1. Can Tetracycline resistance be induced in Pseudomonas fluorescens after 3 weeks?
David S. Abuín & Tania Bembridge
Department of Microbiology
North Carolina State University

2. 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

3. 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
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4. Tetracycline is broad spectrum antibiotic
It can be inactivated by bivalent ions.
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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.

5. The Method. Concentration Push 0.10 - Blue 0.10 to 0.31 – Aqua
Red
0.20 to 0.31 – Dark red
0.20 to Pink

6. Tetracycline resistance at 0.20 mM concentration

7. Tetracycline resistance at 0.42 mM concentration

8. 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

9. 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

10. 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?