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?