• K. M. Anju Division of Crop Protection, Central Tuber Crops Research Institute, Thiruvananthapuram
  • M. M. Archana Central Tuber Crops Research Institute
  • C. Mohandas Central Tuber Crops Research Institute
  • Bala Nambisan Central Tuber Crops Research Institute


Objective: To isolate and identify the bioactive metabolites from the culture filtrates of a bacterium (Bacillus cereus) symbiotically associated with a novel entomopathogenic nematode Rhabditis (Oscheius) species.

Methods: The bacterium was cultured in three different media and the antimicrobial activity was determined by the well diffusion assay. The ethyl acetate extract of the cell free culture filtrate was then purified by silica gel column chromatography and thin layer chromatography. Identification of the active metabolite was done with HPLC, GC-MS and LC-MS.

Results: The cell free culture filtrate of a nematode symbiotic bacterium showed both antibacterial and antifungal activities. Fermentation conditions were standardized and optimum antibacterial activity was observed in tryptic soy broth at 72 h of incubation at 30 °C. When the ethyl acetate extract was purified by silica gel column chromatography and thin layer chromatography, an active fraction was obtained which was subjected to HPLC analysis along with GC-MS and LC-MS leading to the identification of a major compound Bis (2-ethyl hexyl) phthalate. The compound was active against Gram positive bacteria Bacillus subtilis MTCC2756, Staphylococus aureus MTCC902, Gram negative bacteria Escherichia coli MTCC 2622 and fungi such as Aspergillus flavus MTCC277, Candida albicans MTCC183, Fusarium oxysporum MTCC 284, Rhizoctonia solani MTCC 4634.

Conclusion: Bis (2-ethyl hexyl) phthalate was identified as one of the metabolites produced by a nematode symbiotic bacterium associated with a novel entomopathogenic nematode Rhabditis (Oscheius) species. Thus similar compounds isolated from novel entomopathogenic bacteria would pave the way for identifying new drugs for the pharmaceutical and agricultural sector.


Keywords: Entomopathogenic nematodes, EPN, Rhabditis (Oscheius) sp, Bis (2-ethyl hexyl) phthalate, antibacterial, antifungal


Download data is not yet available.


