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

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

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

4. 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 Why Actinomycetes are Interesting 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 Crude extracts Ethyl Acetate, n-Butanol Methanol Assay for antibiotic activity 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 Nystatin Polyene Macrolides

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

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

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

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

13. What I Accomplished This Summer Fractions 1,2 Reverse phase C18 column Fraction 2 NMR, LCMS (inconclusive) 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?