Increased survival of Staphylococcus aureus co-cultured with Gram-negative pathogens against antibiotics

Polymicrobial infections are known to be associated with increased infection severity and poor clinical outcomes. In this study, we investigated the effect of co-infection with Gram-negative bacteria on the survival of Staphylococcus aureus. In vitro time-kill assays were performed on S. aureus monoculture and co-cultures with three Gram-negative bacteria, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, treated with ciprofloxacin and vancomycin. Cultures of Gram-negative bacteria were separated into cell pellets and supernatant and the survival of S. aureus was measured in each fraction. The effect of cell density of Gram-negative bacteria on the survival of S. aureus was also investigated. Intracellular ATP concentration and expression of rsh were examined in surviving S. aureus cells from the in vitro time-kill assays. The survival of S. aureus against ciprofloxacin and vancomycin increased when co-cultured with the Gram-negative bacterial species. Enhanced survival was observed only in co-cultures containing cell pellets, not in cell-free culture medium. With increased cell density of Gram-negative bacteria, the survival of S. aureus tended to increase. Low intracellular ATP concentrations and increased rsh expression were detected when S. aureus was co-cultured with A. baumannii. S. aureus showed increased survival against antibiotics in co-culture with several Gram-negative bacterial pathogens compared to monoculture, which was not attributable to their secreted factors. Increased antibiotic persistence in polymicrobial infections may be a significant cause of antibiotic treatment failure.