Antimicrobial and Enzymatic potential of Streptomyces sp. KAS-1 isolated from the microbiologically unexplored estuary of Kali river ecosystem

In the present study, a Streptomyces sp. KAS-1was isolated from sediment soil samples of the Kali river estuary, Karwar, Karnataka, India. The isolated Actinomycetes strains were screened against pathogenic microorganisms and for the production of hydrolytic enzymes. Later, the strongest strain Streptomyces sp. KAS-1 was taken for further analysis based on primary characterizations. The morphological, physiological and biochemical characterizations of Streptomyces sp. KAS-1 were carried out, followed by molecular characterization through 16S rRNA gene sequencing. The 16S ribosomal RNA gene sequencing and analysis of the phylogenetic tree showed a 98.97% sequence similarity with Streptomyces thermocarboxydus strain SDT64 (KJ018992). Finally, the intracellular methanol extract was characterized through UV-Visible and FTIR spectrophotometer. The Streptomyces sp. is a gram-positive, aerobic, non-motile, spore-forming bacterium and it was designated as Streptomyces sp. KAS-1 and was found to possess a strong antimicrobial activity against pathogenic microorganisms. The characterizations revealed the Streptomyces sp. KAS-1 was moderately thermophilic, neutrophile organism having the capacity to degrade a broad range of carbon sources. The UV-Visible spectrum of methanol extract of Streptomyces sp. KAS-1 revealed the presence of Muconomycin-B and the presence of different functional groups such as aldehydes, amines, and alkyl halides were indicated by the FTIR spectrum.


INTRODUCTION
Actinomycetes are a group of gram-positive, ilamentous bacteria and having high G+C content.
They are physiologically versatile and found in most of the environments, including terrestrial and aquatic habitats (Pathalam et al., 2017). The word Actinomycetes was coined from the Greek language where "atkis" means "a ray" and "mykes" means "fungus." This is why Actinomycetes are also called as Ray fungi due to close similarities with fungal counterparts. However, they even have many dis-similarities with fungi; due to unique properties and thus are restricted into the domain bacteria (Chaudhary et al., 2013). There is an increasing realization of the potential for sediment soil as sources of Actinomycetes that produce useful bioactive compounds. (Okami, 1986), reported that Actinomycetes of freshwater origin produce novel bioactive substances. However, the focus is increas-ing towards novel regions, estuarine ecosystems, and extreme environments for isolating novel bioactive strains, especially Actinomycetes, which produce nearly 80% of all known antibiotics.
Additionally, the microbial pro iles also serve as an indicator of freshwater, estuarine ecological health. Microbes are the factors for a multitude of human diseases, but also equally owe a great extent to human medicine, disease treatment, and control. Marine actinobacteria are the rich source for the potent novel secondary metabolites and the majority of these compounds are derived from a single genus Streptomyces .
The bioactive secondary metabolites from the Actinomycetes are endowed with novel chemical structures with strong biological activities. Marine habitats provide more chances for isolating new species of Streptomyces with unique chemical structures repelling many microbial diseases and cancers (Janardhan et al., 2014). Streptomyces are more useful in the ield of pharmaceutical industries because of their outstanding capacity to produce antibiotics, enzymes, antimicrobial metabolites, mainly such as aminoglycosides, macrolides, polyenes, glycopeptides, polyketides, betalactides and actinomycins (Ding et al., 2019).
Actinomycetes of marine and estuarine habitats are less explored because of the dif iculties in the collection and isolation processes involved when compared to terrestrial Actinomycetes (Kamjam et al., 2017). An estuary is a semi-enclosed coastal body of water estuaries act as a transition zone between two aquatic ecosystems, namely marine and freshwater. They are affected by both marine in luences, such as waves, tides, a mixture of saline water, and sediments. As a result, they may contain many biological niches within a small area, and so are linked with large biological diversity (Parthasarathi et al., 2012;Rosmine and Varghese, 2016). Estuaries provide some of the most productive habitats on earth because of the accumulation and availability of nutrients along with adequate light conditions that enhance the production of phytoplankton in the water (Cloern et al., 2014).
The result of an intensive screening program carried out over the past many decades is that there is a growing problem of a rediscovery of already known bioactive compounds (Genilloud, 2018). An approach to address this problem is to expand the source of Actinomycetes by carrying out an ecological assessment of environments other than terrestrial soils. There is a growing interest in the Streptomyces from estuarine ecosystems and it was found that the majority of the isolates were Streptomycetes, indicating that the estuary soil is a suitable source of Actinomycetes to screen for production of novel bioactive compounds (Dharumadurai et al., 2010).
Our review of the literature showed that there was no isolation study of Actinomycetes from the Kali river estuarine sediment samples of Karwar, Uttara Kannada (U.K) until today. Therefore a systematic screening program was undertaken and a Streptomyces sp. KAS-1 was isolated from sediment samples of the Kali river estuarine area on the south coast of India. Taxonomic characterization was carried out based on 16S rRNA sequence analysis in combination with morphological, physiological, hydrolytic enzymes and biochemical characterizations. The antibacterial and antifungal activity was also investigated.

