Title: Identification of Antibiotics Produced by Microorganisms from the Indonesian Black Water Ecosystem
1Identification 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)
2Broad Significance of Antibiotic Screening
- Antibiotic resistance
- Overuse, misuse
- Consequences for big pharmaceutical companies
- Loss of profitability
- Reduction in antibiotic programs
3Introduction
- 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
4Why 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.
5Where 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
6How 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
7How 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
8Characterization of Crude Extracts
- HPLC coupled with UV spectroscopy and mass
spectrometry - Search AntiBase database
Tetramycin A
Polyene Macrolides
Nystatin
9Bio-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
11What 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
12What 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)
13What 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
14Thank 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
15Questions?