Antimicrobial Profile of Streptomyces Viridis MSL Isolated from Laterite Soils *M

M. Vijayalakshmi et al. / Journal of Pharmacy Research 2011,4(8),2615-2618 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Antimicrobial profile of Streptomyces viridis MSL isolated from laterite soils *M. Vijayalakshmi and M. Raja Hima Bindhu Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur-522510, Andhra Pradesh, India Received on: 17-05-2011; Revised on: 12-06-2011; Accepted on:16-07-2011

ABSTRACT Streptomyces viridis MSL isolated from the laterite soils of Acharya Nagarjuna University campus was tested for its antimicrobial activity. Growth pattern, cultural and biochemical characteristics of the strain were also studied. Bacillus cereus, B. megaterium, B. subtilis, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, P. solanacearum, Salmonella typhi, Shigella flexneri, Staphylococcus aureus, Vibrio cholera and Xanthomonas campestris were used as test bacteria and fungal pathogens employed for testing include Alternaria sp., Asperigillus niger, Botrytis cinerea, Candida albicans, Fusarium solani, F. oxysporum and Verticillium alboatrum. A very good antimicrobial activity was observed and hence this isolate was considered for the discovery of bioactive compounds.

Key words: Streptomyces viridis, antimicrobial profile, bioactive compounds.

