Abstract
Staphylococcus aureus is the main cause of osteomyelitis and forms biofilm and staphylococcal abscess communities (SACs) in humans. While S. aureus has several toxins with specificity for human targets and working with human host cells would be preferred, for SACs no in vitro models, two-dimensional (2D) or three-dimensional (3D), have been described in literature to date. Advanced 3D in vitro cell culture models enable the incorporation of human cells and resemble in vivo tissue more closely than conventional 2D cell culture. Therefore, the aim of this study was to develop an in vitro model of SACs by using a 3D system. The model should allow for studies into antibiotic tolerance and S. aureus - human host cells interactions.
With a clinical isolate (S. aureus JAR) or a lab strain (S. aureus ATCC 49230-GFP), SACs were grown in a collagen gel (1.78 mg/ml, Gibco) supplemented with 200 µl human plasma at 37 °C. Transmission and scanning electron microscopy was used to obtain a detailed overview of SACs, whereas immunofluorescent stainings were done to determine whether the pseudocapsule around SACs consist of fibrin. Antibiotic tolerance of SACs was assessed with 100× the minimal inhibitory concentration (MIC) of gentamicin (Roth). Bacterial clearance of non-establised SACs and established SACs with or without pseudocapsule was determined by exposure to differentiated PLB neutrophil-like cells (differentiation with 1.25% DMSO and 5% FBS for 5 days; dPLB) or primary neutrophils isolated with lymphoprep from fresh heparin blood. Degradation of the pseudocapsule was done with 7.5 µl/ml plasmin (Sigma). Colony forming unit (CFU) counts were performed as quantification method. Statistical analysis was performed with the ANOVA multiple comparison test or, when data was not normally distributed, with a Mann-Whitney U test.
We have developed a 3D in vitro model of SACs which after overnight growth were on average 200 micrometers in diameter, consisted of 8 log10 CFUs and were surrounded by an inner and outer fibrin pseudocapsule. The in vitro grown SACs tolerated 100× the MIC of gentamicin for 24h and did not significantly differ from control SACs (p=0.1000). dPLB neutrophil-like cells or primary neutrophils did not clear established in vitro SACs (p=0.1102 and p=0.8767, respectively). When the fibrin pseudocapsule was degraded by the enzyme plasmin, dPLB neutrophil-like cells or primary neutrophils caused for a significant decrease in total CFU compared the SACs that did had a pseudocapsule (p=0.0333 and p=0.0272, respectively).
The in vitro SACs model offers a tool for host-pathogen interaction and drug efficacy assessments and is a valuable starting point for future research.
