Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria

1. Cell inoculation

1. Begin with a culture of A549 human epithelial cells in Dulbecco's modified Eagle medium (DMEM) high glucose with phenol red, supplemented with 10% fetal bovine serum (FBS) without antibiotics.
2. Observe every well of the 24-well plate by low magnification microscopy to ensure that the human epithelial cells are healthy and growing as expected.
3. Remove and discard the spent cell culture medium from the 24-well plate.
4. Add 500 µL of the bacterial suspension of Staphylococcus aureus prepared in DMEM without phenol red, for inoculation to each well with 100% confluent cells.
5. Incubate the cells for 2 h at 36 ± 1 °C and 5% CO₂.
   NOTE: it is recommended to use three wells of the plate for each condition to be tested (triplicate) and to perform at least three independent experiments. The delay of incubation can be adapted according to the experimental aim.

2. Quantification of intracellular bacteria with improved enzyme protection assay (iEPA)

1. Prepare 7 mL of 4x lysis buffer with 3.5 mL of 2% Triton X-100 in sterile water and 3.5 mL of trypsin-EDTA.
2. Prepare a lysostaphin stock solution at 10 mg/mL in acetate buffer and aliquot 25 µL into cryovials. Store at -80 °C for up to 6 months.
3. Prepare 250 µL of a fresh lysostaphin working solution at 1 mg/mL by mixing 25 µL of the lysostaphin stock solution (10 mg/mL) and 225 µL of 0.1 M Tris-HCl. Store at 4 °C for up to 48 h.
4. Prepare 6.25 mL of complete infection medium supplemented with lysostaphin by adding 6 mL of complete infection medium to 250 µL of the lysostaphin working solution.
5. Add 250 µL of complete infection medium supplemented with lysostaphin into each well and gently agitate the plate by swivelling the plate by hand.
6. Incubate the cells for 1 h at 36 ± 1 °C in 5% CO₂ to let the lysostaphin kill the extracellular bacteria.
7. At the end of the incubation time, add 10 µL of proteinase K at 20 mg/mL into each well to inactivate the lysostaphin.
8. Incubate the cells for 2 min at room temperature.
9. Add 250 µL of 4x lysis buffer to lyse the cells by osmotic shock.
10. Incubate the cells for 10 min at 36 ± 1 °C.
11. Mix thoroughly by pipetting up and down ten times all over the bottom of the well to ensure that the cells are fully lysed and homogenized.
12. Use an automatic spiral plater to determine the S. aureus load of each well.
13. Incubate the agar plates for 18-24 h at 36 ± 1 °C.
14. The next day, count the number of colonies with a colony counter to calculate the intracellular S. aureus load of each well.