Isolation, Identification and Analysis of Novel Metabolites from Soil Samples (Microbial Co Culture) of Cardamom Plantations Western Ghats, India

Padmini V. Sekar¹, V. Deepa Parvathi², R. Sumitha*

¹Student, Dept of Biomedical Sciences, Faculty of Biomedical Sciences and Technology,

Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai 600116, India.

²Assistant Professor, Dept of Biomedical Sciences, Faculty of Biomedical Sciences and Technology,

Sri Ramachandra, Institute of Higher Education and Research, Porur, Chennai 600116, India.

³Assistant Professor, Dept of Biomedical Sciences, Faculty of Biomedical Sciences and Technology,

Sri Ramachandra, Institute of Higher Education and Research, Porur, Chennai 600116, India.

*Corresponding Author E-mail: sumithamadhu79@gmail.com

ABSTRACT:

Growing microbial resistance in clinically important microorganisms are of immediate interest in the field of pharmaceutical research. Plants, animals and microbes have been source of various antibiotics for decades. Research on these aspects is extensive and newer compounds have been identified from both terrestrial and marine sources. In recent times, there is emergence of co-culture technique that has proved to produce novel compounds due to the stress induced activity when two strains are grown in the same medium with competition for survival. Studies have shown successful outcomes in co-culture of organisms of the same species including Bacteria-Bacteria, Fungi-Fungi and Fungi-Bacteria. This study aimed at isolating specific microbes from the soil of Cardamom plantation from the Western ghats and obtaining pure culture of Pseudomonas sp. The isolated strains were subjected to morphological and molecular characterization by 16s rRNA sequencing. These strains were further co cultured as a combination to isolate the stress induced metabolites that would be produced due to competition. The extraction and the isolation of the crude metabolite from the co-culture were further evaluated for antimicrobial activity against human pathogenic bacteria. The study revealed the isolation and species identification of bacterial strains Pseudomonas nitritireducens and Micrococcus endophyticus from the soil samples. The co-cultured bacterial strains on extraction by organic solvents of hexane and dichloromethane showed promising activity against both gram positive and gram-negative bacteria on comparison with the other extracts. Further qualitative analysis revealed the presence of saponins and alkaloids in the active extracts.

KEYWORDS: Co-culture technique, Bacteria-Bacteria, Cardamom plantation, 16s rRNA sequencing and antimicrobial activity.

INTRODUCTION:

Origin of antibiotics started from prehistoric culture included a variety of naturally herbs, mouldy fungus and even cow dung was used for curing deadly diseases. “Antibiotic Age” progressed when Alexander Fleming discovered Penicillin which was successful in treating a wide range of clinically important and infectious diseases thus increasing life expectancy. Most antibiotics are specific metabolites derived from soil-dwelling bacteria and fungi. Hence this discovery paved way for newer metabolites like streptomycin and vancomycin isolated from soil microbes which were used as drugs for bacterial infections.¹ Rapid emergence of microbial resistance is a serious threat for the treatment of fatal diseases. The resistance to antibiotics demonstrated by microorganisms has evolved mainly due to the overuse of medication, inappropriate recommendation, and irregular completion of an antibiotic course and the availability of few new antibiotics to replace the resistant ones. The process of resistance develops due to horizontal transfer of resistant gene or spontaneously mutated gene by the genetic element called plasmid among exposed microbes, consequently leaving the resistance to be evolved by natural selection. Multidrug resistant microorganisms are of serious concern and have been placed as foremost priority to channelize research for the discovery of newer antibiotics.^2,3 The primary metabolites produced during cellular metabolism are typically used for cell growth and activities while secondary metabolites are secreted for its own protection possessing biological importance. For very long periods plants, animals and microbes have been source of antibiotics that are widely used and extensively researched. The study on various aerial parts of the plants has found a new area of research in the field of pharmaceutics.⁴ A recent discovery introduced the concepts of co-culture systems which promotes the production of novel metabolite due to the stress induced strive of microbes on survival. Microbial co-culture refers to symbiotic or competitive association of microbes in an environment. This association leads to the formation of metabolites as a product of interaction that might have biological properties with clinical applications. Generally, these co-culture systems survive commonly in the marine habitats as the marine derived natural products are promising. There are various studies proven to show that the co-cultivation led to effective production of secondary metabolite which possess a competitive activity against various microorganism.⁵ The present study aimed at isolation, identification and testing the novel metabolite from active crude isolates from microbial co culture of soil samples from Cardamom Plantations (Western Ghats, India).

