Isolation, Identification and Evaluation of Antibacterial Activity of Actinomycetes from Soil Sample of Adilabad, Telangana State (India)
Eerla Rakesh1, Thampu Rajakomuraiah2*
1,2Department of Microbiology, Kakatiya University, Warangal-506009, Telangana State, India
*[email protected], [email protected]
ABSTRACT
Actinomycetes are fungus-like filamentous bacteria that are Gram-positive and facultatively anaerobic. They are the top natural antibiotic producers. A vast variety of microorganisms with effective sources of antimicrobials are found in the Adilabad forest area of Telangana State, India, due to the climatic and geographical variety. The goal of this research was to isolate, identify, and test putative antimicrobial- producing actinomycetes from soils in the Adilabad forest region. Actinomycetes were recovered from eighty- one soil samples taken from various places in Adilabad. A morphological investigation and biochemical testing were used to identify the isolates. Temperature and pH were also used to classify them. The perpendicular streaking method was used to screen for antimicrobial activity against several test organisms, including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, and the agar well diffusion method was used to screen for antimicrobial activity against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Streptomyces spp. were found in 54.3 percent of the isolates, Amycolatopsis in 14.8 percent, Micromonospora in 9.87 percent, Microbacterium in 14.8 percent, and Saccharoppolyspora spp.in 13.5 percent. In the main screening, 52 percent of actinomycete isolates were determined to be powerful antibiotic producers, with 38.31 percent effective against Gram-positive test organisms and 14 percent effective against Gram-negative test organisms. During secondary screening, isolate J5 (Streptomyces spp.) had the strongest broad-spectrum antibacterial activity. Distinct isolates created a total of six different kinds of isolate colours, with the brown colour being the most predominant. The relationship between antimicrobial production and elevation, pH, and colour was determined to be minor. This discovery might be important for future research on broad-spectrum antibiotics for medicinal purposes.
Keywords
Actinomycetes, Adilabad, Streptomyces spp, anti-bacterial, Amycolatopsis
Introduction
Isolating new antibiotics that are effective against resistant pathogenic bacteria from undiscovered environments across the globe remains a major research focus. There is a growing dilemma as a result of the emergence of antibiotic-resistant microbial infections and the toxicity of numerous commonly used antibiotics [1,2]. About 17000 antibiotics have been identified from various microbiological sources as a consequence of the thorough screening [3]. Actinomycetes are well-known as a source of bioactive molecular compounds, since these organisms, particularly Streptomyces, supply more than half (70%) of the naturally occurring antibiotics widely employed in the pharmaceutical sector [4].
Around 23 000 bioactive secondary metabolites generated by bacteria have been identified, with Actinomycetes producing over 10,000 of these chemicals, accounting for 45 percent of all bioactive microbial metabolites known [5]. Streptomyces species create roughly 7600 chemicals among Actinomycetes. Streptomycetes are the principal antibiotic-producing organisms used by the pharmaceutical industry because several of their secondary metabolites are powerful antibiotics [6].
last two decades [7]. As a result, overcoming the issue of antibiotic resistance is critical in the effort to isolate actinomycetes in novel environments and identify high-efficacy antibiotics. The Indian Western Ghats area is being studied for its rich and diverse microbial diversity. Several antimicrobial-active actinomycetes strains have been identified and described from soil samples in India [8]. However, there is a scarcity of information on isolated actinomycetes strains with antibiotic potential from Telangana's Adilabad forest area.
The objective of this work was to isolate, identify, and assess the antibacterial activity of an actenomycetes strain acquired from a soil sample in Adilabad, Telangana, India, in order to optimise it.
Materials and Methods Sample collection and processing
Soil samples were taken at a depth of 8-14 cm in the Kawal Wildlife Sancutary and Jannaram forest in Adilabad district, Telangana State, India. Samples were collected and transferred to clean zip lock sterile polythene bags, which were then transported to the actinomycetes isolation laboratory and kept at 4 °C, using sterile procedures. To remove pebbles and debris, the obtained soil samples were carefully mixed and put through a 2 mm filter. After that, each sample was dried overnight at 27 °C to kill any microorganisms.
Isolation of Actinomycetes
Soil samples were serially diluted before being plated on Hi media's Actinomycetes Isolation Agar medium and incubated at 28°C for 6 days [9]. The colonies were isolated and purified on yeast extract-malt extract agar medium treated with 50 g/mL Nystatin as an antifungal agen [10].
