BackgroundStaphylococcus aureuscauses a wide variety of infections, many of which are chronic or relapsing in nature. Antibiotic therapy is often ineffective againstS. aureusbiofilm-mediated infections. Biofilms are difficult to treat partly due to their tolerance to antibiotics, however the underlying mechanism responsible for this remains unknown. One possible explanation is the presence of persister cells—dormant-like cells that exhibit tolerance to antibiotics. Recent studies have shown a connection between afumC(fumarase C, a gene in the tricarboxylic acid cycle) knockout strain and increased survival to antibiotics, antimicrobial peptides, and in aDrosophila melanogastermodel.ObjectiveIt remained unclear whether aS. aureushigh persister strain would have a survival advantage in the presence of innate and adaptive immunity. To further investigate this, afumCknockout and wild type strains were examined in a murine catheter-associated biofilm model.ResultsInterestingly, mice struggled to clear bothS. aureuswild type and thefumCknockout strains. We reasoned both biofilm-mediated infections predominantly consisted of persister cells. To determine the persister cell population within biofilms, expression of a persister cell marker (Pcap5A::dsRED) in a biofilm was examined. Cell sorting of biofilms challenged with antibiotics revealed cells with intermediate and high expression ofcap5Ahad 5.9-and 4.5-fold higher percent survival compared to cells with lowcap5Aexpression. Based on previous findings that persisters are associated with reduced membrane potential, flow cytometry analysis was used to examine the metabolic state of cells within a biofilm. We confirmed cells within biofilms had reduced membrane potential compared to both stationary phase cultures (2.5-fold) and exponential phase cultures (22.4-fold). Supporting these findings, cells within a biofilm still exhibited tolerance to antibiotic challenge following dispersal of the matrix through proteinase K.ConclusionCollectively, these data show that biofilms are largely comprised of persister cells, and this may explain why biofilm infections are often chronic and/or relapsing in clinical settings.