In Vitro Screening and Identification of Bioactive Compound Producing Marine Actinomycetes from Thoothukudi Coastal Water
B. Rajalinga Malathi1*, S. Abirami2, C. Gayathri3
1,2,3Department of Microbiology, Kamaraj College, Thoothukudi, India. (Affiliated to ManonmaniumSundaranar University, Tirunelveli)
Email: 1[email protected](Affiliation msuniv.ac.in)
ABSTRACT
The coastal region water has been widely explored as the chief sources of microorganisms, especially actinomycetes, that are capable of producing a wide variety of bioactive molecules. The majority of the marine actinomycetes produce their value-added metabolites. The adversary effect of bioactive compounds of marine actinomycetes against various pathogens was pathetic. In our study, 10 different strains of actinomycetes were isolated from seawater. Among these two strains (MAT1 and MAT2) showed better inhibitory activities against the selected pathogens. Based on 16S rRNA gene sequence analysis this strain was most closely related to Streptomyces13581T (96.4 % sequence similarity) species and its phylogenetic analysis showed that the isolated strains were confirmed as Streptomycesarmeniacus and Streptomycesalkalithermotolerans. Itis one of the important alkaliphilicactinomycete strains. This work was analyzed for its physiological and biochemical characteristics of isolated actinomycetes and also to analyze the application of bioactive compounds in the biomedical field.
Keywords
Bioactive compounds, Marine Actinomycetes, Human pathogens, etc
INTRODUCTION
The ocean is a huge saltwater body that covers about two-thirds of the Earth's surface. Marine ecosystems consequently have a greater diversity than their terrestrial system. The marine environment attracts many researchers because of its rich biodiversity. Many mysteries were hidden with aquatic flora and fauna in the marine environment. In the marine environment, organisms are not distributed evenly throughout the seawater. Variations in characteristics of the marine environment create different habitats and influence the production of bioactive compounds. When compared to the terrestrial species, marine organisms producing bioactive compounds are unique and composed of many peculiar characteristics (Wagner- Dobleret al.,2002). Marine actinomycetes have a wide spectrum of biological activities such as antimicrobial, plant growth hormones, immunosuppressive, neurotoxic, and cytotoxic actions (Ireland et al.,2000). They are all well known for their secondary metabolite production.
Streptomycesare Gram-positive, aerobic, mycelial bacteria, known to produce a wide variety of bioactive compounds which are industrially and pharmaceutically relevant. Bioactive compounds present in the marine actinomycetes possessing various beneficial compounds such as Abyssomicin, Diazepinomicin, Frigocyclinone, Lynamicins, and Marinopyrroles. Essramycin is a novel antibiotic isolated from Streptomyces sp. This compound is antibacterially active against pathogens. (El-Gendyetal., 2008). Marine actinomycetes produce bioactive compounds that inhibit the growth of human pathogens (Viswanathanet al.,2017). The aim of the present study to screen the production of bioactive compounds from marine actinomycetes and their antibacterial activity against human pathogens.
MATERIALS AND METHODS STUDY AREA AND SAMPLE COLLECTION
Thoothukudi district is located in the South East of Tamil Nadu state. The district covers an area of 4621sq.km and its geographical coordinates are 8.7642° N, 78.1348° E. It is located about 590 km south of Chennai and 190 km northeast of Thiruvananthapuram and by Tirunelveli district on the West and South West. In this study, the marine water sample was collected from different places of Thoothukudi coastal area such as pearl city beach, Roche park, Harbour beach, and Muyaltheevu. Samples were collected from 5 to 15 cm depth and kept in sterile polyethylene bags and preserved in the laboratory for further analysis.
ISOLATION OF MARINE ACTINOMYCETES
Starch casein agar medium was prepared and sterilized at 121°C in 15 lbs pressure for 15 min.
