Can Tetracycline resistance be
induced in Pseudomonas
fluorescens after 3 weeks?
David S. Abuín & Tania Bembridge
Department of Microbiology
North Carolina State University
Inspirations
The rise of multiple drug
resistant organisms.
Dr. Richard Lenski of
Michigan State University
• E. coli Long-term
Experimental Evolution
experiment reaching
50,000 generations
Pseudomonas fluorescens
‒ an environmental bacteria
that inhabits soil, water and
plant surfaces.
‒ beneficial for plants by
‒ inhibiting pathogens
‒ aiding nutrient absorption
‒ degrading environmental
pollutants.
‒ Gram negative
Tetracycline is broad spectrum
antibiotic
It can be
inactivated
by bivalent
ions.
• Tetracycline antibiotics are protein synthesis inhibitors
• They prevent the binding of tRNA to the mRNA-ribosome
complex.
• They bind to the 30S ribosomal subunit in the mRNA
translation complex
• Tetracycline inhibits cell growth by inhibiting translation.
The Method.
1.5
0.10
Concentration
Push
1.1
OD
0.10 - Blue
0.10 to 0.31 – Aqua
0.20
1.3
0.9
0.10 mM (11-7)
0.20 -> 0.31
0.20 mM (11-7)
0.20 - Red
0.20 to 0.31 – Dark red
0.20 to 0.42 - Pink
0.7
0.10 to 0.31 mM (11-7)
0.20 to 0.31 mM (11-7)
0.5
0.20 to 0.42 mM (11-7)
0.10- > 0.31
0.20 -> 0.42
0.42 mM (11-7)
0.3
0.42
0.1
0:00:00
12:00:00
24:00:00
36:00:00
Time
48:00:00
60:00:00
72:00:00
1.5
1.3
0.20 mM
Tetracycline
1.1
OD
3rd trial
(Nov 7)
0.9
0.7
0.20 mM (11-7)
0.20 mM (10-31)
2nd trial
(Oct 31)
0.20 mM (10-24)
0.5
1st trial
(Oct 24)
0.3
0.1
0:00:00
12:00:00
24:00:00
36:00:00
Time
Axis Title
48:00:00
60:00:00
72:00:00
Resistance Genes
Pseudomonas
tet(A) (B) (C) (E) (G) (M) (34) (L) (X) (42)h
Encodes a membrane
protein that actively pumps
tetracycline out of the cell
Encodes proteins that protect
the ribsomes
Conclusion
• Data is inconsistent to definitively
demonstrate directed evolution had occurred.
• Possible contamination due to continuous
shaking in the Bioscreen C detector as well as
during transfer process.
• Contamination of each trial of
LB only media
Further Direction
• Test hypothesis with an anaerobic organism to
limit possible contamination.
• Determine the pathways which antibiotics use
to destroy microorganisms and how the
coping mechanisms that survivors use.
• Is there a much faster way to induce directed
mutation in favor of antibiotic resistance?