A Morphological, Biochemical and Biological Studies of Halophilic Streptomyces sp. Isolated from Saltpan Environment

Problem statement: Dermatophytes have developed resistance to the exi sting antifungal antibiotics. As a part of our continuous search we had isolated, identified and characterized actinomycetes from the halophilic environment havin g a tagonistic activity against the dermatophytes namely Trichophyton, Microsporum and Epidermophyton. Approach: Actinomycetes were isolated from the soil sample collected from the Ennore salt pan region, Chennai, India and screened for antidermatophytic secondary metabolite production b y well diffusion method. Four dermatophytes Trichophyton rubrum [MTCC 3272], Trichophyton mentagrophytes [MTCC 7687], Microsporum gypseum [MTCC 2819] and Epidermophyton floccosum [MTCC 7880] were used to study its susceptibility to the isolated actinomyce tes. Actinomycetes which showed antidermatophytic activity were subjected to cultural characterizatio n with respect to aerial and substrate mycelia colo r, diffusible and melanin pigment production and the g rowth of the organisms on different media. Further the micro morphological characteristics such as spo re surface ornamentation and spore chain morphology determined by Scanning Electron Microscopic (SEM) a nalysis also suggested that the isolates belonged to the genus Streptomyces. The isolates were also tested for utilization of various carbon and nitrogen sources, degradation of complex compounds, sensitiv ity to antibiotics and inhibitory compounds. Results: All the 3 isolates exhibited different cultural an d morphological characteristics. Based on the cultural characters and morphology they were assign ed to the genus Streptomyces. The three isolates produced an inhibition zone of 30-31 mm on an avera ge, utilized a wide range of carbon and nitrogen sources, degraded almost all the complex compounds a exhibited a broad spectrum of antibiotic resistance. They were designated as Streptomyces sp. DKDVIT1, Streptomyces sp. DKDVIT2 and Streptomyces sp., DKDVIT3. Conclusion: The Streptomyces sp. isolated from the Ennore saltpan of Bay of Bengal exhibited potential antidermatophytic act ivity. The extraction and characterization of secon dary metabolites from these Streptomyces may be used as a lead compound/therapeutic agent f or dermatophytosis.


INTRODUCTION
Actinomycetes are gram-positive bacteria, free living, saprophytic bacteria widely distributed in soil, water and colonizing plants showing marked chemical and morphological diversity but form a distinct evolutionary line of organisms [1] . Actinomycetes are potential source of many bioactive compounds [2][3][4][5] which have diverse clinical effects and important applications in human medicine [6] .
Members of the actinomycetes genus especially Streptomyces sp. have long been recognized as prolific producers of useful bioactive compounds, providing more than half of the naturally occurring antibiotics discovered to date and continuing to be a major source of many types of antibiotics and other class of biologically active secondary metabolites that may ultimately find application as anti-infectives, anticancer agents or other pharmaceutically useful compounds [7][8][9] . Some Streptomyces sp. produces antibiotics during sporulation [10] .
Most actinomycetes were believed to be terrestrial; however, some strains have also been found to occur in marine environments. Some actinomycetes were isolated from a marine environment which required seawater for growth and these strains were designated marine actinomycetes [11] . Oceans account for more than 70% of the earth's surface and the microorganisms growing in marine environments are metabolically and physiologically diverse from terrestrial organisms [12] .
Dermatophytes are a group of closely related fungi that have the ability to invade keratinized tissue such as skin, hair and nail of humans and animals to produce an infection [13] . The relation to a dermatophyte infection may range from mild to severe as a consequence of the hosts' reactions to the metabolic products of the fungus. These organisms are classified into genera of Trichophyton, Microsporum and Epidermophyton [14] . Dermatophytes, like most filamentous fungi of ascomycetous affinity have a secondary metabolism characterized by the production of substantial quantities of distinctive metabolites [15] . India is a large subcontinent with remarkably varied topography, located within the tropical and subtropical belts of the world. Its ambiance is conducive to the acquisition and maintenance of mycotic infections [16] .
The prevalence of antimicrobial resistance among pathogens is increasing at an alarming rate worldwide [17] . Antibiotics such as imidazole compounds that are currently employed to treat dermatophytic infections possess a series of limitations such as undesirable side effects or rapid development of resistance [18] . As a consequence, new antifungal agents preferably naturally occurring with novel mechanisms of action and free from any side effects are needed to improve the treatment of superficial fungal infections [19] .
The present study evaluates micro and macro morphological, cultural, biochemical and biological characteristics of Streptomyces sp. recovered from the soil sample collected at the Ennore saltpan of the Bay of Bengal, Chennai.

