Dynamic interactions between cephalexin and macrophages on different Staphylococcus aureus inoculum sizes: a tripartite in vitro model

The bactericidal activity of an antimicrobial drug is generally assessed by in vitro bacterial time-kill experiments which do not include any components of the immune system, even though the innate immunity, the primary host defence, is probably able to kill a large proportion of pathogenic bacteria in immunocompetent patients. We developed an in vitro tripartite model to investigate the joint action of C57Bl/6 murine bone-marrow-derived macrophages and cephalexin on the killing of Staphylococcus aureus. By assessing the bactericidal effects on four bacterial inoculum sizes, we showed that macrophages can cooperate with cephalexin on inoculum sizes lower than 10 CFU/mL and conversely, protect S. aureus from cephalexin killing activity at the highest inoculum size. Cell analysis by flow cytometry revealed that macrophages were rapidly overwhelmed when exposed to large inoculums. Increasing the initial inoculum size from 10 to 10 CFU/mL increased macrophage death and decreased their ability to kill bacteria from six hours after exposure to bacteria. The addition of cephalexin at 16-fold MIC to 10 and 10 CFU/mL inoculums allowed the macrophages to survive and to maintain their bactericidal activity as if they were exposed to a small bacterial inoculum. However, with the highest inoculum size of 10 CFU/mL, the final bacterial counts in the supernatant were higher with macrophages plus cephalexin than with cephalexin alone. These results suggest that if the bacterial population at the infectious site is low, as potentially encountered in the early stage of infection or at the end of an antimicrobial treatment, the observed cooperation between macrophages and cephalexin could facilitate its control.