Accurate and semi-automated analysis of bacterial association with mammalian cells

• Bacterial interaction with mammalian cells can be measured with the gentamicin protection assay.
• This assay can be time consuming and is not lent to high throughput.
• An inducible GFP report construct was generated.
• In concert with antibody labelling this permits differential detection of bacteria.
• In a high content assay this permits efficient enumeration of invasive bacteria.

To efficiently and accurately quantify the interactions of bacteria with mammalian cells, a reliable fluorescence microscopy assay was developed. Bacteria were engineered to become rapidly and stably fluorescent using Green Fluorescent Protein (GFP) expressed from an inducible Tet promoter. Upon application of the fluorescent bacteria onto a monolayer, extracellular bacteria could be discriminated from intracellular bacteria by antibody staining and microscopy. All bacteria could be detected by GFP expression. External bacteria stained orange, whereas internalised bacteria did not. Internalised bacteria could thus be discriminated from external bacteria by virtue of being green but not orange fluorescent. Image acquisition and counting of various fluorophore-stained entities were accomplished with a high-content screening platform. This allowed for semi-automated and accurate counting of intracellular and extracellular bacteria.

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