INTRODUCTION Actinomycetes are Gram positive bacteria frequently filamentous and sporu- supplemented with 100 µg/mL nystatin, 50 µg/mL streptomycin and incu- lating with DNA rich in G+C (55-75%).[1] They are the most fruitful source for bated at 28-30°C for 7 days. The predominant strain was subcultured and the production of bioactive secondary metabolites. More than 9000 biological preserved on YMD agar slants at 4°C. [3] active molecules have been isolated from actinomycetes yielding more than 60 pharmaceutical agents using in medical or agricultural fields [2] while terres- Cultural, Physiological and Biochemical Characteristics: trial actinomycetes have been the main stay for antibiotic discovery efforts The strain was grown on seven International Streptomyces Project media and during the past five decades. five non-ISP media to determine the cultural characteristics such as color of aerial mycelium and substrate mycelium, pigment production and spore for- Fungal plant diseases are often controlled by fungicides, however, extended mation.[4] The strain was examined for morphological, biochemical and physi- use of fungicides has not only created problem of fungicidal resistance and ological and cultural caharacteristics.[5, 6, 7, 8, 9] The morphological characteris- increased contamination of the environment, but also have adverse mamma- tics were assessed using scanning electron microscopy (SEM: Model- JOEL- lian health disorders. In response to environmental and health concerns there JSM 5600, Japan) of 4-day cultures grown on ISP2 medium (YMD) at various has been increasing interest in using eco-friendly methods and some natural magnifications. products like ‘Rhizovit’ from Streptomyces rimosus and ‘Mycostop’ from Streptomyces griseoviridis are used to control a wide range of fungi like Pythium Identification of the culture based on 16srDNA sequence: spp., Rhizoctonia solani, Fusarium spp., Phytophthora spp. and Alterna- The strain was grown in YMD broth for 3 days and was centrifuged at 10,000rpm ria spp. The emergence of drug resistant pathogens and the increase in for 20min and the pellet was used for the extraction of DNA.[10] PCR mixture diseases affecting the immune system have greatly intensified the need to consisted of 2.5 µl of 10× buffer, 3.5 µl of MgCl2 (25 mM), 2 µl of dNTP (0.4 investigate new bioactive metabolites for potential pharmaceutical and indus- mM), 1 µl of 16S rDNA actino specific Primer - forward (10 pmol/µl), 1 µl of trial applications. 16S rDNA actino specific Primer- reverse (10 pmol/µl), Taq polymerase (2 U/ µl) and 2 µl template DNA. PCR amplification was carried out as follows: As a part of effort to replace the chemical fungicides with biocontrol agents initial denaturation step at 94 ºC for 3 min followed by 30 cycles of denatur- and to introduce novel bioactive compounds to combat multiple drug resistant ation at 94°C for 1 min, annealing at 65°C for 1min and extension at 72°C for pathogens, we have selected the laterite soils of Acharya Nagarjuna University 1 min, with a further 5min extension at 72°C. The PCR product was purified campus, Guntur as a source for the isolation of potent actinomycetes with with Agarose Gel DNA Purification Kit (SoluteReady® Genomic DNA purifi- broad spectrum antimicrobial activity. cation kit, PCR Master Mix, Agarose gel electrophoresis consumables and Primers are purchased from HELINI Biomolecules, Chennai, India). The 750 MATERIALS AND METHODS: bp 16S rDNA sequence was determined with 16S rDNA actino specific forward and reverse primers. The deduced 16s rDNA sequence was compared with the Soil sample: sequences in GenBank (http://www.ncbi. nlm.nih.gov/) using the Basic Local Soil samples were randomly collected from Acharya Nagarjuna University Alignment Search Tool (BLAST) then aligned with the related reference se- campus, Guntur. The collected soil samples were air dried at room temperature quences retrieved from NCBI GenBank databases using the Clustal W method. for 48h to reduce the bacterial contaminants. Phylogenetic and molecular evolutionary analyses were conducted using Mo- lecular Evolutionary Genetic analysis (MEGA) version 4.0. [11] Isolation of actinomycetes: Ten fold serial dilutions of soil samples were done. Serial dilution agar plate Growth pattern of the strain: technique was employed to isolate the actinomycetes on Yeast extract malt To determine the growth pattern, the strain was inoculated into 500 ml flasks extract dextrose (YMD) agar medium. The medium was adjusted to pH 7.0 and containing 100 ml YMD broth. The flasks were incubated at 28 ±2°C for 0.1 mL of serially diluted soil sample was spreaded on the YMD agar medium optimum yields on a rotary shaker at 180 rpm. At every 24 h interval, the flasks were harvested; the growth of the strain was measured by weighing the dry weight of the biomass of the strain and antimicrobial metabolites production was determined in terms of their antimicrobial spectrum. The culture *Corresponding author. filtrates were extracted with ethyl acetate and antimicrobial activity of crude [12] Prof. M. Vijayalakshmi extract was determined by agar well diffusion method. M. Sc., Ph. D. Fermentation and Extraction: Department of Botany and Microbiology The fermentation was carried out in 1L Roux bottles for 120h at 30 °C. The broth was harvested with the filtration of biomass through whatman filter Acharya Nagarjuna University, paper no. 42 (Merck, Mumbai, India). The culture filtrate was extracted twice Guntur – 522510, A. P., India with an equal volume of ethyl acetate, and the combined organic layers were concentrated with a Rotavac. The deep brown concentrated semi solid com- pound was used as the crude bioactive extract.

