Can Tetracycline resistance be induced in Pseudomonas fluorescens after 3 weeks?

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 Dr. Richard Lenskiof Michigan State University • E. coli Long-term Experimental Evolution experiment reaching 50,000 generations The rise of multiple drug resistant organisms.

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

4. Tetracycline is broad spectrum antibiotic It can be inactivated by bivalent ions. • Tetracycline antibiotics are protein synthesis inhibitors • They prevent the binding of tRNAto 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 0.20 - Red 0.20 to 0.31 – Dark red 0.20 to 0.42 - 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?