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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
RT: 0.00 - 30.14
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Time (min)
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uAU
24.11
1.79
9.75
2.47
11.14
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
Channel A UV 8319-EAM
RT: 0.00 - 29.99
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29Time (min)
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Relat
ive A
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ance
29.91
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26.65
27.8024.66
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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m /z
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Rel
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e Ab
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533.07
453.47
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?