MATERIALS AND METHODS
Sample collection: Marine soil samples were collected at the Ennore saltpan region of the Bay of Bengal at a depth of 5-15 cm into sterile polyethylene bags, transported to the laboratory and stored in the refrigerator at 4°C until use.

Isolation of actinomycetes:
Starch caesin agar supplemented with seawater, was used for the isolation of actinomycetes in situ. The samples were appropriately diluted with sterilized seawater and 0.1 mL was spread on the media. After incubation for 1 week at 30°C, the isolated actinomycetes colonies were selected and purified [20] .

Biological characterization of actinomycetes:
The purified actinomycetes colonies were screened for antidermatophytic activity against Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum gypseum and Epidermophyton floccosum by well diffusion method [21] . The isolates were grown in ISP1 broth until adequate turbidity was achieved. 100 µL of the actinomycetes broth culture was placed in the wells made on Sabouraud dextrose agar plates (pH 5.6) seeded with the test fungal cultures. The plates were incubated at 28°C and observed for inhibition zone after 3-4 days.

Macro and micro morphological characterization:
The potential isolates were inoculated on Starch caesin agar, incubated at 30°C for 7 days and were examined for cultural characteristics such as colony size, shape and margin; aerial and substrate mycelium color and diffusible pigment production. The optimum media for the growth of the isolates was determined by inoculating the cultures on solid media such as Starch caesin agar, ISP1 agar, ISP7 agar, Marine Zobell agar, Actinomycetes isolation agar and Seawater agar [22][23] .
The spore chain morphology and spore surface ornamentation of the isolates were evaluated by SEM analysis (S3400, 20 KV, 5.00 µm). The cultures were grown on Starch caesin agar for 7 days at 30°C.
Biochemical characterization: Utilization of various substrates as carbon and nitrogen source by inoculating the isolates in ISP1 broth supplemented with the respective substrates for carbon and nitrogen and incubating for 7 days at 30°C. The production of melanoid pigment was determined for all the 3 isolates by inoculating on tryptone yeast extract iron agar (ISP-6) and tyrosine agar (ISP-7) and incubating at 30°C for 7 days [23] .
The pectinolytic and chitinolytic activity of the isolates was determined by inoculating the isolates on Starch caesin agar supplemented with pectin and chitin respectively, incubating at 30°C and observing the plates for zone of clearance after 7 days [23] .
Degradation of starch, caesin, gelatin, esculin, tyrosine, adenine and guanine was evaluated using Bennett's agar supplemented with the respective compounds. The degradation of starch and gelatin was determined by the presence of zone of clearance around the colonies after flooding the plates with mercuric chloride and iodine respectively. Similarly the degradation of caesin, tyrosine, adenine and guanine was determined by observing the plates for clear zones around the colonies whereas the degradation of Esculin was determined by observing the plates for blackening around the colonies [23] .
The degradation of Tween 80 was determined using Sierra medium [24] . The presence of white precipitate around the colonies after 7 days indicated positive results. Keratinolytic activity of the three isolates was determined by inoculating the cultures in Starch caesin agar containing the chicken feather and horse hair respectively and incubating at 30°C for 7 days [25] . Cellulase activity was determined using Starch caesin agar supplemented with Carboxy Methyl Cellulose (CMC) and incubating for 7 days at 30°C [26] . Lecithinase activity was tested using Egg yolk agar [27] .
The susceptibility of the isolates to various antibiotics was studied on Starch caesin agar plates by placing the various antibiotic discs on the agar surface and incubating at 30°C for 7 days whereas the susceptibility of the isolates to inhibitory compounds was evaluated using Starch caesin agar supplemented with the respective inhibitory compounds [23] .