Then it was supplemented with Amphotericin B 50 μg/l and Tetracyclin 20 μg/l to prevent bacterial and fungal growth. The agar plates were prepared. 1ml of marine water sample was spread over the agar plates. The inoculated plates were incubated at 28°C for seven days. After incubation, the actinomycetes colonies were observed, and then it was purified and stored for further investigation. After that, each colony was tested for physical, morphological, and biochemical characterization and also screened for bioactive production (Kokareet al., 2003).
SCREENING OF BIOACTIVE COMPOUNDS
The qualitative analysis of the functional group of the antimicrobial compounds was done by the method given by (Harindranetal., 1999). Functional group analysis was performed to find out the classes or nature of the group of antimicrobial compounds.
OPTIMIZATION OF BIOACTIVE COMPOUNDS PRODUCTION FROM
ACTINOMYCETES
The nutrient broth was prepared and sterilized. The pH of the broth was adjusted to 6, 7, 8, 9, and 10 using 0.1 N HCl and NaOH. The Actinomycetes cultures were inoculated into the test 35 tubes containing broth and incubated at 28±2°C for 7-14 days. The growth was recorded after incubation by measuring the optical density ( Dubeyet al.,2002). To determine the effect of temperature the nutrient broth was prepared and sterilized. The Actinomycetes cultures were inoculated into the broth. The tubes were incubated at 15, 20, 25, 30, 35, 40, 45, and 50°C for 7- 14 days. After incubation, the growth was recorded and measured by recording its optical density ( Dubeyet al.,2002).
BIOACTIVE COMPOUNDS EXTRACTION FROM ISOLATED ACTINOMYCETES
The bioactive metabolites were extracted using the solvent extraction method of Sunil LaxmanAttimaradet al.(2012). 100ml culture was centrifuged at 12000 rpm for 30 min. 10ml of supernatant was taken in a test tube and mixed with 4 different solvents of ethanol, methanol, phenol, and acetone separately each 2-3 times, and the solvent extracts were allowed for evaporation. After that, the extracts were dissolved in sterile water and then antagonistic activity against selected pathogens was done.
HUMAN PATHOGEN COLLECTION:
The pus sample was collected from a diabetic patient who was suffering from a diabetic foot ulcer from Thoothukudi Government hospital. The sample was collected with a sterile swab and stored in saline and transported to the laboratory.
ISOLATION OF HUMAN PATHOGENS FROM PUS SAMPLE
The sample brought from the laboratory were processed on different specific media for selected pathogens such as Blood agar plates, Pseudomonas isolation agar plates, and Eosin methylene blue agar plates, etc. and the pus sample were streaked in every selective plate and incubated at 37°C for 24 hours. According to the Bergey’s manual biochemical characteristics, isolated colonies were identified.
ANTAGONISTIC ASSAY OF HUMAN PATHOGENS
The isolated human pathogens were inoculated into hundred milliliters of nutrient broth and incubated at 35°C for 24h. The isolated pathogens were swabbed on Mueller Hinton agar and wells (5mm in diameter) were made in plate agar using sterile sharp borer. Then 100μl of each supernatant was added into each well and the plates were incubated at 35°C for 24th. After incubation, a clear zone of growth inhibition was observed, measured in mm, and qualified which was considered as bioactive compound activity (Senet al.,1995).
DETERMINATION OF TOTAL ANTIOXIDANT ACTIVITY
The total antioxidant activity was evaluated by the phosphomolybdenum method described by (Prietoetal., 1999) 1.0 ml of the extract was mixed with 1.0 ml of the standard reagent solution.
The tubes were closed tightly and incubated at 95°C for 90 min in a thermal block. After cooling, the optical density was measured at 695 nm against a blank. The result was expressed as milligram of Ascorbic Acid Equivalence (AAE) per gram of extract.
HYDROXYL RADICAL SCAVENGING ASSAY
Hydroxyl radical scavenging activity was determined by the method given by Klein et al., (1981).
