AiM  Vol.4 No.9 , July 2014
Caerulomycin A—An Antifungal Compound Isolated from Marine Actinomycetes

Actinomycetes have been prolific sources of novel secondary metabolites with a range of biological activities that may ultimately find application as therapeutic compounds. Hence several drug discovery companies are engaged in isolation of novel bioactive metabolites from these microbial sources. Antibiotics form the major class of such bioactive metabolites and have been widely used for treating infectious diseases. One of the most critical problems in clinical practice is the increase of prevalence of drug resistant strains, especially azole resistance among fungi. Due to this, there is a constant need for development of new antifungal antibiotics having novel scaffolds and/or mechanism of action. In our in-house screening program in the quest of novel and superior antifungal compounds, an actinomycetes strain PM0525875 was isolated from a marine invertebrate. The extracts of this microbe showed potent in-vitro antifungal activity against drug resistant fungal strains. The antifungal active peak from the extract obtained by shake flask fermentation was identified by chromatographic and other analytical techniques during bioactivity guided isolation. Later the fermentation conditions were optimized in 30 L fermentor for the production of sufficient amount antifungal compound for complete structural characterization. Consequently the fermented broth extract was subjected to bioactivity-guided fractionation, to isolate the active principle using different preparative chromatographic techniques followed by its characterization. The active principle was characterized to be Caerulomycin A. Minimum inhibitory concentration (MIC) of the compound was found in the range of 0.39 - 1.56 μg/ml against pathogenic fungal test strains. The phylogenetic analysis of producer strain using 16S rRNA sequence showed closest match with Actinoalloateichus cyanogriseus. Herewith we report the isolation of Caerulomycin A from marine invertebrate-associated Actinoalloteichus sp. using optimized medium and fermentation conditions.

Cite this paper
Ambavane, V. , Tokdar, P. , Parab, R. , Sreekumar, E. , Mahajan, G. , Mishra, P. , D’Souza, L. and Ranadive, P. (2014) Caerulomycin A—An Antifungal Compound Isolated from Marine Actinomycetes. Advances in Microbiology, 4, 567-578. doi: 10.4236/aim.2014.49063.
[1]   Martin, G.S., Mannino, D.M., Eaton, S. and Moss, M. (2003) The Epidemiology of Sepsis in the United States from 1979 through 2000. New England Journal of Medicine, 348, 1546-1554.

[2]   Zilberberg, M.D., Shorr, A.F. and Kollef, M.H. (2008) Secular Trends in Candidemiarelated Hospitalization in the United States from 2000-2005. Infection Control and Hospital Epidemiology, 29, 978-980.

[3]   Wisplinghoff, H., Bischoff, T., Tallent, S.M., Seifert, H., Wenzel, R.P. and Edmond, M.B. (2004) Nosocomial Bloodstream Infections in US Hospitals: Analysis of 24,179 Cases from a Prospective Nationwide Surveillance Study. Clinical Infectious Diseases, 39, 309-317.

[4]   Berdy, J. (2005) Bioactive Microbial Metabolites, a Personal View. Journal of Antibiotics, 58, 1-26.

[5]   Singh, S.B. and Pelaez, F. (2010) Terrestrial Microorganisms: Filamentous Bacteria. In: Mander, L. and Liu, H.-W., Eds., Comprehensive Natural Products II, Elsevier, Amsterdam, 109-140.

[6]   Genilloud, O., Gonzalez, I., Salazer, O., Martin, J., Tormo, J.R. and Vicente, F. (2011) Current Approaches to Exploit Actinomycetes as a Source of Novel Natural Products. Journal of Industrial Microbiology Biotechnology, 38, 375-389.

[7]   Gonzalez, I., Ayuso-Sacido, A., Anderson, A. and Genilloud, O. (2005) Actinomycetes Isolated from Lichens: Evaluation of Their Diversity and Detection of Biosynthetic Gene Sequences. FEMS Microbiology Ecology, 54, 401-415.

[8]   Salazar, O., Valverde, A. and Genilloud, O. (2006) Real-Time PCR for the Detection and Quantification of Geodermatophilaceae from Stone Samples and Identification of New Members of the Genus Blastococcus. Applied and Environmental Microbiology, 72, 346-352.

[9]   Bull, A.T., Stach, J.E., Ward, A.C. and Goodfellow, M. (2005) Marine Actinobacteria: Perspectives, Challenges, Future Directions. Antonie Van Leeuwenhoek, 87, 65-79.

[10]   Lam, K.S. (2006) Discovery of Novel Metabolites from Marine Actinomycetes. Current Opinion in Microbiology, 9, 245-251.

[11]   Manivasagan, P., Venkatesan, J., Sivakumar, K. and Kim, S.K. (2013) Marine Actinobacterial Metabolites: Current Status and Future Perspectives. Microbiological Research, 168, 311-332.

