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Isolation and Identification of Antimicrobial Actinomycetes from Non-Agricultural Soil

Oliver Madhale*, Vinay Chougule, Ujwala Mane

Department of Microbiology, Rayat Institute of Research and Development, Satara, Shivaji University, Kolhapur-416004, Maharashtra, India

Abstract: Potential Actinomycetes produced biologically significant secondary metabolites in abundance in non-agricultural farm. Actinomycetes were isolated from soil on a non-agricultural farm in Miraj, Sangli District, Maharashtra, with only six isolates chosen for this study. Streptomyces spp. isolates demonstrated strong antimicrobial activity against human pathogenic strains of Salmonella typhi, Salmonella paratyphi A, and Salmonella paratyphi B pathogens. The isolate INA-53 had excellent antimicrobial activity against enteric fever pathogens zone of inhibition were observed at the highest level (16.0-23.0 mm). According to BLAST analysis, INA - 53 is related to Streptomyces parvulus Actinomycetes and the Actenobacteria class. As a result, the powerful INA - 53 isolate is extremely effective at producing bioactive compounds.

Keywords: non- agriculture soil, streptomyces, actinomycetes, antagonistic activity, antibacterial, 16S rRNA

Received March 04, 2022; Revised April 06, 2022; Accepted April 12, 2022

Citation: Oliver Madhale, Vinay Chougule, Ujwala Mane, Isolation and Identification of Antimicrobial Actinomycetes from Non-Agricultural Soil. Applied Ecology and Environmental Sciences, 10 (4). 219-224. January 2022. doi:10.12691/aees-10-4-6

1. Introduction

Actynomycetes are common in soil and aid in the digestion of resistant carbohydrates such as chitin and cellulose. They are filamentous bacteria capable of producing new antimicrobials. Actinomycetes can produce a wide variety of secondary metabolites with biological significance, including antibiotics, antimicrobials, enzymes, and so on. Actynomycetes were responsible for more than 45 percent of the 22.500 well-characterized biologically active compounds discovered from various sources to date [1]. Streptomyces is the most important genus in the pharmaceutical industry as a potential source of antibiotics, accounting for more than 70% of global antibiotic production and trade [2]. Because of their diverse capabilities in the production of antibiotics and other therapeutic products. Actinomycetes have played an important role in a number of hot areas of pharmaceutical research [3]. Actinomycetes are essential for soil mineralization of organic matter, nutrient immobilization, antibiosis, and the development of plant growth promoters, making them a promising candidate for agricultural use [4]. Actinomycetes are of particular interest among these microorganisms because they are well-known hyperproducers of chemically diverse compounds with diverse biological applications [5] As the demand for new antibiotics grows, new pathogens emerge that cause life-threatening illness and even death. As a result, a new antibiotic is constantly in demand. Because nature is a rich source of novel antibiotics and metabolites [6].

2. Materials and Method

Soil samples were collected from non-agriculture farms throughout the Miraj environment of the Miraj Kupwad Muncipal Corporation in the district of Sangli, Maharashtra. Soil quality, area, cultivation or barren soil, and organic matter, among other factors, all have a significant impact on actinomycetes overgrowth and diversity in soil. Miraj's environment is complex and extremely dynamic. Actinomycetes have been isolated as antibiotic sources in several studies. Even though actinomycetes are abundant in nature, only a small portion of the world's soil has been screened [7] 55 Soil samples were collected from various locations of non-agriculture farms in Miraj environment at Miraj Kupwad Muncipal Corporation, District Sangli, Maharashtra, as a source of actinomycetes. After removing the upper layer of soil about 3.0 cm from the surface, the samples were taken at a depth of 20 cm. Soil samples were collected in polyethylene bags, sealed, and refrigerated for later analysis. Each soil sample was air dried for one week at room temperature before sieving to remove gravel, debris, vegetative material, and dry leaves, this sieved soil was used for further investigation.

Actinomycetes were isolated from soil samples by serial dilution of 1g of dried sieved soil in 10 ml od distilled water using the spread plate technique. 0.2 ml of each dilution was inoculated in the sterilized plates of glycerol asparagine media. To reduce other bacterial and fungal growth, Actinomycetes isolation agar medium was supplemented with 1g/ml Rifampicin antibiotic and 1g/ml Glysoflavin antifungal. For 21 days, all plates were incubated at 28°C. The distinctive appearance of soil Actinomycetes colonies was used to identify their appearance and growth. From the isolation plate, several colonies with varying morphological and cultural characteristics were chosen and streaked for pure culture. Cultures were kept at 4°C until they were harvested [8].

