Un abonnement à JoVE est nécessaire pour voir ce contenu.  Connectez-vous ou commencez votre essai gratuit.
An In Vitro Technique to Study Antimicrobial Treatments on Bacterial Aggregates

An In Vitro Technique to Study Antimicrobial Treatments on Bacterial Aggregates

Transcription

To study the impact of antimicrobials on bacterial aggregates, take Pseudomonas aeruginosa culture in a growth medium that promotes aggregate formation — simulating growth during infection.

The engineered bacteria express a fluorescent protein for aggregate detection under a fluorescence microscope. Image in three dimensions to compute the volume of the aggregates, or the bacterial biomass, and visualize the increase in biomass over time, indicating bacterial growth.

Add colistin — a cationic antimicrobial peptide — at a sublethal concentration that kills susceptible bacteria while selecting the antibiotic-tolerant ones.

Colistin binds to negatively-charged lipopolysaccharides, or LPS, on the bacterial outer membrane, displacing cationic bridges that stabilize the LPS monolayer — damaging membrane integrity.

Colistin enters periplasm and binds to LPS on the cell membrane, disrupting the membrane and resulting in cell lysis. As a counter mechanism, specific genetic mutations in a subset of bacteria neutralize the negative charge of LPS. This modification inhibits the electrostatic interaction of cationic colistin with less anionic LPS — preventing cell death.

Image to visualize decreased biomass with time, indicating the impact of the antimicrobial. Add propidium iodide or PI — a fluorescent dye — that enters through the damaged membrane to bind DNA, labeling only the dead cells.

Perform fluorescence-activated cell sorting, or FACS, separating PI-labeled susceptible cells and fluorescently-labeled antibiotic-tolerant cells. The cells are ready for downstream assays.

Vidéos Connexes

Read Article