An In Vitro Technique to Study Antimicrobial Treatments on Bacterial Aggregates

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.