Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

The enzyme protection assay enable to study the extent of Staph aureus internalization and its ability to survive inside adherent cells, as well as the efficacy of antimicrobial compound. The improved enzyme protection assay greatly simplifies technical handling, and enable to recover internalized bacteria from weakly-adherent cells and cells in suspension. Demonstrating the procedure will be Josselin Rigaill, medical doctor and PhD student from my laboratory.

Two days before the infection assay, seed A549 epithelial cells at a density of 2.0 times 10 to the fifth cells per milliliter per well of a 24-well plate and incubate the cells for 48 hours at 36 degrees Celsius in 5%carbon dioxide, until they reach 100%confluence. On the day of the assay, remove and discard the spent culture medium from the three wells dedicated for counting the A549 cells, and add one milliliter of complete infection medium containing five micrograms per milliliter of Hoechst 33342 and one microgram per milliliter of propidium iodide. Incubate the cells for 30 minutes, then, using a wide-field fluorescence microscope, count the number of cells and calculate the cell viability.

To prepare the bacterial suspension, dispense 25 milliliters of complete infection medium in a tube, and prewarm it at 36 degrees Celsius. Next, adjust the Staphylococcus aureus suspension to an OD of 0.5 in a complete infection medium using a cell density meter, then prepare 20 milliliters of bacterial suspension for cell inoculation by diluting the culture in a complete infection medium to achieve an MOI of one according to the number of cells per well. Next, using an automatic spiral plater, determine the Staphylococcus aureus load of the diluted bacterial suspension to be used for the cell inoculation step, and incubate the agar plates for 18 to 24 hours at 36 degrees Celsius.

The next day, count the number of colonies with a colony counter to calculate the accurate MOI for each strain tested. Before cell inoculation, observe every well of the 24-well plate by low magnification microscopy to ensure that the cells are healthy and growing as expected, then remove and discard the spent cell culture medium from the 24-well plate and add 500 microliters of the bacterial suspension to each well containing 100%confluent cells. Incubate the cells for two hours at 36 degrees Celsius in 5%carbon dioxide.

To quantify the intracellular bacteria with the improved enzyme protection assay, prepare 4x lysis buffer, 2%Triton X-100, trypsin-EDTA, and lysostaphin stock in working solutions as mentioned in the text manuscript. Next, prepare 6.25 milliliters of complete infection medium supplemented with lysostaphin by adding six milliliters of complete infection medium to 250 microliters of the lysostaphin working solution. Then, add 250 microliters of the complete infection medium supplemented with lysostaphin into each well and gently agitate the plate by swiveling the plate by hand.

To allow the lysostaphin to kill the extracellular bacteria, incubate the cells for one hour at 36 degrees Celsius in 5%carbon dioxide, then inactivate the lysostaphin by adding 10 microliters of proteinase K at 20 micrograms per milliliter into each well and incubating the cells for two minutes at room temperature. Next, add 250 microliters of 4x lysis buffer to lyse the cells by osmotic shock, and incubate the cells for 10 minutes at 36 degrees Celsius. After the incubation, pipette the cell lysate up and down several times to ensure that the cells are fully lysed and homogenized, then use an automatic spiral plater to determine the Staphylococcus aureus load of each well and incubate the agar plates for 18 to 24 hours at 36 degrees Celsius.

The next day, count the number of colonies with a colony counter to calculate the intracellular Staphylococcus aureus load of each well. The internalization of two strains of Staphylococcus aureus by A549 epithelial cells is depicted here. Using the enzyme protection assay that includes washes to remove lysostaphin, the mean intracellular loads were 4.46 and 0.49 log cfu per milliliter for the SF8300 wild type and the isogenic mutant lacking the fnbA and B genes, respectively.

Using the improved enzyme protection assay that uses proteinase K to inactivate lysostaphin, the loads were 4.53 and 0.56 log cfu per milliliter, respectively. Intracellular activity of vancomycin, rifampicin, and levofloxacin against Staphylococcus aureus measured using enzyme protection assay is depicted here. The mean intracellular loads were 4.57 log cfu per milliliter for control, 4.51 for vancomycin, 3.03 for rifampicin and 2.91 for levofloxacin.

Intensive washes to remove the enzyme tend to detach the most infected cells, which can give inaccurate results and support the use of the improved protocol. The improved enzyme protection assay make it easier to study Staph aureus internalization with organoid, and can be easily adapted on high content screening system that's used by putting