MATERIALS AND METHODS:

Collection of Soil Sample:

Fertile soil samples were collected from Parathodu, near Mayiladumpara Indian Cardamom Research Institute (ICRI), Idukki District, Kerala at the location 9^o53’15.0” N 77^o09’36.2” E” . The soil samples used for this study were collected specifically from the roots associated with the cardamom plantations in the Western Ghats of Indian. Five different soil samples were collected aseptically 5 - 10cm layers bellow the soil surface and transported for processing.

Identification and Isolation of the Soil Microorganisms:

The collected soil samples were subjected to isolation and identification by plate count agar method. 1gram of each soil sample was weighed and added separately to 10ml of deionized water. The stock mixture was then mixed by vortexing. 1ml of supernatant from five different soil stocks were aseptically transferred to 9ml of deionized for further processing for the plate count agar method. Each soil suspension was serially diluted (10^-1 to 10^-6) aseptically and was labelled. The serial diluted samples were aseptically transferred to sterile plate count agar which was evenly spread. The plates were labelled correspondingly and were incubated for 24 hours and observed for colony formation.

Colony Morphology and Staining Identification:

The numbers of colonies were counted for each dilution and were tabulated respectively. The morphological characteristic identification of the individual colonies was identified and the colony features were noted. Based on the consistency and uniqueness; the colonies were subjected to microscopic staining techniques. The colonies were smeared on the slides aseptically using sterile inoculation loop and were stained by Gram staining and Acid-fast staining methods to confirming cell morphology. The stained smears were examined microscopically (100X).

Preparation of Pure Culture – Isolation in Selective Media:

Two unique colonies were selected for further analysis by inoculating them on selective media namely; Cetrimide Agar (M024) and Yeast Extract Glucose Agar (M963) for cultivation and isolation of the specific microorganisms. The single selected colony was picked and was further streaked on to the agar plates aseptically for supplementary isolation which was incubated for 24 and 48 hours accordingly. The plates were checked for the growth characteristics and were analysed further.

Molecular Characterization by 16S rRNA Sequencing:

The isolated colonies from Cetrimide agar and Yeast extract glucose agar plates were further characterised using 16s rRNA sequencing for confirmation and comparison of the bacterial isolates. Genomic DNA was extracted from overnight grown cultures of the selected bacterial isolates using QIAGEN DNA isolation kit. The overnight culture of the bacterial isolates was diluted 20 folds and was suspended in 100μl of elution buffer (10 mM/L Tris - HCl, pH 8.5) and quantified at 260nm. PCR amplification was performed using 50μl reaction mixture containing 100ng of template DNA, 20μmol of 16S rRNA primers, 200μM of dNTPs, 1.5mM of MgCl2, 1U of Taq DNA polymerase (MBI Fermentas) and 10μl of 10x Taq polymerase buffer. The sequences of 16S rRNA primers were 27f: (5'-AGAGTTTGATCCTGGCTCAG-3') and 1522r: (5′-AAGGAGGTGATCCANCCRCA-3'). Amplification was carried out with an initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 56°C for 45 sec, extension at 72°C for 1 min and final extension at 72°C for 5 min using a thermo cycler (I Cycler; Bio-Rad Laboratories, CA). PCR products were analysed on 1% agarose gel for 16S rRNA amplicons in 1x TBE buffer at 100 V. The amplified product was sequenced using ABI PRISM 3730 Genetic Analyzer (Applied Bio systems).

