorimetrically using Folin-Ciocalteu reagent [20] with minor modifications.Diluted aqueous extract (3.5 mL) was mixed with 0.5 mL of Folin-Ciocalteu reagent and incubated at room temperature for 3 minutes, and 1 mL of saturated Na 2 CO 3 was added to the mixture.Control was maintained using water in place of the bacterial extract.All tubes were incubated at 45°C for 5 minutes, and the absorbance was measured at 760 nm (Cecil CE7200, Cecil Instruments, Milton, UK).All determinations were performed in triplicate.Total phenolic content was quantified using a known concentration of gallic acid.The concentration was expressed as milligram of gallic acid equivalent per gram of extract.Ascorbic acid (1 mg mL -1 ) was taken as a positive control for comparative analysis.

Total flavonoids
The total flavonoid content was determined using the aluminium chloride method having the absorbance maximum at 430 nm.One milliliter of diluted sample was mixed with 1 mL of 2% aluminium chloride methanolic solution, 0.2 mL of 1M potassium acetate, and kept at room temperature for 30 minutes.Control was maintained using water in place of the bacterial extract.The absorbance of the reaction mixture was measured at 430 nm (Cecil CE7200).The total flavonoid content was determined from the standards obtained for quercetin.The concentration of total flavonoids was expressed as milligram of quercetin equivalent per gram of extract.Rutin (1 mg mL -1 ) was taken as a positive control for comparative analysis.

Antioxidant activity by 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method
The antioxidant activity of bacterial aqueous extract was evaluated using the DPPH (2,2'-diphenyl-1-picrylhydrazyl free radical) modified assay [21].This method is based on the reduction of alcoholic DPPH solution in the presence of an antioxidant.For each antioxidant, different concentrations of the extract were tested.The aqueous extract (0.1 mL) was added to 3.9 mL of a 6 × 10 -5 mol L -1 methanol DPPH solution.The control consisted of 0.1 mL water added to 3.9 mL DPPH solution.The reaction mixture was vortexed thoroughly and left in the dark at ambient temperature for 30 minutes.DPPH solution was prepared fresh daily and kept in the dark at 4°C between measurements.All determinations were performed in triplicate.Methanol was used to set zero blank and the decrease in absorbance was read at 517 nm (Cecil CE7200).A solution of methanol DPPH without extract was used as a control.The percentage of radical scavenging was calculated using the equation Where A⁰ is the absorbance of control and A 1 is the absorbance of sample extract or standard.Ascorbic acid (1 mg mL -1 ) was taken at various concentrations as a known antioxidant for comparative analysis.Then percentage inhibitions were plotted against respective concentrations used, and from the graph, IC 50 was calculated.

Reducing power assay
Different concentrations of aqueous extract of bacteria were mixed with phosphate buffer (2.5 mL, 0.2M, pH 6.6) and potassium ferricyanide (2.5 mL, 1%).This mixture was kept at 50°C in a water bath for 20 minutes.After cooling, 2.5 mL of 10% trichloroacetic acid was added and centrifuged at 3,000 rpm for 10 minutes.The upper layer of solution (2.5 mL) was mixed with distilled water and 0.5 mL of freshly prepared ferric chloride solution (0.1%, w/v).The absorbance was measured at 700 nm [22].The control was prepared in a similar manner but excluded samples.Ascorbic acid (1 mg mL -1 ) at various concentrations was used as a positive control.An increase in absorbance of the reaction mixture indicated an increase in reducing power.The extract concentration providing 0.5 of absorbance (IC 50 ) was calculated from the absorbance graph generated at 700 nm against extract concentration.