The reaction mixture contained 1.0 ml ofdifferent concentration of extracts (2-10 mg/ml), 1.0 ml of iron-EDTA solution, 0.5 ml of 0.018% EDTA, 1.0 ml of DMSO (0.85 % in 0.1 M phosphate buffer pH7.4) and 0.5 ml of 0.22% ascorbic acid. The tubes were closed and heated in a water bath at 80-90°C for 15 minutes, the reaction was ended by adding 1.0 ml of ice-cold TCA (17.5
%). To the above reaction mixture, Nash reagent was added and incubated for 15 minutes. The intensity of the color change was measured at 412 nm against blank. Ascorbic acid was used as standard.
% inhibition = [(Control- Test)/control] ×100
NITRIC OXIDE RADICAL SCAVENGING ASSAY
Nitric oxide radical scavenging assay was done by theprocedure described by Green et al.,(1982).
The reaction mixture contained sodium nitroprusside in phosphate saline and various concentration of extracts. The solution was incubated at 25°C for 60 minutes. To the incubated sample 5ml of Griess reagent was added and the absorbance of the chromophore was measured at 546 nm against a blank. The generation of nitrate ions is observed. The standard ascorbic acid and BHT were used for comparison. This activity was determined by evaluating % inhibition.
% inhibition = [(Control- Test)/control]×100
IDENTIFICATION OF BIOACTIVE COMPOUND PRODUCING MARINE
ACTINOMYCETES
The potent actinomycetes strains were phylogenetically identified by 16S rRNA sequencing.
GENOMIC DNA EXTRACTION
The 5 ml nutrient broth containing was inoculated with a single bacterial colony. Harvest the pellet after vigorous shaking of the incubated tube at 360rpm from 1.2 to 1.5 ml overnight culture and centrifuge at 12,000 rpm for 10 minutes and collect the pellet in a fresh tube. Add freshly prepared 570 μ1 of TE buffer and invert the tube and resuspend well with pipetting. Add freshly prepared 30μ1of 10 % SDS, 3μ1of proteinase k, and 3 μ1of RNAse and mixed well. After incubating at 37 °C for 1 hour. Add 80μ1 of CTAB-NaCl +100 μ1 of 5M NaCl and invert the tube and mix well. After incubating the water bath at 65 °C for 10 minutes. Add freshly prepared equal volume of Chloroform: Isoamyl alcohol (24:1) inverting and mixed well. After centrifuging at 12,000 rpm for 10 minutes and collect the supernatant (upper phase) in a 1.5 ml fresh tube.
Add freshly prepared equal volume of Phenol:Chloroform: Isoamyl alcohol (25:24:1) inverting and mixed well. After centrifuging at 12,000 rpm for 10 minutes and collect the supernatant (upper phase) in a 1.5 ml fresh tube. Add 0.6 (600 μ1) ice-cold isopropanol gently inverting and mixed after incubating at -20°C for 10-20 minutes. After centrifuging at 12,000 rpm for 10-15 minutes and discard the supernatant collect the pellet. After adding 500 μ1 70% of ethanol. After centrifuging at 12,000 rpm for 5 minutes discard the supernatant collect the pellet and air dry the pellet. After add 20-25 μ1 DEPC treated nucleus free water or TE buffer at storage at -20°C.
Then the sample gets purified by QIA quick kit method.
GC-MS ANALYSIS OF EXTRACTED BIOACTIVE COMPOUNDS FROM MARINE ACTINOMYCETES
The trace GC ultra and DSQII model MS from Thermo Fisher Scientific Limited was used for the analysis. The injector port temperature was set to 250°C, Interface temperature to 250°C, and the source was kept at 200° C. The oven temperature was set to 70°C for 2 mins, 150°C for 8 mins, and up to 260°C for 10mins. The split ratio was set to 1:50 and the injector was used in splitless mode. The DB-35 MS Nonpolar column was used with the dimension 0.25mm OD x 0.25µm IDx30 meters length produced by Agilent Co., USA. 1ml/min of Helium gas is used as a carrier.
