A Rapid Image-Based Assay for Screening Surface-Induced Bacterial Virulence

Begin with plates containing surface-attached bacteria and planktonic or free-floating bacteria.

Add amoeba cells to both samples.

Place an agarose pad containing Calcein-AM, a cell-permeable dye, over each sample.

Remove excess liquid, allow to dry, and incubate the plates.

In the plate with surface-attached bacteria, surface contact upregulates virulence genes, expressing toxins.

These toxins disrupt the amoeba membrane and change their shape.

Damaged amoeba membrane allows more calcein-AM to enter the cytoplasm, where intracellular esterases cleave them into fluorescent calcein, leading to its accumulation.

In contrast, planktonic bacteria, lacking surface contact, remain non-virulent, leaving amoeba membranes intact. This reduces Calcein-AM entry into the amoeba, limiting fluorescent calcein accumulation.

Healthy amoeba also engulf the non-virulent bacteria.

Under a fluorescent microscope, bright, rounded amoeba cells indicate virulent bacterial interaction, while dim, amorphous amoeba indicate nonvirulent bacterial interaction.

To assay virulence of surface attached bacteria cells, add 10 microliters of amoeba cells from the previously obtained surface-attached bacterial cells.

To isolate the planktonic cells, transfer 10 microliters of the culture to a fresh petri dish.

Next, assay the virulence of planktonic cells by mixing 10 microliters of amoeba cells with the previously obtained planktonic bacteria.

Immediately place the small agar pad on top of the appropriate bacteria amoeba mixture on the petri dish surface.

Then, remove excess liquid by gently pipetting any excess liquid, and allow the dish to dry for 20 minutes.

Cover the dish with the lid and incubate it at room temperature for an additional 40 minutes. Finally, use a fluorescence microscope to acquire images for at least 100 amoeba cells.