Metal chelating activity
Determination of metal chelating activity was based on chelating of Fe 2+ ions by ferrozine, which forms a complex with Fe 2+ ions.Different concentrations of aqueous extract were added to 50 µL of 2 mM FeCl 2 .The reaction was initiated by the addition of 0.2 mL of 5 mM ferrozine, and the mixture was shaken vigorously and left standing at room temperature for 10 minutes.Water was added in place of the bacterial extract in the control.Absorbance of the solution was measured spectrophotometrically at 562 nm [23].The percentage inhibition of ferrozine-Fe 2+ complex formation was calculated as Where A⁰ is the absorbance of control and A 1 is the absorbance of sample extract or standard.NaEDTA (1 mg mL -1 ) was used at various concentrations as positive control.The extract concentration providing 50% inhibition (IC 50 ) was calculated from the graph.

Antagonistic activity
The concentrated aqueous extracts of different isolates were tested using the agar well diffusion method [24] for their antimicrobial activity against selected human pathogenic strains, including Streptococcus pyogenes MTCC 1925, Candida albicans MTCC183, Escherichia coli MTCC730, and Staphylococcus aureus MTCC96.The test organisms were spread on the surface of Müeller-Hinton Agar (MHA) (HiMedia) using a sterile swab stick as described previously [13].Zones of inhibition were measured, and the mean value was obtained [25].Control experiments were carried out under similar conditions using standard drugs: chloramphenicol (30 µg) for antibacterial activity and nystatin (50 µg) for antifungal activity.

Screening for NRPSs
PCR amplification and sequencing of the NRPS gene was carried out in a 25 µL reaction mixture using primers MTF2 5'GCNGGYGGYGCNTAYGTNCC3' and MTR2 5'CCNCGDAYTTNACYTG3' [26] under the following conditions: template DNA denaturation at 94°C for 5 minutes followed by 30 cycles at 94°C for 10 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 30 seconds.The final step was carried out at 72°C for 5 minutes and then 4°C until infinity using PCR Gene Amp 9700 (Applied Biosystems).The DNA template was replaced with sterile water used as a negative control.

Statistical analysis
The experimental results of biological activity tests were expressed as mean ± standard deviation (SD) of three replicates.The results were processed using Microsoft Excel 2007 and Origin 6.0.
The methods are summarized in Figure 1.

Results
The results provided an understanding of the association of bacterial endophytes and epiphytes in the different ethnomedicinal plants (Table 1).The most frequently isolated endophytic bacteria was Bacillus sp., followed by Pseudomonas sp.Phytochemical studies revealed similarity in the amount of total phenolics in both endophytes and epiphytes, in the range of 10.5 ± 0.01 to 16.0 ± 0.005 and 8.1 ± 0.017 to 15.0 ± 0.015 milligrams of gallic acid equivalent per gram of extract, respectively   Both endophytic and epiphytic bacterial extracts were able to reduce the stable radical DPPH to a yellow-colored diphenylpicryl hydrazine.The aqueous extract of Bacillus sp.F21 (associated with Houttuynia cordata) showed the highest radical scavenging activity, with IC 50 of 20 ± 0.012 mg mL -1 (Table 1).The formation of the ferrozine-Fe 2+ complex was found to be incomplete in the presence of endophytic and epiphytic extracts, indicating their capacity to chelate iron.Extract of Paenibacillus uliginis C22 showed the highest iron chelating activity, with IC 50 of 1 ± 0.01 mg mL -1 (Table 1).The metal chelating capability of the isolates showed the lowest EC 50 of 1 mg mL -1 compared to reducing power and DPPH radical scavenging activity (Table 1).Bacteria associated with Centella asiatica, Potentilla fulgens, and Houttuynia cordata showed better antioxidant and antagonistic activities compared to the isolates associated with the other ethnomedicinal plants (Table 2).The antagonistic activity of the culture extracts of endophytes was evident in the agar well diffusion assay (Figure 4).All (100%) of the endophytic isolates showed antagonistic activity, whereas only 20% of the epiphytic isolates showed antagonistic activity against the tested pathogens.Among the different endophytic isolates, B. siamensis C53 and B. subtilis cenB showed the highest antagonistic activity (Figure 4).Amplification of seven plant-associated bacteria NRPS adenylation domain was confirmed via sequencing and BLASTX (translated) analysis.BLASTN (nucleotide) analysis of the PCR products did not reveal any significant matches to the

