In Vitro Staphylococcal Aggregate Morphology and Protection from Antibiotics Are Dependent on Distinct Mechanisms Arising from Postsurgical Joint Components and Fluid Motion

The formation of a bacterial biofilm in the postsurgical joint environment significantly complicates the resolution of an infection. To form a resilient biofilm, incoming bacteria must first survive the initial invasion of the joint space. Considerable progress has been made toward elucidating the mechanism of Staphylococcus aureus aggregation in synovial fluid. In this study, aggregate morphology was assessed following incubation under several simulated postsurgical joint conditions. Using fluorescently labeled synovial fluid polymers, we show that aggregation occurs through two distinct mechanisms: (i) direct bridging between S. aureus cells and host fibrinogen and (ii) an entropy-driven depletion mechanism facilitated by hyaluronic acid and albumin. By screening surface adhesin-deficient mutants (clfA, clfB, fnbB, and fnbA), we identified the primary genetic determinant of aggregation in synovial fluid to be clumping factor A. To characterize this bridging interaction, we employed an atomic force microscopy-based approach to quantify the binding affinity of either wild-type S. aureus or the adhesin mutant to immobilized fibrinogen. Surprisingly, we found there to be cell-to-cell variability in the binding strength of the bacteria for immobilized fibrinogen. Superhigh-resolution microscopy imaging revealed that fibrinogen binding to the cell wall is heterogeneously distributed at both the single cell and population levels. Finally, we assessed the antibiotic tolerance of various aggregate morphologies arising from newly deciphered mechanisms of polymer-mediated synovial fluid-induced aggregation. The formation of macroscopic aggregates under shear was highly tolerant of gentamicin, while smaller aggregates, formed under static conditions, were susceptible. We hypothesize that aggregate formation in the joint cavity, in combination with shear, is mediated by both polymer-mediated aggregation mechanisms, with depletion forces enhancing the stability of essential bridging interactions.IMPORTANCE The formation of a bacterial biofilm in the postsurgical joint environment significantly complicates the resolution of an infection. To form a resilient biofilm, incoming bacteria must first survive the initial invasion of the joint space. We previously found that synovial fluid induces the formation of Staphylococcus aureus aggregates, which may provide rapid protection during the early stages of infection. The state of the host joint environment, including the presence of fluid flow and fluctuating abundance of synovial fluid polymers, determines the rate and size of aggregate formation. By expanding on our knowledge of the mechanism and pathogenic implications of synovial fluid-induced aggregation, we hope to contribute insights for the development of novel methods of prevention and therapeutic intervention.

Automated summary

The formation of a bacterial biofilm in the postsurgical joint environment significantly complicates the resolution of an infection. This study investigated the mechanisms of Staphylococcus aureus aggregation in synovial fluid and identified clumping factor A as the primary genetic determinant of aggregation. The study also found that the size and antibiotic tolerance of the aggregates formed under different conditions varied.