An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells

Take a multi-well plate containing mammalian cell monolayers.

Seed test wells with Herpes simplex virus. Control wells lack the virus.

The virus attaches to cell-surface heparan sulfate and is internalized via endocytosis.

Virus infection triggers heparanase secretion, which cleaves heparan sulfate and unmasks specific fungal receptors.

Co-incubate the cells with Staphylococcus aureus and Candida albicans germ tubes.

In virus-infected cells, heparan sulfate depletion inhibits bacterial interaction while promoting fungal receptor interactions, preventing bacterial-fungal co-localization.

In control cells, bacteria interact with heparan sulfate, while fungi bind to available receptors.

This microbial co-localization initiates adherence — the first step in biofilm formation.

Post-incubation, remove unbound microbes. Overlay with a lysis buffer, releasing bacteria and fungi into the media.

Spread the cell lysate on plates containing growth media. Incubate for colony formation.

A decrease in bacterial colonies with a corresponding increase in fungal colonies indicates virus-induced inhibition of bacterial-fungal co-localization and biofilm initiation.

To carry out a polymicrobial biofilm assay, seed 96-well plates with 200 microliters of 2 times 10 to the 5th HeLa cells per milliliter. Rock the plates at 37 degrees Celsius for 30 to 45 minutes, before incubating in 5% carbon dioxide at 37 degrees Celsius for 18 hours. Use 1x optimum to wash the monolayers. Then, seed with a single strain of HSV. Test only one strain at a time.

Incubate the plates at 37 degrees Celsius in 5% carbon dioxide for three hours. Using 100 microliters of 37 degrees Celsius PBS with magnesium and calcium, wash the infected monolayers. Then, use 37 degrees Celsius HBSS to replace the PBS. Leave 25 microliters of the HBSS in each well.

Next, add GT, and/or S. aureus working suspensions, and incubate the plates at 37 degrees Celsius in 5% carbon dioxide for 30 minutes. Following incubation, aspirate one column of the plate at a time. Immediately refill the wells using 300 microliters of PBS with magnesium and calcium.

After repeating this wash step twice, add 200 microliters at a 1 to 50 dilution of filter-sterilized RIPA lysis buffer to each well.

When working with other strains of bacteria or fungi, make sure that the concentration of RIPA used is sufficient for cell lysis, but still permissive for microbial viability testing.

Rapidly triturate the HeLa cell lysate, and pipette 50 microliters onto mannitol salts and/or fungicidal medium. Then, using a glass rod bent at a 90-degree angle, spread the lysate over the surface of the plate.

After incubating the plates for 18 hours, manually count the number of colonies per plate. Controls consist of S. aureus and/or C. albicans adherence to HSV-uninfected HeLa cells.