Staphylococcus aureus is a prominent pathogen in bone and soft tissue infections where pathophysiology involves abscess formation with staphylococcal abscess communities (SACs). SACs are populations of S. aureus within a fibrin pseudocapsule, which appears to protect the SAC from ingress of immune cells such as neutrophils.
To facilitate the study of SAC interaction with human cells and/or antibiotics in an in vivo-like setting, we developed a 3D in vitro SAC model grown in a human plasma-supplemented collagen gel. The in vitro SACs consisted of on average 8 log10 colony forming units (CFUs) by 24 h and were observed to elaborate a fibrin pseudocapsule by electron and immunofluorescence microscopy.
To determine antibiotic tolerance of the in vitro SACs, bacteria were treated with gentamicin alone, or in combination with rifampicin. The in vitro SACs tolerated 100x the minimal inhibitory concentration (MIC) of gentamicin alone and in combination with rifampicin for 24 h and CFUs were comparable to non-treated SACs (p = 0.0992 and p = 0.0596, respectively), whilst 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 after 24 h of growth. dPLB cells and primary neutrophils were unable to clear mature in vitro SACs and CFUs were comparable the initial CFUs upon neutrophil addition (p = 0.1102 and p = 0.1711, respectively). Plasmin pre-treatment for 2 h removed the pseudocapsule and subsequent dPLB cells and primary neutrophils exposure for 24 h resulted in a significant decrease in CFU within the SACs compared to non-plasmin pre-treated SACs (p = 0.0333 and p = 0.0272, respectively).
The in vitro SAC model mimicked in vivo features and revealed that the fibrin pseudocapsule around SACs offers protection against antibiotics and neutrophil clearance.