Pure cultures were kept at 4 degrees Celsius on ISP-2 slants. The pure isolate's spore mass and mycelium were preserved in a glycerol solution (20 percent, v/v) at -20°C for subsequent investigation (Difco, Hi media, Mumbai, India) [11].
Identification of isolated strains
Cultural, morphological, physiological, and biochemical approaches were used to identify the strains.
Cultural and Morphological Characteristics
After 14 days of cultured at 30°C, the organism was grown on oatmeal agar, glycerol asparagine agar, yeast extract-malt extract agar, inorganic salt starch agar, and starch casein agar medium to determine cultural features. Growing the organism on ISP-4 medium for 7 days and assessing morphological properties of aerial hyphae, spore chain, spore mass, spore surface, colour of aerial and substrate mycelia, and diffusible pigments production. The sugar pattern and diaminopimelic acid (DAP) were determined according to [12].
Physiological and Biochemical Characteristics
The physiological and biochemical characteristics of actinomecetes were studied using standard media from Hi media, Mumbai, India, and enzymatic tests such as the catalase test, Urease test, Casein hydrolysis, starch hydrolysis, gelatin hydrolysis [13], oxidase and nitrate reduction test [14], and production of melanin pigmentation on ISP-6 [15]. Thin layer chromatography was used to evaluate the presence of 2-6 diaminopimelic acid (DAP) isomers in the total cell wall composition of the strain ER7 (S D Fine-Chem Ltd., Mumbai, India).
Extraction of Antimicrobial Principle From isolates
The bioactive principle was produced in a revolving shaker using production medium (C- Medium broth and Spore-inoculated actenomycetes suspension) and incubated at 200 rpm for 7 days at 28+ 20C. The antibacterial principle was extracted with ethyl acetate and separated in a rotavapor after the growth. The resulting residue (crude extract) was diluted in 1 mL ethyl acetate solvent and kept at 4 degrees Celsius.
Antibacterial activity
Test bacteria for antibacterial assay
Four different gram positive bacteria: Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus, four gram negative bacteria:Enterobacter aerogenes, Escherichia coli, Klebseilla pneumoniae, andPseudomonas aeruginosa obtained from the American Type Culture Collection (ATCC),Kakatiya Medical College local region wereutilized for screening against antibiotic producing actinomycetes.
Primary screening
The antibacterial activity of the actinomycetes isolates was tested against bacteria using the perpendicular or cross streak technique. On nutrient agar plates, a single centre stripe was produced using various actinomycetes strains and incubated at 280C for 4-5 days, after which the test pathogens were streaked perpendicular to the actinomycetes streak and incubated at 320C for 24 hours, after which the growth pattern was observed. Actinomycetes were classified as antimicrobial generating strains and used for secondary screening based on their inhibitory zone.
Secondary screening
Each isolate was inoculated in a 100 ml C- medium and cultured in an orbital shaker for eight days at 28±20C and 200 rpm based on the findings of primary screening. After development culture, the broth was centrifuged for 10 minutes at 10,000 rpm, and the supernatant was collected aseptically and combined with an equivalent amount of ethyl acetate and left overnight.
The topmost layer of ethyl acetate was then separated and evaporated at 40°C once more. The antibacterial capabilities of the concentrated solvent were tested against harmful bacteria and fungi.
Agar Well Diffusion Method
Spreading a 24 hour fresh culture of bacteria and fungi on Muller Hinton agar and Sabouraud dextrose agar plates was used for agar well diffusion. Wells were formed using a sterile cork borer (5mm) and each well was filled with 100 microliter of extracts. Negative and positive controls were ethyl acetate and antibiotic discs (oxacillin, nitrofurantoin, and ciprofloxacin, respectively) resepectively. The plates were allowed to stand for a few minutes before being incubated at 37°C for 24 hours without inverting, and the inhibitory zones were measured in millimetres in diameter.
Results and Discussion
Ten soil samples were obtained from two distinct places in the Adilabad district of Telangana State for this research.
Table 1. Description of soil samples along with the total number of isolates and active isolates.
