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
•New emerging diseases
•Consequences for big pharmaceutical companies
•Loss of profitability
•Reduction in antibiotic programs
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
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.
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 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
Crude extracts
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 database
OH
HO
C:\Xcalibur\...\screen-7-7-06\8319-EAM
O
07/08/2006 07:43:11 AM
RT: 0.00 - 30.14
24.11
HO
O
OH
OH
NL:
1.96E6
Channel A
UV
8319-EAM
1800000
O
1600000
1.79
1400000
O
9.75
uAU
1200000
O
O
1000000
2.47
11.14
800000
Tetramycin A
OH
NH2
12.58
6.36
400000
9.37
6.96
3.44
200000
HO
8.16
3.81
600000
0
4.31
1.66
0.30
0
1
2
3
4
22.98
8.55
5.18 5.67
5
15.68
7.57
10.95
11.31 11.91
13.07
6
7
8
9
10
11
12
13.89
13
16.04
14.96
14
15
Time (min)
16
17.68
17
18
19.59
19.81
20.07
22.05
21.62
19.34
19
20
21
22
23
24
25
26
27
28
29
30
RT: 0.00 - 29.99
29.91 NL:
7.78E8
TIC M S
8319-EAM
100
90
80
Relative Abundance
Polyene Macrolides
70
60
29.45
50
25.68
40
26.65
27.80
30
HO
8.35
7.66 8.10
1.92 2.01 2.19 3.19 3.46 4.41 5.09 5.68 6.12 6.43 7.14
10
0.74
O
OH
O
24.66
OH OH OH O
O
O
O
OH NH2
OH
Nystatin
27.46
16.50
20
9.31 9.91 10.55 11.16
12.27 13.08 13.17
15.05
13.98
15.90
16.93
16
17
17.91
20.94
18.12 18.93 19.70 19.92
21.54 22.23
29.03
23.55
23.38 23.85
28.86
0
0
OH
Relative Abundance
HO
OH
OH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Time (min)
18
19
20
21
22
23
24
25
26
27
28
29
8319-EAM #553-559 RT: 24.02-24.28 AV: 7 NL: 6.86E5
T: + p ESI Full ms [ 150.00-2000.00]
533.07
100
80
453.47
60
40
20
475.53 550.93
227.13
192.93
323.33
0
200
300
590.27
435.20
400
500
600
679.47
701.67
700
791.33 831.40
800
900
1064.93
1082.87
1000.53
1000
1100
m/z
1207.00 1258.47 1348.73 1457.40
1200
1300
1400
1500
1615.53
1600
1672.60 1817.87 1872.53 1965.13
1700
1800
1900
2000
Bio-Activity Guided Separation
Crude extract showing
antibiotic activity
Characterization
by TLC
Separation by
column
chromatography
Assay for antibiotic
activity
Pure compounds
Characterization of Pure Compounds
Pure compounds
Assay against pathogenic bacteria
OH
HO
HO
O
Structure determination using
OH
OH
OH
HO
O
O
O
O
Tetramycin B
OH
HO
Polyene Macrolides
HO
HO
O
OH
OH
OH
OH O
HO
OH
O
O
O
OH
Amphoteracin A
O
OH
NH2
OH
O
1H-NMR
O
O
13C-NMR
OH
2D- NMR
Infrared spectroscopy
UV spectroscopy
Mass spectrometry
NH2
OH
OH
OH
O
OH
O
O
OH
O
HO
O
OH
•
•
•
•
•
•
O
HO
NH2
OH
OH
HO
O
O
OH NH2
OH
OH
OH
O
O
What I Accomplished This Summer
ICBB 8230
Grown in V6 media
(50mL culture)
Mycelia were
sonicated and extracted
with methanol
1 liter growth in V6 media
Solvent extraction
Crude extracts
Crude extract showed
25 millimeter zone of inhibition
on all species assayed
No antibiotic activity detected
What I Accomplished This Summer
Crude extract showing
antibiotic activity
Biochromatographic assay showed
least polar compound is active
Silica gel
normal phase
column
Fractions 1,2
(light oil)
NMR, LCMS were inconclusive
(possible impurities)
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
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?