Collection of samples
12 sediment samples were collected from different locations of the Kali river ecosystem of Karwar, Karnataka, India. The sediment samples were collected at approximately 2 meter depth using a sediment sampler and stored in sterile containers at 4 • C.

Isolation of Actinomycetes
To isolate Actinomycetes, the sediment samples were pre-treated by heating at 60 • C for 15 minutes to kill unwanted bacteria and fungi. The standard serial dilution technique was employed to isolate Actinomycetes (Hayakawa et al., 1988). Each sample was serially diluted and 100µL aliquot was spread evenly on starch casein agar (SCA) medium. Antifungal and antibacterial antibiotics supplements (Amphotericin B and Tetracyclin) at 25µg/mL respectively were used for Actinomycetes growth enrichment and also to prevent fungal and bacterial contaminations (Cuesta et al., 2012;Zhang et al., 2005). Later the plates were incubated at 30 • C for 8 days and observed periodically for the growth of Actinomycetes. Finally, the selected Actinomycetes colonies were puri ied and maintained at 4 • C for subsequent studies.

Primary screening of antimicrobial activities
To check the potentiality of isolated Actinomycetes, antimicrobial activity was studied by the perpendicular streak method (Hayakawa et al., 1988;Williams et al., 1983)against pathogenic bacteria and fungi. The test organisms such as Aspergillus fumigatus (MTCC8877), Enterobacter aerogenes (ATCC2822), Staphylococcus epidermidis (MTCC435) and Shigella lexneri (MTCC1457) were streaked at an angle of 90 • to Actinomycetes isolates on nutrient agar (NA) medium. Thereafter, the most active strain, which      inhibited the growth of microbial pathogens during preliminary screening, was selected for further studies.

Studies on Enzyme activity
To study amylase activity, the isolates were grown on starch casein agar medium (pH 7.0 ± 0.5) for 6 days at 30 • C. After incubation, each plate was looded with 1% iodine solution and the formation of the halo zone around colonies was observed (Oumer and Abate, 2018). Esterase activity was studied by streaking Actinomycetes isolates on Tween-80 agar (7.2 ± 0.1) plates and incubating at 30 • C for 6 days. The zone of hydrolysis around colonies was observed and recorded (Rai et al., 2014). Thereafter, pectinase activity was carried out by inoculating isolated Actinomycetes on pectinase screening agar (MP-5 medium, pH 5.5 ± 0.1) plates and incubated at 30 • C for 6 days. Later, after incubation, each plate was looded with potassium iodide solution, the halo zones produced around each colony were observed and noted (Hamedi et al., 2019).