The source was maintained at 200C and below 40 vacuum pressure. The ionization energy was maintained to -70eV. The MS was inbuilt with a pre-filter which reduce the neutral particle. The data system has two inbuilt libraries for matching and searching spectrum.
RESULTS AND DISCUSSION
After incubation morphologically different strains were selected for this study. 10 actinomycete strains were recovered from Starch Caesin Agar plates. Based on the screening of bioactive compounds production, two isolates (MAT1 and MAT2) were selected for this study, and their morphological and physiological characteristics were studied. The physiological characteristics of actinomycete isolates (MAT1, MAT2) were analyzed. The optimum pH for growth was tabulated in Table 1. Similarly, the optimum temperature for the growth of isolated actinomycetes showed in Table 2. The maximum growth of MAT1 and MAT2 actinomycetes was observed in pH 11. The better growth of isolated actinomycetes MAT1 and MAT2 was observed on the temperature 30 º C. Physiological characterization such as the effect of pH, the temperature was observed by Mohan Remyaet al. (2008). Optimum growth was recorded in the pH 8 to 9 and Temperature 30 º C for the strain of Nocardiopsisalba(Daniel et al., 2002).
Controversy, Starch et al. (2003) reported that some actinomycetes grew on inoculated modified nutrient agar plates with different temperature of 37, 45, and 55 º C. Optimum growth was observed at 11%NaCI and its optimum growth temperature was observed at 45º C
TABLE 1: EFFECT OF pH IN ACTINOMYCETES ISOLATES (MAT1 and MAT2) pH MAT1( OD at 595nm) MAT2 (OD at 595nm)
1 3.1 2.7
3 4.9 3.9
5 5.3 4.2
7 5.8 5.1
9 6.8 7.1
11 6.9 7.6
TABLE 2: EFFECT OF TEMPERATURE IN ACTINOMYCETES ISOLATES (MAT1 and MAT2)
TEMPERATURE MAT1 OD at 595nm MAT2 OD at 595nm
30 ºC 7.4 7.1
37 ºC 5.4 5.7
50 ºC 5.4 4.1
60 ºC 5.3 5.0
70 ºC 5.1 4.7
The morphological characteristics of the isolated actinomycetes strain MAT1 were observed as Ash-centered colonies, rod-shaped, and identified as Gram-negative, Similarly, the Actinomycetes isolate MAT2 were observed as Orange pigmented colonies and identified as Gram-negative. The Actinomycetes isolates ( MAT1, MAT2) showed a positive result for the enzyme production (Amylase, protease, and Gelatinase) and negative results for cellulose and lipase production. The isolated pure colonies were subjected to various biochemical characterization and the results were tabulated in Table 3 and Table 4.
TABLE 3: MORPHOLOGICAL CHARACTERISTICS OF ACTINOMYCETES ISOLATES (MAT1 and MAT2)
Sample Colony appearance Shape Gram staining Respiration Amylase Protease Lipase Cellulase Gelatinase
MAT1 Ash centered
powdery Bacilli Gram-negative Aerobe - - - - + MAT2 Orange mucoid Bacilli Gram-negative Aerobe - + - - +
TABLE 4: BIOCHEMICAL CHARACTERISTICS OF ACTINOMYCETES ISOLATES (MAT1 and MAT2)
BIOCHEMICAL TESTS MAT1 MAT2
Indole Production Test + +
Methyl Red Test + -
VogesProskauer test - - Citrate utilization test + +
Catalase test + +
Oxidase test + +
Nitrate reduction test + +
Ammonia test + +
Urease test + +
Sucrose + +
D-Galactose + +
D-Fructose + +
D- Glucose + +
L-Arginine + +
L-Methionine + +
Acid and Gas production + +
The human pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella sp. were isolated from pus samples of diabetic patients wounds and they were identified by Bergey's manual biochemical characterizations. Screening of antimicrobial compound production tests was carried out on the ten marine Actinomycetes. The better results were observed in only two isolates ( MAT1, MAT2). The solvent extraction of bioactive compounds of the two isolates and their antibacterial activity was tested. MAT1 isolate shows maximum inhibition for Pseudomonasaeruginosa in the phenol extract. MAT2 shows maximum inhibition to Pseudomonasaeruginosa in phenol extracts. Among the four solvents of two strains Phenol shows the maximum activity, Ethanol shows moderate activity, Methanol shows the minimum activity and Acetone shows the least activity. The results were tabulated in Table 5.Similarly, Riedlingeret al. (2004) described that isolated Streptomyces and Verrucosissporastrains showed better antimicrobial activity against gram-negative bacteria of human pathogens. The maximum inhibition was observed with actinomycete isolate SJP4 against the pathogens like E.coli,P.aeruginosa, S.aureus, and B.cereus. The actinomycetes isolate showed moderate activity against all 4 test pathogens (Priyankaet al.,(2019). Similarly, the four actinomycetes isolates showed maximum inhibition of Bacillus, Staphylococcus, Escherichia coli, Klebsiella, and Pseudomonas (Oskayet al., 2004; Dhananjeyanet al., 2010; Ozeret al., 2010).