Discussion
This study presents a comprehensive assemblage of endophytic and epiphytic bacteria associated with plants of medicinal relevance and highlights a high level of diversity in the sampled niches.The study revealed the antioxidant and antagonistic activities of plant-associated endophytes and epiphytes.The antioxidant activity of the isolates is due to their redox properties, which play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides [26][27].Lipid peroxidation not only causes loss in food quality, but is also associated with diseases such as carcinogenesis, mutagenesis, ageing, and arteriosclerosis [28].Tissue damage caused in such diseases reveals the deteriorative reaction of active oxygen and free radicals [29].However, the antioxidant ability of the isolates may be used to treat the human body for oxidative damages caused by free radicals and active oxygen.Antioxidant activity is represented by the EC 50 value, which is a widely used parameter to measure free-radical scavenging activity.
A lower EC 50 value indicates a higher antioxidant activity [30].Phenolics or polyphenols and flavonoids are secondary plant metabolites that are ubiquitously present in plants and are observed among isolated plant-associated bacteria.The presence of such metabolites enhances the level of antioxidant [31][32] and antagonistic activities [33][34] of endophytic and epiphytic bacteria, making them good candidates for the isolation and characterization of bioactive compounds.
Endophytic bacteria showed significantly higher antagonistic activity against the tested pathogens compared to epiphytic bacteria.This may be the reason that these two micro-populations colonize different micro-environments on the host.Endophytes indicate a possibility of mimicking mechanisms to produce similar metabolites as their host plants.Among the epiphytic bacteria, Exiguobacterium indicum CC8 and Pantoea eucalypti CEN7E showed good antioxidant activities but relatively poor antagonistic activity, which may be the reason for their inability to enter the internal tissues.
The genetic screening of the NRPS gene increases the likelihood of isolating plant-associated bacteria, which biosynthesize novel pharmaceutical compounds independent of the host plant.In the present study, both endophytic and epiphytic bacteria revealed the presence of the NRPS gene.The isolates showing positive for the NRPS gene also indicated relatively higher antioxidant activity than others.Overall, the endophytic bacteria revealed bioactivity superior to that of the epiphytic bacteria.These findings suggest plant-associated bacterial endophytes as potential sources of natural compounds of therapeutic relevance that could replace their hosts in providing bio-natural products and prevent the exploitation of medicinal plants.Further research is required to optimize and identify compounds that aid in antioxidant and antagonistic properties of endophytic bacteria.

Conclusions
This study identified medicinal plants found to harbor endophytes and epiphytes with bioactive potential, which could be targeted in future investigations.The antioxidant and antimicrobial ability of the isolates could be due to the presence of phenolics, flavonoids, or NRPSs.The antioxidant ability of the isolates may be used to reduce the oxidative damage caused by free radicals and to treat diseases caused by lipid peroxidation.The antimicrobial ability and the presence of NRPSs among the isolates has revealed new antimicrobial agents and could be used in maintaining public health, as drug-resistant pathogens spread rapidly.The study holds significance in the light of the problem facing the future of endophyte biology and natural product discovery related to the rapid depletion of rainforests together with the disappearance of traditionally used medicinal plants, which are the greatest potential resource for acquiring novel microorganisms and their products.

Figure 1 .
Figure 1.A flowchart of the methods adopted in the study

Figure 2 .Figure 3 .
Figure 2. Comparison of total phenolic content between endophytic and epiphytic aqueous extracts at different concentration (the results are average of three readings ± standard error of mean)

Figure 4 .
Figure 4. Comparison of antagonistic activity between endophytes and epiphytes against tested human pathogens (the results are average of three readings ± standard error of mean)

Table 1 .
Antioxidant activity of endophytic/epiphytic bacteria associated with various ethnomedicinal plants

GenBank accession no. Antioxidant activity (IC 50 )
Antioxidant activity: a DPPH assay; b Reducing power; c Metal chelating