Indian Journal of Experimental Biology Vol. 43, April 2005, pp. 342-345

Production of heptaene antifungal antibiotic by Streptomyces purpeojuscus eM 1261

Praveen Kumar Jain & PC Jain*

Department of Applied Microbiology and Biotechnology, Dr H S Gour Vishwavidyalaya, Sagar 470 003 India

Received 2 November 2004; revised 30 December 2004

A strain of Streptomyces purpeojuscus CM 1261 isolated from a sample of compost collected locally was found to possess strong antagonistic activity against 4 human pathogenic fungi i.e., Candida albicans, Aspergillus niger, Microsporum gypseum and Trichophyton sp. The active antifungal compound produced by it was found to be a heptaene group of polyene antifungal antibiotic.

Keywords: Antibiotic, Antifungal activity, Candida albicans, Polyene, Streptomyces purpeojuscus

IPC Code: Int CI7 A61K; C12N

Microorganisms from different ecological niches are now gaining considerable interest because of their vast potential to produce a variety of bioactive molecules l

-3

. Production of antibiotics by actinomycetes is one of the fields of medical, veterinary and public health importance and thus, is a rapidly expanding branch of industrial microbiologl-8

. Rapidly increasing fungal infections in human society particularly in immunodeficient patients are now demanding more number of antifungals in the market. The search of novel antifungal antibiotic producing strains thus, becomes a major thrust area in modern medicine9

• Present work was undertaken to study the cultural, biochemical and physiological properties of a strain of Streptomyces isolated from local compost sample and to understand the nature of bioactive compound produced by it.

Materials and Methods Microorganism - A strain of Streptomyces was

isolated from a compost sample collected from Sagar, India (23° 50° N latitude and 78° 40° E longitude). The compost sample was dried at 45 0 C for 24 hr. The sample (1g) was vortexed in 9.0 ml sterile distilled water and the suspension was further diluted to obtain 10-3 dilution of the test sample. A loop full of this dilution was aseptically streaked on petri plate having

*Correspondent author-Tel: 91(7582) 265517; Fax: 91(7582) 264236 E-mail: micjain@hotmail.com

soil extract agar medium consisting of glucose, Ig; K2HP04, 0.50 g; agar-agar, ISg; soil extract, 100 ml and tap water, 900 ml; (pH-6.8-7.0). The plate was incubated at 28° ±1°C for 14 days. A colony appeared with characteristics of actinomycete morphology was isolated and purified using glucose yeast extract agar medium 10. After ensuring the purity, it was preserved in glycerol (10%) under deep freeze condition and maintained at 4°C as stock cultures on yeast, malt extract, agar mediumll

. The isolated strain CM 1261 was identified as CM 1261 of Streptomyces purpeojuscus by IMTECH, Chandigarh and deposited at Microbial Type Culture Collection and Gene Bank (MTCC 6230), IMTECH, Chandigarh.

Antifungal activity - Antifungal activity of the strain, S. purpeojuscus CM 1261, was tested against Candida albicans, Aspergillus niger, Microsporum gypseum and Trichophyton sp. using 'Cross streak method' as described by Waksman and Lechevalierl2

.

The results have been expressed in terms of growth inhibition and retardation in the growth of test fungi.

Detection of nature of antifungal compound in the culture broth of strain eM 1261

The detection of nature of antifungal compound produced by strain CM 1261 was done using following methods:-

Ergosterol agar plate method - The method is based on the principle of strong affinity between ergosterol and polyene compounds. Presence of ergosterol in antibiotic assay medium resists the

JAIN & JAIN: PRODUCTION OF HEPTAENE ANTIFUNGAL ANTIBIOTIC 343

diffusion of polyene antibiotics, which results in the reduction of zone of inhibition as compared to the medium having no ergosterol in it.