TABLE 5: QUANTIFICATION OF ANTIBACTERIAL ACTIVITY OF
ACTINOMYCETES ISOLATES (MAT1 and MAT2) AGAINST HUMAN PATHOGENS IN DIFFERENT SOLVENT EXTRACTS:
Test organisms Zone of inhibition (mm)
MAT1 MAT2
E M P A E M P A
Staphylococcus aureus 20 16 42 - 20 15 43 -
Pseudomonas aeruginosa 20 17 48 - - - 44 -
Bacillus subtilis 16 19 39 - - 22 36 -
Klebsiella 17 - 39 28 - 14 31 18
The total antioxidant study, hydroxyl radical scavenging activity, NO scavenging activity were carried out for both samples MAT1 and MAT2 and the results were tabulated in Table 6,7, and 8.
In hydroxyl radical scavenging activity 46.67% of inhibition was observed at 0.640 OD at 412nm for MAT2 and 40% of inhibition is observed for MAT1. The maximum inhibition of 24% is achieved for MAT1 in Nitric oxide scavenging activity with 0.380 OD at 546nm and the minimum inhibition of 2% is achieved for MAT2.
TABLE 6: NITRIC OXIDE SCAVENGING ACTIVITY OF ACTINOMYCETES ISOLATES (MAT1 and MAT2)
Sample no OD at 546nm % inhibition
1 0.490 2
2 0.380 24
Control 0.500
TABLE 7: HYDROXYL RADICAL SCAVENGING ACTIVITY OF ACTINOMYCETES ISOLATES (MAT1 and MAT2)
Sample no OD at 412nm % inhibition
1 0.720 40
2 0.640 46.67
Control 1.200
TABLE 8: TOTAL ANTIOXIDANT ACTIVITY (TAA) OF ACTINOMYCETES ISOLATES (MAT1 and MAT2)
Sample Conc µg/ml OD at 695 nm TAA mg/g equivalents of ascorbic acid
Ascorbic acid standard
200 0.050
400 0.350
600 0.380
800 0.630
1000 0.740
1 1000 0.01 0.667
2 1000 0.05 3.333
The 16S rRNA gene sequencing and phylogenetic analysis of isolated actinomycetes revealed that (both strains MAT1 and MAT2 ) were closely related to novel Actinobacteria bacterium and it was confirmed as Streptomyces armeniacus ATCC 15676 chromosome and Streptomyces alkalithermotoleransstrain AC3 16S ribosomal RNA, partial sequence analysis. The Phylogenetic tree of the strain MAT1 and MAT2 were expressed in Figure 1 and Figure 2.