RESULTS
On screening the 100 isolates of actinomycetes for antidermatophytic activity our isolates namely DKDVIT1, DKDVIT2 and DKDVIT3 produced a clear zone of 30-31 mm for T. rubrum and 18-35 mm for M. gypseum (100 µL per well). The inhibition zone produced was compared with that of the standard antifungal agent Fluconazole (10 mg/100 µL per well) ( Table 1).
Cultural properties of the isolates are presented in the ( Table 2). Actinomycetes isolates grew well at 28°C on Starch Caesin agar (optimal medium) producing pinpoint to medium sized, slow growing, powdery, irregular to regular, flat to raised colonies possessing an earthy odour characteristic of actinomycetes. On Starch caesin agar our isolates produced white coloured aerial spore mass and yellow coloured substrate mycelium with no distinct diffusible pigment production. Based on the cultural characters and morphology they were assigned to the genus Streptomyces. The isolates are designated as Streptomyces sp. DKDVIT1, Streptomyces sp. DKDVIT2 and Streptomyces sp. DKDVIT3. The morphology of the aerial hyphae was determined by SEM analysis of the culture surface. The aerial mycelium of the isolates DKDVIT1 and DKDVIT2 formed unfragmented, branched, straight hyphae bearing non-motile spores with smooth surface (Fig. 1A and B) whereas the isolate DKDVIT3 formed unfragmented, branched, looped hyphae showing 2 curves and bearing non-motile spores with smooth surface (Fig. 1C).   The physiological characteristics of the isolates are summarized in the ( Table 3). The melanin pigment producing ability was checked on tryptone yeast extract agar [31] and none of the isolates produced melanin. The isolates DKDVIT1 and DKDVIT3 utilized a wide range of carbon source but did not utilize glycerol whereas isolate DKDVIT2 utilized all the carbon sources. The isolate DKDVIT1 grew utilizing a variety of organic and inorganic nitrogen source except ammonium sulphate, ammonium nitrate and potassium nitrate. Similarly isolate DKDVIT2 and DKDVIT3 utilized a range of nitrogen source except valine and ammonium nitrate. In addition, DKDVIT2 also failed to use ammonium sulphate. All the 3 isolates degraded gelatin, starch, caesin, tyrosine, Esculin, adenine, guanine and Tween 80 but failed to degrade keratin, lecithin, pectin and cellulose.

DISCUSSION
In our screening for novel actinomycetes exhibiting potential antidermatophytic activities, soil samples were collected from the Ennore saltpan region of Bay of Bengal, Chennai. In the course of our continuous search a total of 100 isolates were recovered from the collected soil samples.
Screening for the antidermatophytic activity by well diffusion method of the culture supernatant was carried out for all the 100 pure isolates against the dermatophytes, T. rubrum, T. mentagrophytes, M. gypseum and E. floccosum. Among them the 3 isolates DKDVIT1, DKDVIT2 and DKDVIT3 exhibited potential inhibitory activity against T. rubrum and M. gypseum whereas no activity was detected against T. mentagrophytes and E. floccosum. The antibacterial and enzymatic activities of Streptomyces sp. are well known, however its antifungal property has been reported only against common fungal pathogens. Similar antidermatophytic studies were carried out by Augustine et al. [21] . Their studies confirmed antidermatophytic activity of Streptomyces rochei AK 39 isolated from soil and water samples collected at various locations of Pune, Maharashtra, India.
The colony morphology of our isolates were similar to that described by Locci in his earlier studies [22] which clearly indicates that the isolates under investigation belonged to the genus Streptomyces. All the 3 potential isolates grew well on a range of agar media showing characteristics typical of Streptomyces [28] . Streptomyces have been reported to grow well on Starch caesin agar by earlier workers in this field [29] . Our studies on the micro morphology of the three isolates showed revealed two spore morphology types namely Rectiflexibles and Retinaculiaperti. Stefka et al. [30] has done a similar study but reported a different type of spore morphology where the aerial mycelium formed monopodially branched spore-bearing hyphae with the shape of hooks, loops, open or compact spirals with 3-6 curves with smooth surface.
In our studies we observed that all the three isolates could degrade almost all available complex substances. Reports on the degradation and enzymatic activity of Streptomyces by Rabah et.al also revealed a much similar result [29,32] .
Studies on resistance to antibiotics by Ibrhaim [32] indicated that Streptomyces isolates used in their studies were sensitive to gentamycin, tobramycin, rifamipcin and penicillin G, tetracycline but resistant against oleandomycin, lincomycine, vancomycin, streptomycin and neomycin. Our investigation also resulted in a similar type of finding which is comparable to that of Ibrhaim [32] . Also the studies on the resistance to inhibitory substances can be compared to the results reported by Ibrhaim [32] .

CONCLUSION
From the above mentioned results, it may be concluded that all the 3 isolates Streptomyces sp. DKDVIT1, Streptomyces sp. DKDVIT2 and Streptomyces sp. DKDVIT3 belongs to the genus Streptomyces and showed strong antidermatophytic activity against T. rubrum and M. gypseum but no activity against T. mentagrophytes and E. floccosum. Further studies on the purification and characterization of the antidermatophytic secondary metabolites are currently in progress.