Soil
Samples Location Physical characteristics of soil No. of actinomycete pH Color Consistency
K1
Kawal Wildlife Sanctuary
7.46 Gray Moist 3
K2 6.87 Brown Moist 6
K3 6.55 Light
Brown Dry 7
K4 6.88 Brown Dry 8
K5 6.32 Brown Moist 2
J1
Jannaram Forest
6.34 Black Dry 3
J2 5.58 Black Dry 13
J3 5.78 Gray Dry 11
J4 5.23 Brown Dry 19
J5 5.90 Gray Moist 9
On the basis of physiological observation and biochemical testing, a total of 81 actinomycetes isolates were isolated in various parts of Adilabad district, as shown in Figure-1 and Table- 1.When compared to the Kawal Wildlife Sanctuary, the Jannaram forest zone has a lot more actinomycetes.Variation in occurrence was detected across actinomycetes, with 12 isolates identified in Amycolatopsis, 8 isolates in Micromonospora, 6 isolates in Microbacterium, 11 isolates in Saccharoppolyspora, and 44 isolates in Streptomyces, the most among all genera (Table-2).
Figure 1. Genera of isolates based on physiological observation and biochemical tests.
When compared to other actenomycetes, J5 sample was discovered as having more Streptomyces spp. (22.7 percent) and was given in Table-2.
Table 2. Variation of different actenomycetes species identified in different locations
Soil Samples
Actenomycetes species
Amycolatopsis Micromonospora Microbacterium Saccharoppolyspora Streptomyces
K1 1 0 1 1 2
K2 0 0 0 0 1
K3 2 1 0 0 2
K4 0 0 0 2 4
K5 1 2 0 1 2
J1 0 1 1 2 3
J2 4 3 0 2 7
J3 1 0 2 1 8
J4 1 0 0 1 5
J5 2 1 2 1 10
Total 12 8 6 11 44
The 44 Streptomyces isolates from 10 samples that were discovered were subsequently submitted to primary screening, as shown in Table-3. During initial screening using the perpendicular streaking technique, four isolate samples from 44 Streptomyces isolates from ten samples demonstrated antibacterial activity against the test pathogens. Streptomyces isolates were shown to be active against both Gram-positive and Gram-negative test organisms in 52 percent (23/44)
Table 3. Primary screening results of the isolates.
Sample
Gone of inhibition (mm) Gram positive bacteria
Zone of inhibition (mm) Gram negative bacterial B.
cereus B.
subtilis S.
aureus M.
luteus
E.
aerogenes E.
coli
K.
pneumoniae
P.
aeruginosa
K1 0 4 4 2 0 0 0 0
K2 4 6 2 0 4 4 7 4
K3 8 5 2 0 0 0 3 4
K4 0 5 0 0 4 6 5 9
K5 6 10 12 10 8 11 9 10
J1 7 11 10 9 10 9 9 12
J2 3 4 0 0 6 4 8 9
J3 5 7 11 10 11 11 10 9
J4 8 4 4 8 5 7 4 4
J5 14 11 8 16 13 12 9 18
In the secondary screening, the J5 Streptomyces sp sample demonstrated zone of inhibition against the test organisms, as shown in Table-4, among 23 active isolates from four samples (K5, J1, J3, and J5). Except for K. pneumonia, one of the four Streptomyces spp. samples (J5) had the greatest activity against all gram-positive and gram-negative bacteria.
Table 4. Secondary screening results of the isolates
Sample
Gone of inhibition (mm) Gram positive bacteria
Zone of inhibition (mm) Gram negative bacterial B.
cereus B.
subtilis S.
aureus M.
luteus
E.
aerogenes E.
coli
K.
pneumoniae
P.
aeruginosa
K5 9 9 5 7 7 7 10 11
J1 3 6 3 9 8 8 11 9
J3 12 12 7 6 13 17 11 9
J5 20 20 16 14 18 12 4 11
The goal of the research was to find antimicrobial-producing actinomycetes in different areas in Adilabad district, Telangana State, India. A total of ten soil samples were examined from two different sites. Eighty-one actinomycete strains were recovered and identified, with Streptomyces (54.3%), Amycolatopsis (14.8%), Micromonospora (9.87%), Microbacterium (14.8%), and Saccharoppolyspora (13.5%) being the most common (Figure-1). The soil sample obtained from the Jannaram woodland zone had the most actinomycetes. The capacity of microbes to survive in formerly harsh and demanding environments may be related to their capacity to adapt to the environment and develop resistant structures such as spores [16].