1. Hoffmann MP, Frodsham AC. Natural Enemies of Vegetable Insect Pests. Cooperative Extension. Cornell University, Ithaca, NY; 1993.
2. Akhurst RJ. Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae. J Gen Microbiol 1982;128:3061-5.
3. Barbercheck ME, Kaya HK. Interaction between Beauveria bassiana and the entomopathogenic nematodes, Steinernema feltiae and Heterorhabditis heliothidis. J Invertebr Pathol 1990;55:225–34.
4. Chen G, Dunphy GB, Webster JM. Antifungal activity of two Xenorhabdus spp and Photorhabdus luminiscens spp of Heterorhabditis megidis. Biol Control 1994;4:157-62.
5. Han R, Ehlers RU. Trans-specific nematicidal activity of Photorhabdus luminescens. Nematology 1999;1:687–93.
6. Hu K, Li J, Webster JM. Nematicidal metabolites produced by Photorhabdus luminescens (Enterobacterioaceae), bacterial symbiont of entomopathogenic nematodes. Nematology 1999;1:457–69.
7. Bowen DJ, Ensign JC. Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl Environ Microbiol 1998;64:3029–35.
8. Mohandas C, Sheela MS, Mathews S, Naveen Raj DS. Rhabditis (Oscheious) Spp (Nematoda: Rhabditidae), a new pathogenic nematode of crop pests. National Sympo Green Pesticides Insect Pest Management 2004;6:51-2.
9. Deepa I, Mohandas C, Makesh Kumar T, Siji JV, Prakash Krishnan BS, Binoy Babu. Identification of new entomopathogenic nematodes (EPNs) based on sequence of D2-D3 expansion fragments of the 28S r RNA. J Root Crops 2010;36:227-32.
10. Deepa I, Mohandas C, Siji JV. Molecular characterization of novel symbiotic bacteria from entomopathogenic nematodes. National seminar on climate change and food security: challenges and opportunities. Tuber Crops 2011;24:399-404.
11. Berdy J. Bioactive microbial metabolites a personal view. J Antibiot 2005;58:1–26.
12. Brachmann AO. Isolation and identification of natural products and biosynthetic pathways from Photorhabdus and Xenorhabdus. Dissertation, der Universität des Saarlandes; 2009.
13. Fang XL, Feng JT, Zhang WG, Wang YH, Zhang X. Optimization of growth medium and fermentation conditions for improved antibiotic activity of Xenorhabdus nematophilia TB using a statistical approach. Afr J Biotechnol 2010;9:8068-77.
14. Maxwell PW, Chen G, Webster JM, Dunphy GB. Stability and activities of antibiotics produced during infection of the insect Galleria mellonella by two isolates of Xenorhabdus nematophilus. Appl Environ Microbiol 1994;60:715-21.
15. Ji D, Yi Y, Kang G, Choi Y, Kim P, Baek N, et al. Identification of an antibacterial compound, benzylideneacetone, from Xenorhabdus nematophila against major plant-pathogenic bacteria. FEMS Microbiol Lett 2004;239:241-8.
16. Chen G. Antimicrobial activity of the nematode symbionts, Xenorhabdus and Photorhabdus (Enterobacteriacea) and the discovery of two groups of antimicrobial substances, Nematophin and Xenorxides, Dissertation, Simon Fraser University; 1996.
17. Uyeda M, Suzuki K, Shibata M. 3315-AF2, a cell aggregation factor produced by Streptomyces spstrain No. A-3315. Agric Biol Chem 1990;54:251-2.
18. Al-Bari MA, Bhuiyan MS, Flores ME, Petrosyan P, Garcia-Varela M, Islam M. A Streptomyces bangladeshensis sp. nov., isolated from soil, which produces bis (2-ethylhexyl) phthalate. Int J Syst Evol Microbiol 2005;55:1973-7.
19. El-Sayed MH. Di-(2-ethylhexyl) phthalate, a major bioactive metabolite with antimicrobial and cytotoxic activity isolated from the culture filtrate of newly isolated soil Streptomyces (Streptomyces mirabilis Strain NSQu-25). World Appl Sci J 2012;20:1202-12.
20. Hoang VLT, Li Y, Kim S. Cathepsin B inhibitory activities of phthalates isolated from a marine Pseudomonas strain. Bioorg Med Chem Lett 2008;18:2083–8.
21. Stefanov K, Konaklieva M, Brechany EY, Christie WW. Fatty acid composition of some algae from the black sea. Phytochemistry 1988;27:3495–7.
22. Sastry VMVS, Rao GRK. Dioctyl phthalate, and antibacterial compound from the marine brown alga–Sargassum wightii. J Appl Phycol 1995;7:185–6.
23. Chen CY. Biosynthesis of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) from red alga–Bangia atropurpurea. Water Res 2004;38:1014-8.
24. Namikoshi M, Fujiwara T, Nishikawa T, Ukai K. Natural abundance 14C content of dibutyl phthalate (DBP) from three marine algae. Mar Drugs 2006;4:290–7.
25. Amade P, Mallea M, Bouaicha N. Isolation, structural identification and biological activity of two metabolites produced by Penicillium olsonii Bainier and Sartory. J Antibiot 1994;47:201–7.
26. Kavitha A, Prabhakar P, Vijayalakshmi M, Venkateswarlu Y. Production of bioactive metabolites by Nocardia levis. MK-VL_113. Lett Appl Microbiol 2009;49:484–90.
27. Gourlay T, Samartzis I, Stefanou D, Taylor K. Inflammatory response of rat and human neutrophils exposed to di-(2-ethyl-hexyl)-phthalatephthalate-plasticized polyvinyl chloride. Artif Organs 2003;27:256-60.
28. Oie L, Hersoug LG, Madsen JO. Residential exposure to plasticizers and its possible role in the pathogenesis of asthma. Environ Health Perspect 1997;105:972-8.
29. Lee KH, Kim JH, Lim DS, Kim CH. Anti-leukemic and anti-mutagenic effects of di-(2-ethylhexyl) phthalate isolated from Aloe vera Linne. J Pharm Pharmacol 2000;52:593-8.
392 Views | 1110 Downloads
How to Cite
Anju, K. M., M. M. Archana, C. Mohandas, and B. Nambisan. “AN ANTIMICROBIAL PHTHALATE DERIVATIVE FROM BACILLUS CEREUS, THE SYMBIOTIC BACTERIUM ASSOCIATED WITH A NOVEL ENTOMOPATHOGENIC NEMATODE, RHABDITIS (OSCHEIUS) SP”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 7, no. 9, July 2015, pp. 238-42,
Original Article(s)