Journal of Pharmacy Research Vol.4.Issue 8. August 2011 2615-2618 M. Vijayalakshmi et al. / Journal of Pharmacy Research 2011,4(8),2615-2618 Antimicrobial assay: as the salt concentration has a profound effect on the production of antibiotic The antimicrobial activity of the metabolite produced by the strain was deter- from microorganisms due to its effect on the osmotic pressure to the medium. mined by agar well diffusion method. Nutrient agar (NA) and Czapek-Dox [16] However, the growth was gradually retarded from 1.5% and no growth was (CD) agar media were used for culturing the test bacteria and fungi respec- observed above 3% of sodium chloride The strain utilized a wide range of tively. NA medium (100ml) was sterilized at 15lbs pressure (121oC) for 15min, carbon sources but the growth was very good when glucose, starch, arabinose cooled and inoculated with 0.2ml of test bacterial suspension. After thorough and fructose were used. The isolate grew utilizing a variety of organic and mixing, the seed medium was poured into petri plates under aseptic conditions. inorganic nitrogen sources. The strain was sensitive to the majority of antibi- After solidification of agar medium, wells of about 6mm diameter were punched otics tested but showed resistance to streptomycin, gentamicin and vancomy- into it with sterilized cork borer. In case of antifungal assay, Spore suspension cin. of test fungi was mixed with the cooled, molten CD agar medium and poured Table 2: Antimicrobial spectrum of into petri dishes. The crude extract dissolved in ethyl acetate at a concentra- The spore morphology of the Streptomyces viridis MSL under opti- tion of 50ppm was added to each well. Adding only ethyl acetate to the wells strain was studied by SEM. The ar- mized conditions rangement of spores was in spiral served as control. The plates were incubated at 37 °C for 24h for bacteria, 24- Test organism Zone of fashion and the surface of the 72 h for yeast and filamentous fungi and the diameter of the inhibition zones Bacteria inhibition (mm) was measured. [13] spore was rough (Fig. 1a and 1b) and hence it can be placed in the Bacillus cereus 22 spira group of Streptomycetaceae B. megaterium 18 Test Organisms: [17] B. subtilis 16 Bacillus cereus, B. megaterium, B. subtilis, Escherichia coli, Klebsiella sp., family. These results were con- Corynebacterium diphtheriae 19 Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Staphylococ- firmed by the identification of the Escherichia coli 17 culture by 16s rDNA sequencing. Klebsiella sp. 20 cus aureus, Vibrio cholerae, Xanthomonas campestris were used as test bacte- Pseudomonas aeruginosa 22 ria. Alternaria sp., Asperigillus niger, Botrytis cinerea, Candida albicans, The 750 bp 16S rDNA sequence Salmonella typhi 20 Fusarium solani, F. oxysporum and Verticillium alboatrum were the test fungi was determined with 16S rDNA Serratia marcescens 11 actino specific forward and reverse Shigella flexneri 19 employed. Staphylococcus aureus 23 primers. The deduced 16s rDNA Vibrio cholerae 22 RESULTS AND DISCUSSION: sequence was compared with the Xanthomonas campestris 15 During our search for the isolation of potent actinomycete strains from the sequences in GenBank (http:// Fungi www.ncbi. nlm.nih.gov/) using Aspergillus niger 25 laterite soils of Acharya Nagarjuna University campus, one actinomycete Botrytis cinerea 14 strain was found to be predominant. ISP - 2 in combination with nystatin and BLAST then aligned with the re- Candida albicans 18 streptomycin was used for the selective isolation of actinomycetes reducing lated reference sequences retrieved Fusarium oxysporum 16 from NCBI GenBank databases F. solani 16 unwanted microorganisms like bacteria and fungi. Various pretreatment meth- Verticillium alboatrum 12 ods of soil were also reported for the selective isolation of actinomycetes such using the Clustal W method. Phy- Alternaria sp. 09 logenetic and molecular evolu- as Physical, Chemical, combination of physical and chemical and Enrichment The strain exhibited higher antibacterial activity com- techniques [14] and using several selective media like YMD agar, Water-yeast tionary analyses were conducted pared to antifungal activity. However, large zone of extract-agar, Casamino Acids-yeast extract-glucose-agar, Low-nutrient min- using MEGA version 4.0. Accord- inhibition was found against A. niger (25mm). Table 1: Physiological and Biochemical characteristics of the predominant strain isolated from laterite soils: Carbon utilization (w/v)*:

Glucose + + + Sodium Chloride tolerance (%)*: Xylose + 0-1 + + + Sucrose + + 1.5 + + Raffinose + 2-3 + Starch + + + 3.5 - 7 - Galactose + + + Biochemical characteristics: Maltose + + Indole P Arabinose + + + Methyl red N Fructose + + Voge’s Proskaeur P Lactose + Citrate utilization P