Phylogenetic Analysis:

The sequences of the 16S rRNA genes were compared against the sequences available from GenBank using the BLAST program and were aligned using CLUSTAL W software.^6,7 Distances were calculated according to Kimura’s two-parameter correction.⁸ Phylogenetic trees were constructed using the neighbour-joining method. Bootstrap analysis was done based on 1000 replications. The MEGA4 package was used for all analyses.

Nucleotide Blast N (Blast N):

Basic Local Alignment Search Tool (BLAST) was used for comparing the biological sequences of amino acids, proteins or nucleotides from database to locate the similarities and identify the query sequence. The nucleotide specific BLAST N search engine was selected to enter the query sequence. The FASTA format file of the query sequence was selected and entered in the query sequence section. Database of the genome required was selected from the options. The BLAST N program was set to Mega BLAST N to select the highly similar sequences from the available data and the sequence was submitted for comparison.⁹

Co Culture Initiation:

The isolated Pseudomonas Actinomycete strains were cultured and maintained in Cetrimide broth and Yeast extract glucose broth (Himedia), respectively at 37°C. Inoculum preparation for co-culture was initiated. 1ml of culture from each broth was aseptically transferred to the freshly prepared 100ml of Nutrient broth (Himedia), and incubated at 37°C in shaker for 5 days respectively to initiate the co-culture activity.¹⁰

Extraction of Active Crude Metabolites:

The co-culture initiation was arrested on the 5th day and the turbid broth was checked for contamination. 100ml of broth culture was centrifuged at 10,000rpm for 20 min to separate the biomass. The culture supernatant was used for sequential extraction. The non-polar to polar solvents (n-hexane, dichloromethane, ethyl acetate, chloroform and methanol) were used for the extraction in sequential manner. The solvent was added to the supernatant in 1: 1 ratio and agitated for 45 min with homogenizer. The solvent was separated from broth by separating funnel. The crude fractions were then centrifuged at 5000rpm for 15 min to remove traces of the fermentation broth.¹⁰The crude extract was then evaporated using a heating mantle. The crude extracts were collected and dried for further process.

Antibiotic Sensitivity Test by Well Diffusion Method:

The crude extracts were tested for antibacterial activity by agar well diffusion method. The susceptibility testing was done against gram negative Escherichia coli ATCC 12435, gram positive Staphylococcus aureus ATCC 6538. The culture initiation was done by inoculating the fresh nutrient broth with the strains and incubating it overnight at 37^oC. Using McFarland’s turbidity as a standard, the inoculum was prepared (5 x 105 CFU/ml). Muller Hinton agar plates were prepared, from a commercially available dehydrated base according to the manufacturer's instructions. 100µl of the medicinally important bacterial organisms were platted on the Mueller-Hinton agar medium using sterile cotton swab. A well with a diameter of 6 to 8mm was punched aseptically with a sterile cork borer. Wells were cut and defined concentration (20, 40, 60, 80, and 100µg/ml) from the crude extract (1mg/ml) were tested for antibiotic sensitivity and compared with Ciprofloxacin (1mg/ml) as positive control and water as negative control.¹¹ The antibacterial activities for the defined concentrations were performed in triplicates and the values were calculated statistically by standard deviation (SD) method.

Qualitative Analysis of the Active Crude Metabolites:

Qualitative analysis reveal a group of compounds to which the metabolites might possibly belong. The reagents required for the analysis were freshly prepared. Qualitative analysis was performed for the active crude to reveal phenols, reducing sugars, flavones, glycosides, saponins, alkaloids, anthraquinones, quinones, proteins, amino acids, tannins and steroids.^12,13

RESULTS:

Collection of Soil Samples:

Five different soil samples were collected aseptically from the roots of Cardamom Plantations from Parathodu, Udumbanchozha Taluk, near Mayiladumpara Indian Cardamom Research Institute (ICRI), Idukki District, Kerala at the location 9^o53’15.0” N 77^o09’36.2” E

Colony Morphology and Staining Identification:

The colonies after the incubation were counted and the morphological characters were identified. The morphological appearances for the five different soil samples were characterized as white, irregular, smooth, colonies however few colonies appeared to be yellow, irregular, undulate, and smooth for the respective dilutions. Gram staining and Acid-Fast staining was performed to study the morphology from various colonies. Gram Staining revealed a series of gram-negative bacilli and gram-positive cocci from the isolated samples. Samples stained with acid fast stain revealed partial uptake of the stain.

