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)

Broad Significance of Antibiotic Screening

•Antibiotic resistance•Overuse, misuse

•Consequences for big pharmaceutical companies•Loss of profitability•Reduction in antibiotic programs

•New emerging diseases

IntroductionIntroduction

Why actinomycetes are of Why actinomycetes are of interest to usinterest to us

How the bacteria are screened How the bacteria are screened for bioactivityfor bioactivity

How the compounds are separated How the compounds are separated and characterizedand characterized

What I accomplished this summerWhat I accomplished this summer Wrap upWrap up

•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.

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

How Antibiotic Activity is How Antibiotic Activity is FoundFoundReceive bacterial strains glass vials

From Indonesian Center forBiotechnology and Biodiversity

Growth on agar plates

Cultivation in different growth media

Liquid fermentation

Crude extracts

Ethyl Acetate, n-ButanolMethanol

Assay for antibiotic activity

LCMS

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

Characterization of Crude Extracts

•HPLC coupled with UV spectroscopy and mass spectrometry

•Search AntiBase databaseC:\Xcalibur\...\screen-7-7-06\8319-EAM 07/08/2006 07:43:11 AM

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24.11

1.79

9.75

2.47

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8.163.81 12.586.36 9.37 15.68

22.987.576.96

8.55 19.5910.95 11.313.44 11.915.67 16.045.18 22.0519.814.31 17.6813.89 14.9613.07 19.341.660.30 20.07 21.62

NL:1.96E6

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29.91

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27.46 29.0316.5020.94 23.5517.9115.05 28.8619.70 19.92 23.8522.2313.17 23.3815.90 16.939.31 13.98 21.5418.128.35 18.9313.0812.279.918.10 10.55 11.167.667.146.436.125.682.011.92 2.19 5.094.413.463.190.74

NL:7.78E8

TIC M S 8319-EAM

8319-EAM #553-559 RT: 24.02-24.28 AV: 7 NL: 6.86E5T: + p ESI Full m s [ 150.00-2000.00]

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533.07

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679.47475.53 550.931064.93227.13

701.67 1082.87 1615.53590.27192.93 791.33 831.40323.33 1207.00 1672.601258.47 1965.13435.20 1000.53 1872.531817.871348.73 1457.40

Tetramycin A

Nystatin

Polyene Macrolides

Crude extract showing antibiotic activity

Separation by column

chromatogra

phyAssay for antibiotic

activity

Pure compounds

Bio-Activity Guided Separation

Characterizationby TLC

Characterization of Pure Compounds

Pure compounds

Structure determination usingAssay against pathogenic bacteria

Tetramycin B

Amphoteracin A

Polyene Macrolides

• 1H-NMR• 13C-NMR•2D- NMR•Infrared spectroscopy•UV spectroscopy•Mass spectrometry

What I Accomplished This Summer

Grown in V6 media(50mL culture)

Mycelia weresonicated and extracted

with methanol

Crude extract showed25 millimeter zone of inhibition

on all species assayed

1 liter growth in V6 media

Solvent extraction

Crude extracts

No antibiotic activity detected

ICBB 8230

What I Accomplished This Summer

Silica gelnormal phase

column

Crude extract showingantibiotic activity

Fractions 1,2

(light oil)

Biochromatographic assay showedleast polar compound is active

NMR, LCMS were inconclusive(possible impurities)

What I Accomplished This Summer

Fractions 1,2

Reverse phase C18 column

Fraction 2NMR, LCMS (inconclusive)

Negative result on antibiotic activity assay

*Somewhere in this process the compound has become inactive

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

Questions?