2.1. Screening of Antibiotic Producing Actinomycetes

To study antimicrobial activity Salmonella typhi, Salmonella paratyphi A, and Salmonella paratyphi B pathogens ware used to test antibiotic production in actinomycetes. For preliminary analysis, the cross streak method was used. The isolates were streaked on nutrient agar plates Pathogens were streaked perpendicular to the isolates stripe and incubated at 28°C for 24hrs. The inhibition zone was measured after 24 hours, and a control plate was considered normal growth of bacteria without actinomycetes isolates.

2.2. Bioactive Compounds

Actinomycetes isolates were selected for bioactive compounds using submerged fermentation. A 5 mL sterile water suspension was added to the slant. The 5 ml of inoculum medium was transferred to another flask containing 45 ml of processing glycerol asparagine broth and incubated at28⁰C at 300 rpm on a rotary shaker for 6 days Following incubation, 10ml of the broth was placed in centrifuge tubes and centrifuged for 15 minutes at 4000 rpm to obtain a clear supernatant for studying the antibacterial activity using the agar well diffusion method [9].

2.3. Antibacterial Activity

Antibacterial activity was tested using the agar well diffusion method. The spread plate technique was used to inoculate the test organisms on Nutrient agar medium. With the help of cork borer well were ware created in the agar plate, the plate was filled with 0.5ml of antibacterial metabolite and refrigerated for 2 hours to allow the bioactive metabolite to diffuse. The plates were then incubated at 37°C for 24 hours. The zone of inhibition was studied for its antibiotic activity.

2.4. Identification of the Potent Actinomycetes

2.4.1. Cell-Wall Analysis

The composition of actinomycetes' cell walls varies greatly between groups and is taxonomic in nature. The major cell wall distinguishes these filamentous bacteria based on specific characteristics. The potent antimicrobial actinomycete isolate was studied for its sugar and amino acid pattern by using paper chromatography technique

Chromatography for Amino acid in cell wall of Potent Actinomycete

The amino acid analysis was performed on INA – 53, a potent actinomycete isolate. 1.5 ml 6N HCl was applied to a 2.0 ml test tube containing 2 mg dry cell mass. For 18 hours, the test tube was sealed and held at 100°C.

The content was filtered through Whatman filter paper No. 1 after 18 hours. At 50°C, the cell extract was dried. The dried cell extract was then mixed with 200µl of distilled water and dried at 50⁰C once again.

This process was carried out three times. The dried cell extract was collected and kept in aliquots in 200µl distilled water. This was used to analyze amino acids. 5µl of cell extract was put on the Whatman filter paper No.1 baseline.

For roughly 4 hours, paper chromatography was used. The chromatography paper was air dried before being developed with 0.2 percent Ninhydrin in acetone for 3 minutes at 100°C.

Chromatography for Sugars in cell wall of Potent Actinomycete

A test tube containing 25g of dry cell mass and 1.5 ml of 1 N H₂SO₄ was sealed. For 2 hours, the test tube was heated in a boiling water bath. The cell hydrolysate was transferred to a 15 ml centrifuge tube after cooling.

Dropwise additions of saturated Barium hydroxide were made until the pH was adjusted to between 5.2 and 5.5. For 15 minutes, the tube was centrifuged at 5000 rpm. The supernatant was collected and evaporated in a 50 mL beaker.

The residue was dissolved in 200 ml of distilled water and collected in a microfuge tube. The mixture was then centrifuged for 15 minutes at 5000 rpm to remove any insoluble material. 5µl of hydrolysate were applied to the Whatman filter paper's baseline. No. l.

The paper chromatography was conducted for around 4 hours. Sugar spots were created by spraying with Ninhydrin reagent and heating for 4 minutes at 100°C.