Figure 3: Colony morphology of isolated Streptomyces sp. KAS-1
The isolated microorganisms ability to hydrolyze DNA was studied according to (Pradhan et al., 2015;Rai et al., 2014) by inoculating them on the DNase medium (pH 7.5 ± 0.5). After an incubation period of 6 days at 30 • C, the DNase enzyme producing organisms were selected based on a clear zone around them. The Actinomycetes strain, which showed the best production of all hydrolytic enzymes, was selected for further studies.

Morphological, physiological and biochemical characterization
The most potent strain Streptomyces sp. KAS-1 was studied through morphological, physiological and biochemical analysis. For morphological characterization, the Streptomyces sp. KAS-1 was observed for shape, size, and pigmentation through the coverslip culture method (Kawato and Shinobu, 1959;Williams and Davis, 1965). Gram staining and acidfast staining were done according to the method of (Fawole and Oso, 2004). Likewise, physiological characterizations were done by observing growth and tolerance in the range of temperature from 20 • C to 70 • C and pH in the range of 5.0 to 8.0. Salt tolerance was recorded by growing the isolate in the SCA medium supplemented up to 7% sodium chloride (NaCl) concentration. Further, biochemical characterization was done according to (Gottieb, 1976). Streptomyces sp. KAS-1 was characterized for hydrolysis of starch, urea, casein, gelatin, esculin, arbutin and carbon source utilization test for D-glucose, sucrose, D-lactose, D-mannitol, trehalose, raf inose, galactose and xylose. Hydrogen sul ide production and nitrate reduction tests were also done according to the method .

KAS-1
Streptomyces sp. KAS-1 was grown in medium and the total genomic DNA was extracted. The 16S rRNA gene was ampli ied using two eubacterial primers; forward primer: 5'-AGAGTTTGATCMTGGCTCAG-3'and reverse primer 5'-TACGGYTACCTTGTTACGACTT-3'. On the other hand, for PCR ampli ication; thermal cycling conditions were programmed as follows: initial denaturation at 96 • C for 5 minutes; followed by denaturation at 96 • C for 30 seconds, hybridization at 50 • C for 30 seconds and elongation at 60 • C for 90 seconds. The electrophoresis of the PCR products was accomplished on 1% agarose gel with 500 base pair DNA ladder as a size reference (Arasu et al., 2008). The DNA data was sequenced, compiled, analyzed and matched with the Gene Bank database using the basic local alignment search tool (BLAST). Furthermore, the aligned DNA sequence was employed and a phylogenetic tree was constructed through the neighbor-joining method using the software MEGA 7.0 (Altschul et al., 1990;Tamura et al., 2007).

Production and extraction of bioactive compounds
Streptomyces sp. KAS-1 was grown for the production of bioactive compounds in a rotary shaker (150 rpm) at 33 • C for 14 days. After fermentation, the biomass was separated by centrifugation at 8000 rpm at 4 • C for 20 minutes and washed three times with sterile distilled water. The crude intracellular extract was prepared according to the method of (Saravana et al., 2014). In short, the harvested biomass was taken in an autoclaved mortar and ground thoroughly. Methanol was added in 1:1 ratio (w/v) and the mixture was shaken vigorously overnight. Later, the mixture was centrifuged at 5000 rpm at 4 • C for 30 minutes and the obtained supernatant was concentrated through evaporation using a rota-evaporator at 40 • C.

Ultraviolet-visible (UV-Vis) spectroscopy
The crude intracellular methanol extract of Streptomyces sp. KAS-1 was analyzed qualitatively using Jasco V-670 UV-Visible spectrophotometer for the possible recognition of compounds.

Fourier transform infrared (FTIR) spectral analysis
The biological functional groups present in the methanol extract were interpreted by FTIR spectroscopy. Here, the sample was prepared by grinding a small pinch of KBr with methanol extract and thin discs were made through hydraulic pressing. Further, the FTIR spectrum of the active extract was detected in the range of 400 cm −1 to 4000 cm −1 using a Nicolet 6700 FTIR spectrophotometer.

