Identification and Characterization of Planktonic Biofilm-Like Aggregates in Infected Synovial Fluids From Joint Infections

Abstract

Recent in vitro studies reported the exceptional ability of some bacterial species to form biofilm-like aggregates in human and animal synovial fluids (SF), but evidences from infected clinical samples are still lacking. In this study, we investigated whether this bacterial phenotype was present in infected SFs collected from joint infections and if it was maintained in in vitro settings. SFs sent for culture to the Laboratory of Microbiology of our institute were directly analyzed by means of confocal laser scanning microscopy (CLSM), and the infective agents were isolated for further in vitro tests. Moreover, sterile SF was collected from patients who did not receive previous antibiotic therapy to investigate the formation of bacterial aggregates, together with biofilm and matrix production on a titanium surface. Finally, antibiotic susceptibility studies were performed by using bovine SF. Four Staphylococcus aureus, one Staphylococcus lugdunensis, and one Prevotella bivia strain were identified in the infected SFs. The CLSM analysis showed that all staphylococci were present as a mixture of single cells and bacterial clumps surrounded by an exopolymeric substance, which comprised SF-derived fibrin, while all P. bivia cells appeared separated. Despite that, differences in the ability to aggregate between S. aureus and S. lugdunensis were observed in clinical SFs. These different phenotypes were further confirmed by in vitro growth, even though the application of such ex vivo approach lead all staphylococci to form exceptionally large microbial aggregates, which are several folds bigger than those observed in clinical samples. Planktonic aggregates challenged for antibiotic susceptibility revealed a sharp increase of recalcitrance to the treatments. Although this is still at a preliminary stage, the present work confirmed the ability of staphylococci to form free-floating biofilm-like aggregates in infected SF from patients with joint infections. Furthermore, the obtained results pointed out that future in vitro research on joint infections will benefit from the use of human-or animal-derived SF. Even though this approach should be carefully validated in further studies comprising a larger microbial population, these findings pose new challenges in the treatment of infected native and prosthetic joints and for the approach to new investigations.

Bacterial adhesion on spinal implants: An in vitro study of “hot spots”

“…On the contrary, neutrophils are unable to phagocytize surface attached microcolonies exceeding 10 µm of diameter 22 and studies reported that mature biofilms exposed to subinhibitory concentrations on antibiotics were rapidly induced to increase the biomass, the antibiotic resistance, and expression of extracellular polysaccharide. 23,24 While in vitro studies conducted in the presence of synovial fluids revealed an altered biofilm formation on the underlying titanium surfaces, probably induced by host proteins, 25,26 preliminary studies in our lab does not support the same for serum (data not shown), which, on the contrary, does not promote sessile growth as much as synovial fluids or lab medium. However, this protocol was designed to easily map biofilm aggregates attached to those surfaces more susceptible to bacterial colonization.…”

A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation

“…Importantly, these biofilms are not found attached to any biotic or abiotic surfaces but are found as free-floating clusters. Free-floating aggregates of S. aureus with biofilm-like characteristics, including heightened antibiotic resistance and the presence of an exopolymeric matrix, have been isolated from synovial fluid of joint infections ( 10 ), chronic wound infections ( 35 ), and mucosal secretions in cystic fibrosis infections ( 36 ). Clearly, the traditional in vitro models of surface-attached biofilms will not be appropriate for understanding CA-MRSA chronic infections in these clinical scenarios.…”

“…However, infrequent but careful studies in the past 15 to 20 years have reported that in vivo biofilms considerably depart from the in vitro canonical surface-attached model in that in certain clinical settings, these biofilms are found as unattached small aggregates (50 to 100 μm) and typically dispersed in mucus or compromised soft tissue ( 5 , 6 ). Aggregates of Pseudomonas aeruginosa , Staphylococcus aureus , Escherichia coli , Staphylococcus lugdunensis , and Mycobacterium abscessus with increased tolerance to antibiotics and embedded in a biofilm matrix have been isolated from the lungs of cystic fibrosis patients; chronic wounds; and middle ear, infectious arthritis, and periprosthetic joint infections ( 7 – 10 ). These aggregates of cells show active group behavior rather than just representing a passive collection of individual cells: they demonstrate phenotypic attributes of biofilms, including the presence of an encasing matrix, antibiotic tolerance, and host immune evasion.…”

Free-Floating Aggregate and Single-Cell-Initiated Biofilms of Staphylococcus aureus

“…S. aureus forming free-floating aggregates in human and bovine SF and growing to a macroscopic size in 24 h have been reported in many in vitro studies [ 12 , 17 , 18 , 19 , 21 , 34 ]. The formation of aggregates provides the bacteria with enhanced tolerance to antibiotics and promotes surface colonization and biofilm formation [ 35 , 36 ]. Therefore, we hypothesized that the aggregates that form in SF provide early protection to bacteria entering the surgical site, allowing them time to attach to the implant surface, leading to biofilm formation.…”