Derek E. Moormeier and Kenneth W. Bayles, University of Nebraska Medical Center, Omaha, NE
Staphylococcus aureus biofilm growth is known to potentiate pathogenesis of infective endocarditis and medical implanted-related infections by conferring resistance to the host immune response and antibiotic therapy. Previously, the S. aureus cidABC and lrgAB operons were shown to play significant roles in mediating cell death and the release of extracellular DNA (eDNA) during biofilm maturation. Although the exact mechanisms regulating these operons are still being defined, it appears the expression of these operons is mediated by metabolic alterations in overflow metabolism (i.e. excess glucose and hypoxic conditions). Due to the regulatory roles of the cid and lrg operons in cell death, we hypothesized that only a subpopulation of cells would express these operons during biofilm development. Therefore, we examined gene expression using single and dual transcriptional reporter fusion constructs and correlated expression to cell death/eDNA release via staining with propidium iodide (PI). To monitor expression and staining in real-time, we utilized the BioFlux1000 automated shear flow system to acquire serial epifluorescent images over 18 hr of biofilm.