Discovery and Identification
of Antibacterial Agents for
Dental Use
Adam Kirkpatrick
HHMI: Summer 2005
Dr. Bill Gerwick
Tooth Decay


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Est. 90% of U.S. have
at least one cavity
2nd only to common
cold
$68 billion annually
Potential Outcomes

Useful molecule

Could help defeat dental disease

Dental caries (“cavities”)
Gerwick Lab Research
Organism
Crude Extract
Prefractionated
Extract
Pure Compound
Molecular
Structure
My Research
Pure Compound
Test
Antimicrobial Pure
Compound?
Procedure

Obtain bacteria

The Mutans Streptococci
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S. mutans
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Broad term
7 species of Streptococcus
Cause dental caries (“cavities”)
ATCC # UAB 308
Choose compounds
Culture the bacteria

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Freezer stock
Working stock
Photograph from
http://www.mrcophth.com/finalmrcophthmcqss/16parasitolo
gy/1strep-mutans.jpg2
Antibiotic Susceptibility Testing
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Bacteria “lawn”
Impregnate disc
Plates
Controls
Incubate
Zones of inhibition
Photograph from http://www.austin.cc.tx.us/microbugz/48antibio.html
Results
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
21 pure compounds tested
1 antimicrobial pure compound

Allolaurinterol
Allolaurinterol Properties


Structure
Name of Source Organism

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Found

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
Martensia
Laurencia
Curacao
1991
Other known activity


Anti-cancer
Antibiotic resistant bacteria
OH
Br
Photograph from
http://www.biol.tsukuba.ac.jp/~inouye/ino/r/Martensia.GIF
Current Project
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Other strains?

Toxic?

Mouthwash?
My Research – 2nd Facet
Crude and Prefractionated
Extracts
Test
Antimicrobial Crude and
Prefractionated Extracts?
Purify
Pure Compound
Test
Antimicrobial Pure
Compound?
Structure
Fractionation Technique

Vacuum Liquid Chromatography (VLC)
Pre-fractionation of Crude Extracts to Produce Reduced
Complexity, Higher Titer Screening Materials
Crude Extract (preferred 2.0 gms)
Size of VLC (for 2.0 gm, use 350 mL VLC funnel)
50 mg for parallel
crude extract testing
Silica gel
cm wide x
cm high
Volume of solvent = 300 mL of each
20% E/H
100% H
A
C
10% E/H
B
100% E
60% E/H
E
40% E/H
D
100% M
G
80% E/H
F
I
25% M/E
H
Extract in the
middle of column
Silica Gel
TLC grade
Use Course Porosity
Vacuum Filter Funnel
Enter # mg
per fraction
Reverse Phase HPLC Traces from
Pre-fractions A-I
Most Polar
Least Polar
Purification Scheme
A
VLC
HPLC
B
C
D
F
E
G H I
TLC
Solid Phase Extraction
Chromatography (SPEC)
Characterization of Purified
Compounds
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
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Mass Spectrometry
IR Spectroscopy
1H, 13C and 2-Dimensional NMR
Spectroscopy
Photograph from
http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Gra
phics/MolStruct/L-leucine.jpg3
Results
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30 organisms tested
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9 fractions, 1 crude extract
300 total
89 antimicrobial crude extracts and
fractions
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No isolated compounds
Newly discovered molecule?
Results
Fraction
Number
Organism
Name
Collected From
ZOI Diameter
(mm)
1303 E
Red Alga
Papua New
Guinea
13.5
0988 D
Laminaria
Hokkaido, Japan
13
1512 A
Brown Tufts
Madagascar
14.5
1500 A
Symploca
Papua New
Guinea
12
1460 H
Red Brown Fuzzy
Papua New
Guinea
11, very clear
Other Potential Research
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Other bacterial strains

Human toxicity

Antimicrobial interaction
Photograph from
http://www.answers.com/main/content/wp/en/thumb/c/cb/400pxCellMembraneDrawing.jpg
Acknowledgements
Howard Hughes
Medical Institute
Dr. Bill Gerwick
Dr. Kevin Ahern
Dr. Taifo Mahmud
Acknowledgements
Carla Sorrels
Mirjam
Musafija-Girt
Dr. Roger Linington
Eric Andrianosolo
Harry Gross
Jennifer Kepler
Aishu Ramakrishnan
You!