Slow-growing, aerobic, glabrous, and folded with a variety of colourful aerial and substrate mycelia, all of the isolates were found to be slow-growing, aerobic, and glabrous. On starch casein agar, the majority of them generated colours in brown, yellow, blue, bluish green, and
purple (SCA). In one investigation, the isolate AR-ITM02 (Streptomyces spp.) displayed pigment-producing capabilities solely on SCA, which might be related to a sufficient quantity of nutrients in the medium [16]. In another study, 5 different pigments were extracted from actinomycete isolates, purified, and tested for antimicrobial activity, and it was discovered that all of the pigments inhibited the growth of E. coli, Staphylococcus aureus, Bacillus subtilis, and Salmonella typhi, with the exception of the yellow pigment, which inhibited only Gram-negative bacteria.
When the entire Streptomyces spp isolates were screened for activity against the test organisms in the present investigation, 52 percent were determined to be active. Antibiotic activity was found in all active isolates from K5, J1, J3, and J5 samples against Staphylococcus aureus, although only J5 demonstrated action against all of the test species. Only 12.82 percent of 117 actinomycete isolates tested positive for antibacterial activity in a study [15]. In another investigation, 6 out of 20 strains were shown to be active against Gram-positive test organisms, but only 4 strains were found to be active against Gram-negative test organisms [17]. Similarly, only 36 isolates out of 106 demonstrated activity against test organisms, with two active just against Gram-negative bacteria, eight active against Gram-positive bacteria, and 26 active against both Gram-positive and Gram-negative bacteria [17].
The difference in sensitivity between Gram-positive and Gram-negative bacteria may be explained by morphological distinctions between these species [18]. Gram-negative bacteria have an exterior polysaccharide membrane that prevents lipophilic solutes from entering the cell wall;
Gram-positive bacteria only have an exterior peptidoglycan layer, which is ineffective as a permeability barrier. Only 10 isolates from the J5 sample demonstrated zone of inhibition during subsequent screening of active isolates using the ethyl acetate extract. The pure ethyl acetate extract of S. sannanensis SU118 culture broth resulted in the recovery of a strong antibacterial agent active against Gram-positive bacteria in a research [18]. In another investigation, the antibacterial activity of the ethyl acetate: methanol extract was shown to be superior to that of the water solvent extract. This might be owing to the active compound's solubilization in organic solvents over aqueous solvents, as well as the higher concentration of the metabolite in the solvent-extracted combination [18].
During primary and secondary screening, the active isolates showed varying levels of activity.
Actinomycete isolates that demonstrate antibiotic action on agar but not in liquid culture are often observed during secondary metabolite screening (16]. This might be due to changes in culture medium substrate composition, inoculum size, or incubation time between primary screening and submerged fermentation. It's also conceivable that the actinomycetes' active chemicals become inactive, chemically changed, or bond to a broth component. Many actinomycetes are also poor fermenters [18].
Temperature is one of the many environmental constraints and growth variables that impact actinomycetes' development and variety. It has a significant impact on organisms' physiology, morphology, sporulation, biochemistry, and antimicrobial metabolite synthesis. The optimal temperature for actinomycetes growth was discovered to be in the mesophilic range in the research.
Conclusions
Diverse strains of actinomycetes with antibacterial activity were isolated from different soil samples in the Adilabad area of Telangana State. Streptomyces (54.3%), Amycolatopsis (14.8%), Micromonospora (9.87%), Microbacterium (14.8%), and Saccharoppolyspora (14.8%) were among the eighty-one isolates recovered from ten soil samples (13.5 percent ). Isolate J5 had the strongest broad-spectrum antibiotic activity from secondary screening. A total of ten isolates demonstrated broad-spectrum antibacterial activity. Existing antimicrobial-producing strains have failed owing to the development of resistance among microbes, hence new antimicrobial- producing strains are urgently needed. The current research is also a little contribution to this requirement. n the search for novel antimicrobial agents, isolates that exhibited broad-spectrum action against the test bacteria might be considered candidates.
Acknowledgment
Authors thank to Head, Department of Microbiology, Kakatiya University, Warangal, Telangana State, India, for his support.
Financial support & sponsorship: None.
Conflicts of Interest: None
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