[12]   Zotchev, S.B. (2011) Marine Actinomycetes as an Emerging Resource for the Drug Development Pipelines. Journal of Biotechnology, 158, 168-175.

[13]   Blunt, J.W., Copp, B.R., Keyzers, R.A., Munro, M.H.G. and Prinsep, M.R. (2013) Marine Natural Products. Natural Product Reports, 30, 237-323.

[14]   Hooper, J. and Parisot, J. (2008) Principles of Assessing Bacterial Susceptibility to Antibiotics Using the Agar Diffusion Method. Journal of Antimicrobial Chemotherapy, 61, 1295-1301.

[15]   Ball, S., Bessell, C.J. and Mortimer, A. (1957) The Production of Polyenic Antibiotics by Soil Streptomycetes. Journal of General Microbiology, 17, 96-103.

[16]   Trevelyan, W.E., Forrest, R.S. and Harrison, J.S. (1952) Determination of Yeast Carbohydrates with the Anthrone Reagent. Nature, 170, 626-627.

[17]   NCCLS (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Second Edition. NCCLS Document M27-A2, Wayne, Pennsylvania.

[18]   NCCLS (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard—Second Edition. NCCLS Document M38-A, Wayne, Pennsylvania.

[19]   McInnes, A.G., Smith, D.G., Wright, J.L.C. and Vining, L.C. (1977) Caerulomycin B and C, New 2,2’-Dipyridyl Derivatives from Streptomyces caeruleus. Canadian Journal of Chemistry, 55, 4159-4165.

[20]   Saitou, N. and Nei, M. (1987) The Neighbor-Joining Method: A New Method for Reconstructing Phylogenetic Trees. Molecular Biology and Evolution, 4, 406-425.

[21]   Felsenstein, J. (1985) Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evolution, 39, 783-791.

[22]   Kimura, M. (1980) A Simple Method for Estimating Evolutionary Rate of Base Substitutions through Comparative Studies of Nucleotide Sequences. Journal of Molecular Evolution, 16, 111-120.

[23]   Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731-2739.

[24]   Antifungals Market to 2017—Generic Erosion of Major Polyenes, Azoles, Allylamines and Echinocandi-
ns to Slow Value Growth (2012).

[25]   Funk, A. and Divekar, P.V. (1959) Caerulomycin, a New Antibiotic from Streptomyces caeruleus. I. Production, Isolation, Assay and Biological Properties. Canadian Journal of Microbiology, 5, 317-321.

[26]   Divekar, P.V., Read, G. and Vising, L.C. (1967) Caerulomycin, a New Antibiotic from Streptomycin caeruleus Baldacci. II Structure. Canadian Journal of Chemistry, 45, 1215-1223.

[27]   Vinothkumar, S. and Parameswaran, P.S. (2013) Recent Advances in Marine Drug Research. Biotechnology Advances, 31, 1826-1845.

[28]   Chatterjee, D.K., Raether, W., Iyer, N. and Ganguli, B.N. (1984) Caerulomycin, an Antifungal Antibiotic with Marked in Vitro and in Vivo Activity against Entamoeba histolytica. Zeitschrift fürm Parasitenkunde, 70, 569-573.

[29]   Blunt, J.W., Copp, B.R., Keyzers, R.A., Munro, M.H.G. and Prinsep, M.R. (2013) Marine Natural Products. Natural Product Reports, 30, 237-323.

[30]   Lin, Q., Zhang, G., Li, S., Zhang, H., Ju, J., Zhu, W. and Zhang, C. (2011) Development of a Genetic Modification System for Caerulomycin Producer Actinoalloteichus sp. WH1-2216-6. Wei Sheng Wu Xue Bao, 51, 1032-1041.

[31]   Zhu, Y., Zhang, Q., Li, S., Lin, Q., Fu, P., Zhang, G., Zhang, H., Shi, R., Zhu, W. and Zhang, C. (2013) Insights into Caerulomycin A Biosynthesis: A Two-Component Monooxygenase CrmH-Catalyzed Oxime Formation. Journal of the American Chemical Society, 135, 18750-15753.

[32]   Trecourt, F., Gervais, B., Mallet, M. and Queguiner, G. (1996) First Synthesis of Caerulomycin C. The Journal of Organic Chemistry, 61, 1673-1676.

[33]   Ranganthan, S., Singh, B.B. and Divekar, P.V. (1968) Synthesis of Caerulomycin. Canadian Journal of Chemistry, 46, 165-166.

[34]   Singla, A.K., Agrewala, J.N., Vohra, R.M. and Singh, J.R. (2007) Use of Bipyridine Compound Caerulomycin A Derivates and Analogs Thereof as Immunosuppressive Agents. US Patent