The detection of polyene antifungal compounds in the samples obtained from culture broth of test strain was done using 'Ergosterol agar plate assay' following the method as described in the Laboratory Manual of MTCCI3. For this, the test actinomycete was grown in Erlenmeyer flasks having 25 ml of beef extract broth medium for 108 hr at 120 rpm on rotary shaker. The extraction of antifungal antibiotic from the cultures was carried out by shaking the content of culture flask with equal volume of methanol (95%) for 1 hr at 120 rpm at room temperature. After extraction the content of the flasks was centrifuged at 7000 rpm for 10 min to obtain cell free extract which was used as test sample for detection of polyene in them. For this Candida albicans seeded Sabouraud's dextrose agar (SDA) plates with and without 0.5% ergosterol were prepared and a well of 6 mm diam was made in the center of the plates with the help of sterile cork borer. Fifty microlitre of the test sample was transferred into the wells and the plates were incubated at 30°C for 48 hr. The plates were then observed for zone of inhibition developed on both the assay plates. Presence of polyene type antibiotics was interpreted by reduction in zone size in presence of ergosterol.

UV visual spectrophotometric method - For spectrophotometric analysis of the antifungal compound produced by strain CM 1261, it was grown on medium containing beef extract, 4g; peptone, 4g; yeast extract, Ig; NaCI, 2.5g; glucose, 109; distilled water, 1000 ml; pH, 7.0. Erlenmeyer flasks (150 ml) containing above medium (25 rnI) were autoclaved and inoculated with 1 ml spore suspension of the test organism. The flasks were incubated on rotary shaker (120rpm) at 28° ± 1°C for 108 hr. After incubation the content of the flask was centrifuged at 7000 rpm for 10 min and the mycelial pellet was obtained and used for extraction of polyenes. For this, the mycelial pellet was extracted with 25 ml of methanol (95%) by shaking at 120 rpm for 1 hr at28° ± 1°C. The content of extraction vessel was centrifuged at 7000 rpm and the presence of polyenes was determined in the supernatant by spectrophotometry. The absorption spectrum of supernatant was recorded at 200-500 nm using UV -visual double beam PC based Spectrophotometer (ECIL model-UV 5704 SS). The presence of polyene and its type was determined on

the basis of characteristic three peak spectra using standards as reported by Riviere et al. 14.

Cultural, biochemical and physiological characterization of S. purpeofuscus CM 1261

To establish cultural identity the test strain was grown on yeast, malt extract, agar medium and characteristics were noted following Shirling and Gottleibll and Raynerl5. For physiological and biochemical characterization its Gram's staining properties, production of pigments, growth on media having 5 different concentrations of NaCI (i.e., 2, 5, 7, 9 and 12%), seven different pH from 4.0 to 10.0 with an interval of 1 pH, effect of different temperatures (i.e., 4°, 15°, 25°, 37°, 42° and 55°C) on growth, utilization of 12 different sugars (i.e., glucose, arabinose, mannitol, xylose, meso-inositol, raffinose, rhamnose, salicin, sucrose, galactose, lactose and maltose) and nine biochemical properties including production of acid and acetoin (MR, VP), indole and H2S production, citrate utilization; starch, casein and gelatin hydrolysis and nitrate reduction were studied following standard methods ll

•16

•17

•

Results and Discussion S. purpeofuscus CM 1261 was found highly

antagonist for all the four test fungi (Table 1). It was found to cause total inhibition in growth of Candida albicans, while it caused growth inhibition and retardation in the growth of Aspergillus niger, Microsporum gypseum and Trichophyton sp. causing 32, 11 and 9 mm zone of inhibition, respectively, when tested by using Cross streak methodl2. The results of ergosterol agar plate assay showed the production of polyene antifungal antibiotic by the present test strain. The test sample obtained from its cultures caused a zone of inhibition of 21 mm against Candida albicans grown on SDA medium without ergosterol, whereas the same amount of sample when tested using SDA medium . containing 0.5% of ergosterol, only 13 mm zone of inhibition was recorded against Candida albicans. This reduction in

Table I-Antagonistic properties of Streptomyces purpeoJuscus CM 1261

Test organism Activity* Candida albicans TIG Aspergillus niger GIR (32) Microsporum gypseum GIR (11) Trichophyton sp. GIR (9) Values given in parentheses. indicate zone of inhibition in rnm. *TIG - Total inhibition in growth, GIR- Growth inhibition and retardation, GR- Growth retardation.