2.4.2. Molecular Identification

By isolating DNA and amplifying the gene coding for 16S rRNA using polymerase chain reaction, the potent strain INA – 53 was identified by 16S rRNA employing molecular method, by use of computer software BioEdit . purified DNA fragments were sequenced to determine the order of nitrogen bases with the length of the sample. to study the sequence for identification using protocol of phylogenetic analysis. Molecular based characterization of 16S rRNA was done based on automated sequencer. [10]

PCR amplification and DNA sequencing

The 16s rRNA sequence was amplified from genomic DNA in a thermal cycler using 27F/243R 5l-AGAGTTTGATCCTGGCTCAG-3l and 533F/1492R 5l-TACGGYTACCTTGTTACGACTT-3l as universal primer sets. Initial denaturation at 94°C for 3 minutes, 35 cycles of 94°C for 1 minute, annealing at 54°C for 1 minute, and extension at 72°C for 2 minutes, and final extension for 10 minutes and hold at 4°C were the cycling conditions used. The obtained sequence was then subjected to phylogenetic analysis. BLAST logarithm was used to find the Streptomyces spp. INA - 53 sequence. MEGA software was used to conduct phylogenetic analysis with the neighboring – neighboring joining logarithm [11].

3. Results

Actinomycetes were isolated and purified by selecting a small number of selected colonies and using pure culture methods to isolate and purify the cultures. Each of the six isolates was assigned a number. The cross streak method was used to screen antibiotic-producing Actinomycetes against three pathogenic bacteria: Salmonella typhi, Salmonella paratyphi A, and Salmonella paratyphi B, as mentioned in Table 1 Three of the six isolates, INA- 45, INA- 46, and INA- 53, demonstrated potent antagonistic activity against all pathogenic strains. Salmonella typhi was inhibited by INA-42 and INA-43, while Salmonella paratyphi B was inhibited by INA-42 and INA-43. A INA-44 had no effect on pathogenic strains.

Table 1. Actinomycetes for antimicrobial activity

Purification of antimicrobial activity

Bioactive compounds were studied in the isolates INA-45, INA-46, and INA-53. They were extracted and purified so that their antimicrobial activity could be tested. Antimicrobial activity was tested using the agar well diffusion method. The effects of extracellular compounds on antimicrobial activity are shown in Table 2. INA- 53 had the highest antibacterial activity against the pathogenic species of the three isolates, ranging from 16.0mm to 24.0mm. INA – 53 was chosen as a potent isolate for phylogenetic and molecular analysis because INA – 45 (7.8mm – 9.0mm) and INA – 46 (8.0mm – 13.5mm) demonstrated moderate activity.

Table 2. Antimicrobial activity of extracellular compounds

Cell wall analysis

Cell wall analysis of INA 53 was carried out with the help of paper chromatography to so the number of amino acids and sugar in the cell wall of the potent INA 53 isolate.

Paper Chromatography for separation of amino acids:-

dried cell mass was immersed in deionized water, mixed well, and utilized to identify amino acids contained in the INA – 53 actinomycetes strain. The above-mentioned method was carried out using the paper chromatography technique.

Figure 1. Paper chromatography for Amino Acids

The total number of amino acids present or generated by the actinomycetes isolate INA – 53 is shown in Figure 1. On the Whatman filter paper, the paper chromatography method revealed four distinct spots. Which interprets the presence of four different types of amino acids produced by the strain of actinomycetes Yellow, brown, light pink, and dark pink colored dots were obtained.

Paper Chromatography for separation of sugars:-

The other half of the dried cell mas was suspended in the deionized water, it was mixed well and used for the detection of different types of sugars present in the actinomycetes strain of INA – 53. The paper chromatography technique was applied for detection of sugars.

Figure 2. Paper Chromatography for Sugars

The total number of sugars present or generated by the actinomycetes isolate INA – 53 is shown in Figure 2. The whatman filter paper exhibited three separate spots on the paper chromatography method, indicating the presence of three different forms of sugars generated by the actinomycetes strain. Brown, pale pink, and red colored dots were obtained.

Identification of Actinomycetes Molecular identification

The insta gene matrix DNA amplification kit was used to amplify DNA, which was then followed by 16S rRNA sequencing. Polymerase chain reaction was used to sequence the 16S rRNA gene of the INA-53 isolate, which was followed by agarose gel electrophoresis.

Figure 3. Amplified 16S rRNA of INA 53 Actinomycete Isolates Run on 1.5% Agarose gel. Lane 1(100 bp ladder)

As in Figure 3 shows a illustration of this. The gel documentation system displayed 1259 basepairs from the investigation of the 16S rRNA gene sequencing of the INA-53 isolate.

According to NCBI's BLAST (Basic Local Alignment Search Tool) search analysis. This method detects similarities in protein or nucleotide sequences and calculates their statistical significance.

