Abstract
Understanding host pathogen interactions is paramount to the development of novel antimicrobials. An important facet of this pursuit is the accurate characterization of pathogen replication within infected host cells. Here we describe the use of a fluorescence-based proliferation assay to identify intracellular populations of replicating bacteria at the subcellular level. Using Staphylococcus aureus as a model Gram-positive bacterial pathogen and macrophages as a model host phagocyte, we demonstrate this assay can be used to reliably identify individual phagocytes that contain replicating bacteria. Furthermore, we demonstrate this assay is compatible with additional cellular probes that enable characterization of cellular compartments in which replicating bacteria reside. Finally, we demonstrate that this assay facilitates the investigation of both Gram-negative and Gram-positive bacteria within host cells.