Isolation of Actinomycetes and primary screening
In this work, bioactive Actinomycetes were assessed from collected sediment samples of the Kali river ecosystem. A total of 23 Actinomycetes strains were selectively isolated from 12 sediment samples. All the isolates showed morphologically distinct colonies on starch casein agar supplemented with Amphotericin B and Tetracyclin. However, out of 23 Actinomycetes, 20 isolates were found to be gram-positive. The Streptomyces sp. KAS-1 showed total inhibition during primary screening against two pathogenic organisms, such as Aspergillus fumigatus (MTCC8877) and Staphylococcus epidermidis, as shown in Figure 1. Rest all organisms showed moderate or no inhibition against the pathogenic microorganisms, reported in Table 1. A similar result was observed by (Subhan et al., 2015), where 33 Actinomycetes were isolated from soil samples, but only 17 isolates showed antimicrobial activity against pathogenic organisms. The Actinomycetes can produce numerous enzymes, which are essential for their living and resistance against other pathogens. It is assumed that there may be an interrelationship between the production of enzymes and habitat, where the organism is living. These enzymes have various industrial applications. Here, the 20 strains of gram positive Actinomycetes were screened for enzymatic activity against amylase, esterase, pectinase and DNase, which was shown in Figure 2, Table 2. The Streptomyces sp. KAS-1 was found to produce all the four enzymes. Hence, the Streptomyces sp. KAS-1 was selected for further analysis based on its ability to produce enzymes and antimicrobial activities. Among the 20 isolates, 14 isolates produced amylase enzyme whereas, the esterase enzyme was produced by 12 isolates. However, the enzyme pectinase was produced by only 9 isolates While, DNase was produced by 9 isolates. Microorganisms utilize starch as a source of carbon, but since the starch molecules are too large and cannot be absorbed through the cell membrane, the organisms produce the extracellular enzyme amylase. The enzyme amylase reacts with starch molecules and breaks down into smaller units, which can be absorbed easily by the cell membrane of microorganisms. When organisms are grown on starch agar medium, they hydrolyze starch with the help of enzyme amylase; thereby produce a transpar-ent halo surrounding the colony. The production of enzymes is dependent on methods of cultivation, the composition of medium, temperature, pH, incubation time, cell growth, nutrient requirements, pH, temperature, etc. Among these parameters, the production of the enzyme is mostly dependent on the pH and temperature of the medium (Ka ilzadeh and Dehdari, 2015). Microorganisms produce extracellular esterase enzymes, which hydrolyze and splits esters in alcohols and acid. Generally, the esterase producing Actinomycetes release fatty acids due to the hydrolysis of tween-80. The fatty acids molecules released from Tween-80 bind to calcium ions present in the medium and produce a halo zone around the colonies (Kumar et al., 2012). Here, 9 isolates out of 20 were able to hydrolyze pectin and used that as a source of carbon. A similar result was published by (Mohandas et al., 2018), where 50 microorganisms isolated from spoiled fruits and vegetables indicated the presence of pectinase enzyme. The enzyme DNase was produced only by 9 Actinomycetes. DNA or Deoxyribonucleic acid is made of four nucleotides and it is a large polymer of nucleotides. Actinomycetes can utilize DNA as a source of nitrogen, carbon, and phosphate. The exo-enzyme DNase secreted by Actinomycetes hydrolyzes and cleaves DNA molecules into free nucleotides and a phosphate group. These nucleotides than enter the cell membrane and can be used as a source of nutrition. The negatively charged DNA in the form of a polymer bind to positively charged methyl green indicator in the medium. After breaking down into free nucleotides, it is no longer bound to methyl green and forms a clear zone around the DNase producing organism.