FIGURE 1: Phylogenetic tree of the strain MAT1
SEQUENCING: 18280-MAT1-16sR
FIGURE 2: Polymerase chain reaction- Phylogenetic tree of the strain MAT2
SEQUENCING:18280-MAT2-16sF
The GCMS analysis was made to detect the compounds present in the samples MAT1 and MAT2. Cetane, Isohexadecane, Fuctosterol, Hexadecanoic acid, 2,3,4 Trimethylheptanes, 1,6,6- Trimethyl-7-(3-oxobuttenyl 3,8 dioxatricyclo (5,1,0,0,2,4) octane – 5-one were the compounds present in the MAT1. Isopytol, 3-Mercapto 2(H)-pyridinone, Cetane, Isohexadecane, Fuctosterol, Neophytadiene, 9,12 – Octadecadienoic acid (ZC) – (CAS) were the components identified from MAT2. The components of MAT1 and MAT2 were tabulated in Figure 3 and Figure 4.
Interpretation of mass spectrum of GC-MS was done using the database of National Institute Standard and Technology ( NIST4 and WILEY 9). The spectrum of the known component was compared with the spectrum of the known components and was stored in the inbuilt library.
Priyankaet al.(2019) reported that GCMS analysis of bioactive compounds extracted from actinomycetes isolates SJP4 was identified as 4,8-diaza-2,9-dibenzoyl-5,6- diphenyl-2,8- decadienedioic acid diethyl ester and 1-[1,2,4]triazol-1-ylethanone
The statistical analysis of ANOVA talks about MAT, solvent, and the relationship between them.
Since the sig value is less than 0.05 (0.014) in the case of both MAT and Solvent, it indicates that the hypothesis is rejected. There is a difference between all MAT concerning the effect that it has upon the bacteria. Similarly, there is a difference between all the solvents about the effect that it has upon the bacteria. But in the case of the combination of MAT and solvent, the sig value is 0.855 which is higher than 0.05, and therefore the hypothesis is not rejected. Therefore, there is no interaction effect between the solvent and MAT. The Estimated Marginal Means describes the interaction effect between MAT and the solvents. About both the MATs, the interaction effect of P is high compared to other solvents. For example, the mean value of P is 42.000 when it interacts with MAT1. Similarly, the mean value of P is 38.5.000 when it interacts with MAT2.
Therefore it can be concluded that P has the highest effect upon the bacteria. Among all the MATs, the interaction between MAT1 and P has resulted in the highest mean value of 42.000 and therefore it is the most effective solvent combination.
STATISTICAL ANALYSIS (SPSS) OF HUMAN PATHOGENS
H0 – All strains of actinomycetes affect the pathogens at the same mm level.
H0 – All Solvent affects the pathogens in the same mm level.
H0 – There is no interaction effect between MAT and Solvent.
SAMPLE SOLVENT MEAN STANDARD DEVIATION N
MAT1 E 18.25 2.062 4
M 13.00 8.756 4
P 42.00 4.243 4
A 7.00 14.000 4
TOTAL 20.06 15.716 16
MAT2 E 5.00 10.000 4
M 5.75 11.500 4
P 38.50 6.137 4
A 4.50 9.000 4
TOTAL 13.44 17.139 16
ESTIMATED MARGINAL MEANS:
Sample Solvent Mean Std Error 95% Confidence Interval Lower bound Upper bound
MAT1 E 18.25 3.949 10.311 26.189
M 13.00 3.949 5.061 20.939
P 42.00 3.949 34.061 49.949
A 7.00 3.949 -.939 14.949
MAT2 E 5.00 3.949 -2.939 12.939
M 5.75 3.949 -2.189 13.689
P 38.50 3.949 30.561 46.439
A 4.50 3.949 -3.439 12.439
The antimicrobial activity of the different actinomycete isolates against various test organisms was determined and the data were subjected to ANOVA and the means were compared using Tukey's HSD at a significance level of p < 0.05 using Sigma Stat 3.5 (Deepika Sharma et al.,2011).
CONCLUSION
The present study includes the antibacterial, antioxidant properties, and GCMS analysis of the bioactive compounds present in the Streptomyces strains. Purification and structural analysis of the active compounds from these strains may prove to be novel bioactive compounds.
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