Preparation of Pure Culture – Isolation In Selective Media:

Two unique colonies were observed to be grown on the selective media namely Cetrimide Agar (M024) and Yeast Extract Glucose Agar (M963) showing green pigmented colonies and white translucent colonies respectively. The colonies from the selective media were further confirmed by molecular characterisation.

Molecular Characterisation by 16s Rrna Sequencing:

The colonies from the plates were cultured and amplified using PCR for the extraction of genomic DNA as shown in (Fig. 1 and 2). The gene sequences of the isolates were obtained to compare with the available database to identify the species.

Gene Sequence of Pseudomonas Isolate:

Using the genes analysed, it was compared with the BLAST N bioinformatics tool to identify the similarities between the isolate and the sequences available in the database. From the comparison with the available database, phylogenetic tree was framed to identify the strain of the isolate. The isolate was confirmed by comparing the available database gene sequence and was identified to be Pseudomonas nitritireducens as shown in (Fig. 3).

Figure 1. Isolation of Genomic DNA

1. Cetrimide isolate; 2. Yest extract glucouse agar isolate

Figure 2. PCR Amplification Profile

a- DNA Ladder; b- Cetrimide isolate; c- Yest extract glucouse agar isolate

Table 1. Antibiotic Sensitivity of active crude extracts from Co Cultured Bacteria Against Escherichia coli and Staphylococcus aureus

S. No.	Pathogen	Zone of the inhibition (mm) Hexane (concentration µg/ml)					Positive control Ciprofloxacin (100 µg/ml)	Negative control
S. No.	Pathogen	20	40	60	80	100	Positive control Ciprofloxacin (100 µg/ml)	Negative control
1.	Escherichia coli	10 ±0.15	12±0.5	24±0.26	35±0.05	42 ±0.17	46 ±0.1	0
2.	Staphylaccocus aureus	2±0.15	8±0.05	10±0.26	14±0.05	16±0.17	27 ±0.1	0

S. No.	Pathogen	Zone of the inhibition (mm) dichloromethane (concentration µg/ml)					Positive control Ciprofloxacin (100 µg/ml)	Negative control
S. No.	Pathogen	20	40	60	80	100	Positive control Ciprofloxacin (100 µg/ml)	Negative control
1.	Escherichia coli	12±0.15	16±0.05	24±0.26	26±0.05	38±0.17	46 ±0.1	0
2.	Staphylaccocus aureus	4±0.15	8±0.05	12±0.26	16±0.05	18±0.17	27 ±0.1	0

Qualitative Analysis of The Crude Extracts:

Qualitative analysis was done for the active crude extracts that were found to be metabolically active namely hexane crude extract revealed the presence of saponins, alkaloids and dichloromethane extract exposed alkaloids.

STATISTICAL ANALYSIS:

All the experiments were performed in triplicates and the values were calculated statistically by standard deviation (SD) method.

DISCUSSION:

Secondary metabolites from the microbes have been the centre of attention for research due to their conferred antagonistic activity on other microorganisms.¹⁴Co culture system of microbial cultivation has proved to be a dynamic process for the production of novel metabolites with biomedical applications. The present study was performed by collecting different soil samples from Cardamom Plantations at Parathodu, Udumbanchozha Taluk, near Mayiladumpara Indian Cardamom Research Institute (ICRI), Idukki District, Kerala at the location 9o53’15.0” N 77^o09’36.2” E. The samples were collected aseptically and processed for isolation and identification of soil microorganism by using standard procedures. The microbes were isolated by plate count method and the preliminary identification was done studying the colony morphology and staining techniques (Gram staining and Acid-fast staining) was done to identify the range of microorganisms present in each of the soil samples from which colonies were selected for pure culture. The selected colonies were further subjected for isolation on selective media namely Cetrimide agar and Yeast Extract Glucose agar respectively.