Glycerol + H2S production P Mannitol + Nitrogen source (w/v)*: Yeast extract + + + * Growth of the strain measured as dry Peptone + + weight of the mycelium ‘+ + +’ - good Tryptone + + + growth; ‘+ +’ - moderate growth; ‘+’ - Asparagine + + Tyrosine + weak growth; ‘-‘indicates negative/no Ammonium nitrate + growth; S – Sensitve; R – Resistant; P – The strain exhibited heavy sporulation with spiral arrangement. The culture was 4 – day old Sodium nitrate + + Positive; N – Negative grown on YMD agar slants. Potassium nitrate + Enzymatic activity Fig. 1(a) Spiral spore chains of Streptomyces viridis MSL Pectin P Starch P Casein P Chitin P Keratin P Cellulose P Growth in the presence of antibiotics: Ampicillin S Streptomycin R Chloramphenicol S Gentamicin R Rifampicin S Penicillin S Neomycin S Vancomycin R Tetracycline S eral salts-agar, Low-nutrient mineral salts-cellobiose-agar and Cellulose-yeast extract-peptone-compost extract- agar. Media low in organic nutrients were found to be good for isolating large numbers of actinomycetes from soils. [15] YMD broth was selected as the production medium as the strain exhibited good growth in the medium compared with the other ISP and non – ISP media tested. The physiological and biochemical characteristics of the isolate are summarized in the Table 1. The isolated strain was Gram positive and it has The figure represents the texture of the spores as rough. The spiral arrangement of the spores shown positive results for indole, Voge’s – Proskaeur, citrate utilization and is visible. Fig. 1(b) Rough surface of spores of Streptomyces viridis MSL H2S production tests. Sodium chloride tolerance of the strain was also studied

Journal of Pharmacy Research Vol.4.Issue 8. August 2011 2615-2618 M. Vijayalakshmi et al. / Journal of Pharmacy Research 2011,4(8),2615-2618

25 HQ385919.1| Streptomyces sp. ERI-CPDA-1 55 AB184690.1| Streptomyces tuirus

30 HQ132790.1| Streptomyces labedae 79 HM138703.1| Streptomyces albogriseolus

0 HQ850404.1| Streptomyces akiyoshiensis 98 HQ538731.1| Streptomyces iakyrus 49 EU124570.1| Streptomyces ghanaensis

42 JF728873.1| Streptomyces roseorubens AB184462.2| Streptomyces roseorubens HM481474.1| Streptomyces mutabilis AB184063.2| Streptomyces fradiae Streptomyces anthocyanicus

The evolutionary history was inferred using the Maximum Parsimony method.

Fig. 2 Phylogenetic tree of Streptomyces viridis MSL

Journal of Pharmacy Research Vol.4.Issue 8. August 2011 2615-2618 M. Vijayalakshmi et al. / Journal of Pharmacy Research 2011,4(8),2615-2618 700 REFERENCES: 1. Ho, C., Lo, C., Lai, N., Cheah, H. and Wong, N. (2002) Actinomycetes isolated from 600 Wt.of biomass (mg/100ml) soil samples from the cocker range Sabah; ASEAN Rev. Biodiversity Environ. Conserv. 9: 1-7 500 2. Demain, A. L. (2009) Antibiotics: natural products essential to human health. Med. Res. Rev. 29: 821–842 400 3. Williams, S.T. and Cross, T. (1971) Actinomycetes. In: Methods in Microbiology, Booth, C. (Eds.). Academic press, London. 300 4. Pridham, T. G., Anderson, P., Foley, c., Lindenfelser, L. A., Hesseltine, C. W. and Benedict, R. G. (1957) A selection of media for maintenance and taxonomic study of Streptomyces. Antibiotics Ann., 57: 947-953. 200 5. Kampfer, P., Kroppenstedt, R. M. and Dott, W. (1991) A numerical classification of the genera Steptomyces and Streptoverticillium using miniaturized physiologi- 100