Phylogenetic analysis

The findings of the 16S rRNA sequencing were analyzed, and the identification of the actinomycetes isolate INA – 53 was done based on the phylogenetic analysis. The 16S ribosomal RNA gene Sequence was as follows.

GAGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAAT ACCGGATACTGACCCTCACGCATTGACCTGGGCATCTGTGAGGTTCGAAAGC TCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGCATTGACCTCCAAG GCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAG ACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGG GCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTAACCTCTT TCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCT AACTACGTGCCATAATACGTAGGGCGCGAGCGTTGTCCGGAATTATTGGGC GTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTT AACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGA TCGGAATTCGTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCG GTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGT GGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGC ACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGT GCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAAC CTTACCAAGGCTTGACATACACCGGAAACGTCCAGAGATGGGCGCCCCCTTG TGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTT GGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCC TTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGG TGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGC TACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCA AAAAGCCGGTCTCAGTTCGGATTCGGAGTCGCTAGTAATCGCAGATCAGCATT GCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC

As shown in Figure 4. The INA-53 isolate of actinomycetes showed 96% similarity towards the phylogenitic strains of Streptomyces parvulus.

Figure 4. Phylogenetic Tree Based on 16S rRNA Gene Sequence Analysis, Showing the Relationship of Active Actinomycetes Isolate INA – 53

4. Discussion

Soil samples were collected from various locations from the Miraj non-agricultural farm, the samples were serially diluted. Actinomycetes isolated from non-agricultural farm soil samples are a valuable resource [12]. All six actinomycetes isolates were tested for their ability to produce bioactive compounds. Three of the six isolates tested positive for all pathogenic strains tested, INA 45, INA 46 and INA 53.

The antimicrobial activity of extracellular bioactive compounds produced by different isolates was tested against pathogenic strains. All three isolates, INA 45, INA 46, and INA 53, yielded significant results that were comparable to other studies. [13]. INA – 53's antimicrobial activity against the pathogenic species studies ranged from 14.0mm to 24.0mm. As a result, screening such species for new bioactive compounds has an advantage, and it indicates that unexplored environments, such as underutilized farms, are rich in producing novel actinomycetes capable of producing new bioactive compounds, including antibiotics [14].

Actinomycetes produce useful bioactive compounds and have antagonistic activity against three pathogenic microbe strains, including Salmonella typhi, Salmonella paratyphi A, and Salmonella paratyphi B. In molecular studies, the use of the 16S rRNA gene is simpler but more effective in identifying new Streptomyces strains [15]. INA – 53 was chosen as a potent isolate for molecular identification among the three isolates studied to investigate the genetic diversity of different Streptomyces species due to its strong antimicrobial activity. A molecular analysis based on 16S rRNA sequences was performed to confirm the identity of the isolate a complete phylogenetic tree study was carried out and showed INA - 53 as Streptomyces parvulus. According to the phylogenetic tree, Streptomyces parvulus. INA 53 is related to Streptomyces inkyrus and Streptomyces piloviolofuscus, both of which are streptomyces. The current study is similar to the work of another researcher, who identified strain ACTK2 as Streptomyces flavogriseus from a soil sample in Karnataka state, India, as a bioactive actinomycetes strain capable of producing an antimicrobial secondary metabolite. As a result, the actinomycete Streptomyces parvulus INA – 53 from the Miraj non-agricultural farm can produce extracellular potent bioactive compounds that could be used to make a variety of antimicrobials.

5. Conclusion

Resistant pathogens, particularly acquired multidrug resistant strains, are becoming more common, posing a major public health concern worldwide. As these infections spread rapidly, there is an urgent need for antimicrobial discovery and high-quality treatments. Particularly in tertiary care hospitals, where antibiotic resistance is a major concern. Antimicrobial agents have been discovered in a variety of environments and have proven to be extremely effective in the treatment of a wide range of infectious diseases.

As a result, economically significant secondary metabolites, actinomycetes, were isolated and purified from the Miraj non-agricultural farm. Streptomyces parvulus. INA – 53 was discovered to have antimicrobial activity against a wide range of pathogenic bacteria. Identifying the bioactive compounds that cause antimicrobial activity. Some actinomycetes may be useful antibiotics. As a result, more intensive research is needed to screen the isolates for antibiotic production.

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