344 INDIAN J EXP BIOL, APRIL 2005

zone of inhibition in the presence of ergosterol indicated the production of polyene antifungal compound by the test strain.

Identification of the group of polyene antifungal compound produced by test strain was done by recording UV -visual absorption spectra of the methanolic extracts of its mycelia. The presence of characteristic three peak spectra confirmed the production of polyene antifungal compound by the test strain, whereas the presence of three distinct peaks at 360.0, 379.5 and 402.5 nm indicated that antifungal compound belonged to heptaene group of polyene antibiotics (Fig. 1).

The strain was found to grow rapidly on yeast, malt extract, agar medium. The culture showed the presence of opaque, convex mycelial growth with irregular margins. Colour of the aerial mycelium was dull white to olivaceous buff and the substrate mycelium was white to cream colour. Diffusible pigments were not formed. The results of biochemical and physiological characterization of strain CM 1261 are presented in Tables 2 and 3. The organism grew well at pH 6.0 to 10.0, but at pH 5.0, growth was weak positive. The test strain showed its growth at 15° to 42° C. The strain was also found to tolerate 2 to 9% concentrations of NaCI in the growth medium, but grew poorly on medium having 12% NaCI concentration. It grew well in presence of potassium tellurite (0.001 %), but the growth was observed poor in the presence of 0.1 % of phenol. No growth was recorded in presence of sodium azide (0.01 %).

The strain was found to utilize glucose, arabinose, maltose, xylose, salicin, galactose, lactose and mannose as carbon sources. The utilization of

3.00

2.25

fl 1.50

c: III .c 0 0.75 II) .c «

0.00

0.75 200.0 260.0 320.0 380.0 «0.0 500.0

Wavelength nm

Fig. I - Ultraviolet absorption spectrum of antifungal compound produced by Streptomyces purpeojuscus CM 1261 in methanol (95%).

Table 2--Biochemical and physiological characters of S. purpeofuscus CM 1261

Gram staining + ve

H2S production - ve

MR - ve

VP - ve

Indole - ve

Citrate utilization - ve

Starch + ve

Caseine + ve

Gelatine + ve

Nitrate reduction - ve

Growth in the presence of NaCI (%)

2 + ve

5 + ve

7 + ve

9 + ve

12 W+ve

Growth atpH

4 - ve

5 W+ve

6 + ve

7 + ve

8 + ve

9 + ve

10 + ve

Growth at temperature (0C)

4 - ve

15 + ve

25 + ve

37 + ve

42 + ve

55 - ve

Growth in presence of

Phenol (0.1 %) W+ve

Sodium azide (0.01 %) - ve

Potassium tellurite (0.001 %) + ve

+ ve- positive; W + ve- weak positive; - ve- negative

raffinose, rhamnose and sucrose as carbon source by this strain was found doubtful, whereas meso-inositol was not utilized. The strain S. purpeofuscus CM 1261 differs from the type strain of S. purpeofuscus (ATCC 23952) in its some of the cultural characteristics and carbon utilization behaviour17 and hence described in the present study. The living culture of Streptomyces

JAIN & JAIN: PRODUCTION OF HEPTAENE ANTIFUNGAL ANTIBIOTIC 345

Table ~tilization of different carbon sources by S. purpeofuscus CM 1261

Carbon source Utilization*

Glucose ++

Arabinose +

Mannitol +

Xylose +

Meso-inositol

Raffinose ±

Rhamnose ±

Salicin +

Sucrose ±

Galactose ++

Lactose +

Maltose ++

++, strongly positive; +, positive; ±, doubtful ; -, negative. * After 14 days incubation at 28° ± 1°C temperature.

purpeoJuscus CM 1261 has been deposited at Microbial Type Culture Collection and Gene Bank, IMTECH, Chandigarh, India with an accession number MTCC 6230 and in the research laboratory of Department of Applied Microbiology and Biotechnolgy, Dr H S Gour Vishwavidyalaya, Sagar, India.

Acknowledgement . The author (PCl) is thankful to University Grants

Commission, New Delhi, for funding the project.

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