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Identification of Antibiotics Produced by Microorganisms from the Indonesian Black Water Ecosystem

Identification of Antibiotics Produced by Microorganisms from the Indonesian Black Water Ecosystem. John L. Turner. Dr. Mark Zabriskie (College of Pharmacy). Professor: Mark Zabriskie (College of Pharmacy). Broad Significance of Antibiotic Screening. Antibiotic resistance Overuse, misuse.

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Identification of Antibiotics Produced by Microorganisms from the Indonesian Black Water Ecosystem

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  1. Identification of Antibiotics Produced by Microorganisms from the Indonesian Black Water Ecosystem John L. Turner Dr. Mark Zabriskie (College of Pharmacy) Professor: Mark Zabriskie (College of Pharmacy)

  2. Broad Significance of Antibiotic Screening • Antibiotic resistance • Overuse, misuse • New emerging diseases • Consequences for big pharmaceutical companies • Loss of profitability • Reduction in antibiotic programs

  3. Introduction Why actinomycetes are of interest to us How the bacteria are screened for bioactivity How the compounds are separated and characterized What I accomplished this summer Wrap up

  4. Why Actinomycetes are Interesting • Gram positive, filamentous, soil bacteria found all over the world • Actinomycetes are known to make many bioactive compounds in the form of secondary metabolites • Secondary metabolites are thought to be used by the bacteria to communicate with other organisms in the soil, as a means of chemical protection, as well as other non-essential functions We may be able to adopt these compounds for our own antibiotic use.

  5. Where Our Actinomycetes Come From Indonesian Black Water Ecosystem • Odorless red-black water • Low pH (3) • High levels of toxic metals • (Mn, Cu, Pb) • Humic acid, hydrogen sulfide, phenol

  6. How Antibiotic Activity is Found Receive bacterial strains glass vials From Indonesian Center for Biotechnology and Biodiversity Growth on agar plates Cultivation in different growth media Liquid fermentation Ethyl Acetate, n-Butanol Methanol Assay for antibiotic activity Crude extracts LCMS

  7. How the Extracts are Tested for Antibiotic Properties • A 20 microliter quantity of the extracts is placed on a sterile paper disks • The paper discs are placed on cultures of various bacteria and fungi and incubated overnight • Examine for inhibition the next morning

  8. Characterization of Crude Extracts • HPLC coupled with UV spectroscopy and mass spectrometry • Search AntiBase database Tetramycin A Polyene Macrolides Nystatin

  9. Bio-Activity Guided Separation Crude extract showing antibiotic activity Characterization by TLC Separation by column chromatography Assay for antibiotic activity Pure compounds

  10. Characterization of Pure Compounds Pure compounds Assay against pathogenic bacteria Structure determination using • 1H-NMR • 13C-NMR • 2D- NMR • Infrared spectroscopy • UV spectroscopy • Mass spectrometry Tetramycin B Polyene Macrolides Amphoteracin A

  11. What I Accomplished This Summer ICBB 8230 Grown in V6 media (50mL culture) 1 liter growth in V6 media Mycelia were sonicated and extracted with methanol Solvent extraction Crude extracts Crude extract showed 25 millimeter zone of inhibition on all species assayed No antibiotic activity detected

  12. What I Accomplished This Summer Biochromatographic assay showed least polar compound is active Crude extract showing antibiotic activity Silica gel normal phase column Fractions 1,2 (light oil) NMR, LCMS were inconclusive (possible impurities)

  13. What I Accomplished This Summer Fractions 1,2 Reverse phase C18 column NMR, LCMS (inconclusive) Fraction 2 Negative result on antibiotic activity assay *Somewhere in this process the compound has become inactive

  14. Thank You • Dr. Mark Zabriskie • Dr Phil Proteau, Dr. Serge Fotso,Dr. Ling Zhang, Dr. Kerry McPhail, Diana Ragland • Undergraduate Research, Innovation, Scholarship & Creativity (URISC) • Howard Hughes Medical Institute (HHMI) • Dr. Kevin Ahern

  15. Questions?

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