The molecular characterization was performed to confirm the isolated strain by 16s rRNA sequencing. Results revealed that the isolated strains were Pseudomonas nitritireducens and Micrococcus endophyticus respectively. These strains were then co-cultured with aeration for 5 days after which a sequential extraction was performed for isolation of active metabolite from the crude extract. Previous studies on co-culture techniques for isolation of novel bioactive metabolites from microorganisms have demonstrated the significance of cultivation of three major categories namely co-cultivation of different fungi, fungi with bacteria and bacteria with bacteria has given a significant load of novel compounds with biomedical potentials.¹⁵ The extraction was performed by using the non-polar to polar solvent system (Hexane, Dichloromethane, Ethyl Acetate, Chloroform and Methanol) respectively. Previous studies have demonstrated extraction methods of natural metabolites which are a significant step for the standardization, as they can be utilized in the removal of desirable soluble constituents. A variety of solvents system with diverse polarity must be employed for extraction of different compounds which gives a high extent of accuracy which has been proved to be effective in the extraction of the antimicrobial metabolite extracted from the plant sources.¹⁶

In the present study, the extracted crudes were further subjected for antimicrobial sensitivity testing. The analysis of the crude extracts was performed, defined concentration were subjected for their antimicrobial sensitivity against gram positive Staphylococcus aureus ATCC 6538 and gram-negative bacilli Escherichia coli ATCC 12435 using Ciprofloxacin as positive control. In comparison, hexane and dichloromethane extracts had good antimicrobial activity against both the strains. Earlier studies have proved that secondary metabolite having antimicrobial activity had been advanced by co cultivation of microbial strains. Many such compounds possessing antimicrobial activity has also been isolated namely alkaloid derivatives possessing antimicrobial activity isolated from co-cultivated fungi and actinomycetes has been reported.¹⁷ Another study also reported the isolation of three novel metabolite and a known metabolite with antibacterial activity by co-cultivation of Fusarium tricinctum with the bacterium Bacillus subtilis.¹⁸ A recent study hypothesized on the isolation and identification of dominant soil microbial isolates which produced synergistic and antagonistic effect. Various bacterial soil isolates were isolated and investigated using 16S rRNA sequencing which was then co cultured and monitored. The screening of the synergistic and antagonistic activity was performed for the co-cultured isolates the results indicated that the presence of the Pseudomonas sp in the co-culture may be a promising antibiotic producer. ^{19, 20} In the present study, the active crude extracts were further subjected for the qualitative analysis for confirmation on chemical composition. Active hexane extract contained saponins and alkaloids and the crude active dichloromethane extract contained alkaloids. Further purification by column chromatography is required to precisely identify the bioactive compounds.

CONCLUSION:

The purpose of this study was to isolate a novel metabolite by co culturing dominant microorganisms from the soil samples of Cardamom Plantations. The investigation has revealed that the strain during the co-cultivation has undergone an antagonistic activity resulting in the production of a secondary metabolite with a significant activity on comparison. Hence, further purification and identification of the compounds from the crude is a translational requirement to bring the compounds that are metabolically active and clinically important to the bedside.

ACKNOWLEDEMENT:

We take this opportunity to thank the Department of Microbiology, Sri Ramachandra Institute of Higher Education and Technology (DU), Porur, Chennai for providing us with the bacterial strains.

CONFLICT OF INTEREST:

The authors have no conflicts of interest to declare.

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Received on 15.02.2021 Modified on 11.06.2021

Research J. Pharm. and Tech. 2022; 15(5):1953-1959.

DOI: 10.52711/0974-360X.2022.00325