Three-Dimensional <i>In Vitro</i> Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

Abstract

<i>Staphylococcus aureus</i> is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of <i>in vitro</i> models. We describe a three-dimensional <i>in vitro</i> model of SACs grown in a human plasma-supplemented collagen gel. The <i>in vitro</i> SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The <i>in vitro</i> SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature <i>in vitro</i> SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The <i>in vitro</i> SAC model mimics key <i>in vivo</i> features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the <i>S. aureus</i> pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.