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.