Post on 23-Feb-2016
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Can Tetracycline resistance be induced in Pseudomonas fluorescens after 3 weeks?
David S. Abuín & Tania BembridgeDepartment of Microbiology
North Carolina State University
Inspirations
Dr. Richard Lenski of Michigan State University • E. coli Long-term
Experimental Evolution experiment reaching 50,000 generations
The rise of multiple drug resistant organisms.
‒ an environmental bacteria that inhabits soil, water and plant surfaces.
‒ beneficial for plants by ‒ inhibiting pathogens‒ aiding nutrient absorption‒ degrading environmental
pollutants.
‒ Gram negative
Pseudomonas fluorescens
Tetracycline is broad spectrum antibiotic
• 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.
It can be inactivated by bivalent ions.
The Method.
0:00:00 12:00:00 24:00:00 36:00:00 48:00:00 60:00:00 72:00:000.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0.10 mM (11-7)
0.20 mM (11-7)
0.10 to 0.31 mM (11-7)
0.20 to 0.31 mM (11-7)
0.20 to 0.42 mM (11-7)
0.42 mM (11-7)
Time
OD
0.10
0.10- > 0.31
0.20
0.20 -> 0.31
0.20 -> 0.42
0.42
Concentration Push0.10 - Blue0.10 to 0.31 – Aqua
0.20 - Red0.20 to 0.31 – Dark red0.20 to 0.42 - Pink
Tetracycline resistance at 0.20 mM concentration
0:00:00 12:00:00 24:00:00 36:00:00 48:00:00 60:00:00 72:00:000.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5
0.20 mM (11-7)
0.20 mM (10-31)
0.20 mM (10-24)
Axis Title
OD
0.20 mM Tetra-cycline
1st trial(Oct 24)
2nd trial(Oct 31)
3rd trial(Nov 7)
Time
Tetracycline resistance at 0.42 mM concentration
Resistance Genes
Encodes a membrane protein that actively pumps tetracycline out of the cell
Pseudomonas tet(A) (B) (C) (E) (G) (M) (34) (L) (X) (42)h
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?