Staphylococcus aureus Floating Biofilm Formation and Phenotype in Synovial Fluid Depends on Albumin, Fibrinogen, and Hyaluronic Acid

Abstract

Biofilms are typically studied in bacterial media that allow the study of important properties such as bacterial growth. However, the results obtained in such media cannot take into account the bacterial localization/clustering caused by bacteria–protein interactions in vivo and the accompanying alterations in phenotype, virulence factor production, and ultimately antibiotic tolerance. We and others have reported that methicillin-resistant or methicillin-susceptible Staphylococcus aureus (MRSA or MSSA, respectively) and other pathogens assemble a proteinaceous matrix in synovial fluid. This proteinaceous bacterial aggregate is coated by a polysaccharide matrix as is characteristic of biofilms. In this study, we identify proteins important for this aggregation and determine the concentration ranges of these proteins that can reproduce bacterial aggregation. We then test this protein combination for its ability to cause marked aggregation, antibacterial tolerance, preservation of morphology, and expression of the phenol-soluble modulin (PSM) virulence factors. In the process, we create a viscous fluid that models bacterial behavior in synovial fluid. We suggest that our findings and, by extension, use of this fluid can help to better model bacterial behavior of new antimicrobial therapies, as well as serve as a starting point to study host protein–bacteria interactions characteristic of physiological fluids.

Host Environment Shapes S. aureus Social Behavior as Revealed by Microscopy Pattern Formation and Dynamic Aggregation Analysis

“…According to this mechanism, small planktonic aggregates would rapidly reach and stick to the surface, while larger aggregates would take longer to reach the surface and attach to it due, for instance, to the “cellular crowd” hindrance in the medium or to its viscosity. This mechanism is partially confirmed by the observation that sessile biofilm grown in BSF presented a similar extracellular matrix phenotype to planktonic aggregates formed above it [ 1 , 13 ]. In other words, we propose that in physiological media, planktonic and sessile multicellular communities are not developed independently but are rather two interlinked phases of the same developmental process.…”

“…Because of their subtle symptomatology and treatment recalcitrance, PJIs are true clinical challenges. Biofilm formation is thought to be a key player of this type of bacterial recalcitrance [ 10 , 12 , 13 , 27 ]. Our previous investigation, based on ex vivo analysis [ 1 ], suggested that staphylococcal behavior is sharply different in physiological fluids as compared with commercial broths, particularly in two aspects: the presence of planktonic aggregates and a considerable phenotypic reorganization of the sessile biofilm.…”

“…In their absence, cells must produce intercellular adhesion factors, whose expression is probably conditioned to extreme environmental stresses, such as starvation [ 32 , 33 ]. On the other hand, staphylococci express an impressive arsenal of surface adhesins, which allows them to scavenge host-derived polymeric molecules and to exploit them as an extracellular matrix [ 1 , 8 , 11 , 13 , 21 ]. Moreover, other mechanisms are known to be involved in bacterial aggregation and biofilm formation, such as the homophilic interactions of surface-expressed proteins [ 34 , 35 ].…”

“…A recent microscopic investigation from our group analyzing infected synovial fluids [ 1 ] confirmed S. aureus as the primary infection agent and corroborated in vitro findings describing its rapid aggregation in floating multicellular aggregates when inoculated in synovial fluids [ 8 , 9 , 10 , 11 , 12 ]. Evidence reported in the literature suggests that such phenotype is induced by host-environment-derived molecules, mainly polymers, and may not involve the expression of biofilm genes during its development [ 13 , 14 , 15 ]. Concomitantly, the appearance of planktonic aggregates has been associated with a marked phenotypic reorganization of the sessile biofilm forming on the surface exposed to the growing medium [ 1 , 8 , 16 ].…”

Rapid aggregation of Staphylococcus aureus in synovial fluid is influenced by synovial fluid concentration, viscosity, and fluid dynamics-with evidence of polymer bridging

“…In recent years, synovial fluid-induced aggregation of Staphylococcus aureus has been heavily investigated as a bacterial survival mechanism in the development of periprosthetic joint infections (PJI). Numerous in vitro studies have provided novel insights into both the composition and pathogenic attributes of staphylococcal aggregates(1),(2),(3),(4),(5). It is now evident that aggregation occurs rapidly upon contact with synovial fluid and confers considerable recalcitrance to antibiotic administration (1),(3),(6)(7).…”