Detecting Bacterial Virulence Using Amoeba as a Host System

Begin by co-culturing amoebae with mutant strains derived from a single virulent bacterial isolate.

The amoeba engulfs the bacteria, mimicking the phagocytic activity of host immune cells.

Discard the medium and wash with fresh medium to remove extracellular bacteria.

Add a medium containing antibiotics and incubate it to kill the remaining extracellular bacteria.

Wash with fresh medium, then add medium supplemented with antibiotics. Next, add heat-killed feeder bacteria, then incubate.

The amoeba engulfs the feeder bacteria for nourishment.

During incubation, virulent mutant strains survive and divide within the amoeba.

Next, add a detergent solution and incubate to lyse the amoeba cell, releasing intracellular bacteria.

Spot the lysate onto agar medium, dilute with sterile water, and incubate.

Colonies derived from mutant bacterial strains appear on the agar surface.

Variation in colony counts among mutant bacterial strains indicates differential virulence in intracellular survival and replication within the amoeba.

Begin by culturing amoeba Acanthamoeba castellani, or AC, in 30 milliliters of Peptone yeast Glucose medium in 75 square centimeter tissue culture flasks at room temperature. After one hour, use a 25 milliliter pipette to remove the supernatant, and wash the cells three times with 10 milliliters of AC buffer without carbon or nitrogen per wash. After the last wash, detach the amoeba from the bottom of the flask with a single vigorous shake, and adjust the cells in a Falcon tube to a five times 10 to the five amoebae per mil concentration in fresh AC buffer.

Seed five times 10 to the four amoebae per well in a 96-well plate and place the plate at 32 degrees Celsius for one hour without shaking, to allow the floating amoeba trophozoites to adhere to the wells. Meanwhile, warm the bacteria on ice, and add five microliters of the thawed bacteria to each well at the end of the incubation.

After 1.5 hours at 32 degrees celsius, discard the supernatant by inverting the plate onto a stack of sterile gauze in a sterilized metal tray under a fume hood and wash each well three times with 200 microliters of fresh AC medium per well. After the last wash, incubate the co-culture with 20 microliters of fresh medium supplemented with amikacin per well, to eliminate any remaining extracellular mycobacteria for two hours.

Followed by three washes in fresh AC medium, as just demonstrated. Then, add 200 microliters of medium, supplemented with fresh amikacin to each well, followed by the addition of five times ten to the seven heat inactivated Escherichia coli bacteria. After 48 hours, lyse the cells with 10 microliters of 10% sodium dodecyl sulfate per well, for 30 minutes at 32 degrees Celsius, and spread five microliters of the lysate on Columbia Agar plates containing 5% sheep blood.

Further dilute the lysates by adding 25 microliters of sterile water on each drop. When all of the lysates have been plated, seal the plates with plastic paraffin film, and incubate the cultures for three to four days at 37 degrees Celsius. When colonies appear on the plates, photograph the cultures and identify the mutants impaired for intracellular growth by the deposits with the lowest number of bacterial colonies.