Morphological, physiological and biochemical characterization
The Streptomyces sp. KAS-1 isolated from sediment samples was aerobic and gram-positive. The colony of the Streptomyces sp. KAS-1 was irregularly shaped with a rough surface, whereas the elevation of the colony was slightly raised. The branched vegetative mycelium was light brown, while the sporebearing mycelium was shiny greyish. The strain also produced a faint brown pigment after incubation in the SCA medium for 8 to 9 days as shown in Figure 3. The strain was found positive for gram stain and acid-fast staining. This is because all the Streptomyces strains showed a positive result for acidfast staining and gram staining. Our observations were supported with results by (Islam et al., 2014). The strain was found to grow optimally in the range of temperature from 35 • C to 45 • C, but a moderate to no growth was found in the temperature below and above that. This proved that the Streptomyces sp. KAS-1 was a moderately thermophilic organism. These thermophilic Actinomycetes play a signi icant role in the mineralization of nutrients and degradation of organic matter. The pH of the surrounding environment has a great in luence on the growth of Actinomycetes.
The pH, which favored the optimum growth of the Streptomyces sp. KAS-1 was found at pH 7.5, in presence of up to 7% NaCl tolerance with an optimum at 3 to 4%. But moderate growth was observed at pH 7.0 and 8.0; however, there was found no growth at pH below 7.0 and above 8.0. Therefore, Streptomyces sp. KAS-1 can be identi ied as a neutrophile, which is capable of growing in such an environment, where H + ion concentration is almost at equilibrium. Most of the Actinomycetes, especially Streptomyces grow better in pH ranging from 6.0 to 8.0 (Kim et al., 1992). The organism showed optimum growth at 3 to 4% NaCl concentration. Therefore, it is obvious that the Streptomyces sp. KAS-1 was, not a halo-tolerant sp. because it can grow best at low salt concentration. It is noted that a smaller amount of metallic ion or NaCl concentrations in medium maintain an osmotic condition, which is similar to the cytoplasm of the microorganism. However, a high salt concentration leads to osmosis to the microorganisms and tends to denature proteins which, lead to the death of the cell.
The Streptomyces sp. KAS-1 showed a positive result for starch, casein, gelatin, esculin and arbutin. Streptomyces sp. KAS-1 was able to hydrolyze starch, because of the production of enzyme α-amylase, which cleaves starch into simpler molecules and absorb through the cell membrane as a source of carbon. The organism showed a positive result for gelatin liquefaction. It implies that the organism can produce gelatinase, which is a protease enzyme. The organism extracellularly secretes the gelatinase enzyme and use gelatin as a source of protein followed by hydrolysis. Casein is made up of amino acid subunits and the major milk protein. Microorganisms cannot absorb casein through their cell membrane because of its large size. Therefore, the organisms secrete protease enzyme which, degrade casein into free amino acid molecules and make it easily absorbable through cell membranes.
The Streptomyces sp. KAS-1 was negative for the urea hydrolysis test, but it showed a positive result for esculin degradation. The organisms degrade esculin to esculetin, which shows a positive result. The Streptomyces sp. KAS-1 even showed a positive result for arbutin degradation. In addition to this, the organism was positive for hydrogen sul ide production and nitrate reduction tests. Hydrogen sul ide is produced by some microbes while degradation of amino acids containing a sulfur derivative, such as methionine, where as the positive result for nitrate reduction implies the ability to produce nitrate reductase enzyme. The microorganisms require nitrate as an electron acceptor during anaerobic respiration. The carbon sources utilized by the strain Streptomyces sp. KAS-1 were Dgalactose, D-mannitol, trehalose, and D-lactose but no growth observed in sucrose, raf inose, galactose, and xylose. The morphological, physiological and biochemical characterizations of Streptomyces sp. KAS-1 was reported in Table 3. The results obtained from morphological, physiological and biochemical characterizations were compared with Streptomyces species reported in Bergey's manual of systematic bacteriology (Locci, 1989).

