Assessing Intracellular Bacterial Persistence and Antibiotic Resistance in a Mouse Model

Take two groups of euthanized mice with exposed abdominal cavities.

The control group is pre-injected with macrophages infected with methicillin-resistant S. aureus.

The treated group is pre-injected with macrophages carrying the same bacteria and the antibiotic gentamicin.

In both groups, bacteria persist, replicate, and evade host immune responses inside macrophage phagosomes. In the treated group, bacteria also resist the antibiotics and remain viable.

Locate the kidneys, which contain the infected macrophages. Carefully detach the kidneys from the surrounding tissue.

Place each kidney in grinding tubes with buffer to preserve bacterial viability.

Homogenize the tissue to lyse macrophages and release intracellular bacteria.

Transfer the homogenate to tubes and prepare serial dilutions in buffer.

Spot each dilution onto nutrient-rich agar plates.

Incubate until visible colony-forming units (CFUs) develop.

Similar CFUs in both groups indicate gentamicin resistance and intracellular persistence of methicillin-resistant S. aureus within the host.

Place the mouse under an infrared physiotherapy lamp until the tail vein is dilated. Randomly divide the mice into four groups. Intravenously inject three times ten to the power of six CFU Planktonic MRSA252 into the tail vein of the mouse.

After 24 hours, sacrifice the anesthetized mouse and disinfect it thoroughly with 75% ethyl alcohol. Use tweezers with one hand to lift the abdominal skin and with the other hand, cut the skin using ophthalmic scissors. Locate the kidneys in the abdominal cavity and carefully strip them completely.

Transfer the kidneys into a grinding tube containing 1 milliliter of PBS, and grind them until no solid tissue remains. Pour the homogenized tissue into the Eppendorf tube. Perform serial dilutions of the tissue homogenate using PBS.

Spot each dilution onto separate Tryptic Soy Agar plates and incubate them overnight at 37 degrees Celsius. Count the bacterial colonies the next day and analyze the data.