Wt. of biomass (mg/100 ml) cal tests. J. Gen. Microbiol., 137: 1831-1891. 6. Williams S.T, Goodfellow, M., Alderson, G., Wellington, F. M. H., Sneath, P. H. A. 0 and Sackin, M. J. (1983). Numerical classification of Streptomyces and related 24 48 72 96 120 144 168 192 216 genera. J. Gen. Microbiol., 129: 1743-1813. 7. Ruan, J.S. (1977) The basis of taxonomy of actinomycetes. The Chinese Academic Incubation period (h) Press, Beijing. 139-146. 8. Shirling, E.B., Gottlieb, D. (1966) Methods for characterization Log phase of the strain was started from 48h. High growth was obtained at 72h of incubation of Streptomyces species. Int. J. Syst. Bacteriol., 16: 313-340. (603mg) followed by stationary phase upto 144h. Slowly the growth was reduced from 6th day. 9. Hucker, G. J. and Conn, H. J. (1923) Methods of Gram staining. Technical Bulletin of the New York State, Agriculture Experimental Station, 93. Fig. 3 Growth pattern of the Streptomyces viridis MSL 10. Mehling A., Wehmeier, U. F. and Piepersberg W. (1995) Nucleotide sequences of streptomycete 16S ribosomal DNA: towards a specific identification system for ing to the sequence alignment and phylogenetic tree based on the 16S rDNA streptomycetes using PCR. Microbiol., 141: 2139–2147 genes, the strain was closer to Streptomyces viridis and designated as S. viridis 11. Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular Evolu- MSL (Fig. 2). The growth curve was also constructed and the stationary phase tionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and of the strain where secondary metabolites production occurred was identified Evolution 24:1596- 1599. as 4 - 6 days after inoculation into the broth. However, 5th day showed higher 12. Perez, C., Pauli, M. and Bazerque, P. (1990) An antibiotic assay by the agar well antimicrobial activity against the test organisms (Fig. 3). The secondary diffusion method. Acta Biol. Med. Experim. 15: 113–115 metabolites obtained from four-day old culture of Nocardia levis MK-VL_113 13. Ahmed Arwa A., (2007) Production of Antimicrobial Agent by Streptomyces isolated from laterite soils of Guntur showed high antimicrobial activity against violachromogenes. Saudi Journal of Biological Sciences 14: 7-16 [18] 14. Hayakawa Masayuki (2008) Studies on the Isolation and Distribution of Rare the test microbes. The antimicrobial spectrum of the strain under optimal Actinomycetes in Soil. Actinomycetologica 22:12–19 conditions against the test organisms was given in table 2. The inhibition 15. Crawford, D. L. (1993) Isolation and Characterization of Actinomycete Antago- zones against some human pathogens like Salmonella typhi (11mm), Shigella nists of a Fungal Root Pathogen Appl. Env. Microbiol. 59: 3899-3905 flexneri (13.6mm), Corynebacterium diptheriae (14.2mm) and Vibrio cholerae 16. Pelczar, M. J., Chan, E. C. S. and Krieg, N. R. (1993) Microbiology: Concepts and (12.4mm) were more after the optimization of cultural and physiological Applications. 5th ed. McGraw-Hill, USA. conditions of the strain. 17. Pridham, T. G., Hesseltine, C. W. and Benedict, R. G. (1958) A guide for the classification of Streptomyces according to selected groups. Appl. Microbiol. 6: 52-79. 18. Kavitha A and Vijayalakshmi M. 2009. Cultural parameters affecting the produc- ACKNOWLEDGEMENTS: tion of bioactive metabolites by Nocardia levis MK-VL_113. J. Applied Sci. Res., The authors are grateful to University Grants Commission as this work was 5(12):2138 – 2147 supported in part by the UGC Major Research project, New Delhi,India.

Source of support: UGC,India; Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 8. August 2011 2615-2618