Molecular characterization
The genomic DNA of the Streptomyces sp. KAS-1 was extracted and analyzed by 16S rRNA gene sequencing. For the identi ication of microorganisms up to species level, the 16S rRNA gene sequencing is widely being used in the construction of phylogenies because of the slow rate of evolution at that region of gene (Woese and Fox, 1977). The length of the complete gene sequence obtained was 1358 base pair and was submitted to NCBI gene bank with the Accession number MK788355. This sequence was later subjected in search of close relatives through nucleotide blast (BLASTN) in NCBI. The analysis of the 16S rRNA gene sequence of the Streptomyces sp. KAS-1 revealed 98.97% sequence similarity with Streptomyces thermocarboxydus. Further, the reconstruction of a phylogenetic tree was done through the neighbor-joining method with obtained gene sequences. The analysis of the phylogenetic tree revealed the strain Streptomyces sp. KAS-1 fell within the cluster of the genus Streptomyces and formed a branch with Streptomyces thermocarboxydus strain-SDT64 (KJ018992) as represented in Figure 4.

UV absorption analysis of Streptomyces sp. KAS-1 methanol extract
The qualitative UV-visible analysis of methanol extract exhibited maximum UV absorption at 261 nm as shown in Figure 5. The UV absorption at 261 nm reported the tentative identi ication of the compound muconomycin B (Jacqueline and Vittimberga, 1965). Muconomycin B is an aromatic sesquiterpene and a powerful antibiotic compound naturally present in microorganisms. There was a similar result obtained by (Maleki and Omid, 2011), where the ethyl acetate extract of bioactive compounds exhibited maximum UV absorption at the range 217-221nm and showed the presence of broad-spectrum antimicrobial compounds.

FTIR analysis of Streptomyces sp. KAS-1methanol extract
The biological functional groups present in methanol extract of Streptomyces sp.KAS-1 was determined by FTIR analysis, based on the peak values of the infrared spectrum Figure 6, Table 4 . It revealed that the presence of different functional groups based on the absorption peaks at 618 cm −1 and 868 cm −1 due to C-Br stretching alkyl groups and C-Cl stretch alkyl halides respectively, 1044 cm −1 C-N stretch aliphatic amines, 1119 cm −1 C-N stretch aliphatic amines, 1274 cm −1 C-H wag alkyl halides, 1315 cm −1 symmetric stretching aromatics, 1384 cm −1 CH 2 wag modes / C-H rock alkanes, 1415 cm −1 ring-stretching of phenazine constitutional unit , 1471 cm −1 CH 2 bending polyethylene, 1630 cm −1 N-H bend 1 0 amines, 2853 cm −1 C-H stretch alkanes, 2924 cm −1 H-C=O: C-H stretch aldehydes, 2960 C-H stretch alkanes and 3441 O-H stretch alcohols. This result was compared with the similar results of (Fatima et al., 2017).

CONCLUSIONS
The current study shows that the isolated strain extract of Streptomyces sp. KAS-1 from sediment soil samples of Kali river of the Uttar Kannada region has the potential to act against pathogenic microorganisms. Here, we found that sediment soil samples of the Kali River Estuary, Karwar is a good region of biodiversity and has been adequately acceptable due to its vast lora and microbial diversity. Results showed that the strain Streptomyces sp. KAS-1 is dominant against two active test pathogens and showed the production of hydrolytic enzymes for all four enzyme activity tested. Thus, the study on this isolate can further be explored for the development of new antibiotic drugs to treat infectious diseases caused by pathogenic and drug-resistant strains as well as for the production of hydrolytic enzymes in industrial and pharmaceutical purposes.

ACKNOWLEDGEMENT
The inancial support received from the UGC-SAP-DSA-1, Department of Botany, Karnatak University, Dharwad, Karnataka, India, was gratefully acknowledged. The authors also thank USIC, Karnatak University, Dharwad for the necessary instrumentation facility.