Staphylococcus aureus Floating Biofilm Formation and Phenotype in Synovial Fluid Depends on Albumin, Fibrinogen, and Hyaluronic Acid.
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ABSTRACT:
SUBMITTER: Knott S
PROVIDER: S-EPMC8117011 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Rapid synovial fluid-induced aggregation of Staphylococcus aureus is currently being investigated as an important factor in the establishment of periprosthetic joint infections (PJIs). Pathogenic advantages of aggregate formation have been well documented in vitro, including recalcitrance to antibiotics and protection from host immune defenses. The objective of the present work was to determine the strain dependency of synovial fluid-induced aggregation by measuring the degree of aggregation of 21 clinical S. aureus isolates cultured from either PJI or bloodstream infections using imaging and flow cytometry. Furthermore, by measuring attached bacterial biomass using a conventional crystal violet assay, we assessed whether there is a correlation between the aggregative phenotype and surface-associated biofilm formation. While all of the isolates were stimulated to aggregate upon exposure to bovine synovial fluid (BSF) and human serum (HS), the extent of aggregation was highly variable between individual strains. Interestingly, the PJI isolates aggregated significantly more upon BSF exposure than those isolated from bloodstream infections. While we were able to stimulate biofilm formation with all of the isolates in growth medium, supplementation with either synovial fluid or human serum inhibited bacterial surface attachment over a 24 h incubation. Surprisingly, there was no correlation between the degree of synovial fluid-induced aggregation and quantity of surface-associated biofilm as measured by a conventional biofilm assay without host fluid supplementation. Taken together, our findings suggest that synovial fluid-induced aggregation appears to be widespread among S. aureus strains and mechanistically independent of biofilm formation. IMPORTANCE Bacterial infections of hip and knee implants are rare but devastating complications of orthopedic surgery. Despite a widespread appreciation of the considerable financial, physical, and emotional burden associated with the development of a prosthetic joint infection, the establishment of bacteria in the synovial joint remains poorly understood. It has been shown that immediately upon exposure to synovial fluid, the viscous fluid in the joint, Staphylococcus aureus rapidly forms aggregates which are resistant to antibiotics and host immune cell clearance. The bacterial virulence associated with aggregate formation is likely a step in the establishment of prosthetic joint infection, and as such, it has the potential to be a potent target of prevention. We hope that this work contributes to the future development of therapeutics targeting synovial fluid-induced aggregation to better prevent and treat these infections.
| S-EPMC8557890 | biostudies-literature
Project description:Periprosthetic joint infections (PJIs) are a devastating complication that occurs in 2% of patients following joint replacement. These infections are costly and difficult to treat, often requiring multiple corrective surgeries and prolonged antimicrobial treatments. The Gram-positive bacterium Staphylococcus aureus is one of the most common causes of PJIs, and it is often resistant to a number of commonly used antimicrobials. This tolerance can be partially attributed to the ability of S. aureus to form biofilms. Biofilms associated with the surface of indwelling medical devices have been observed on components removed during chronic infection, however, the development and localization of biofilms during PJIs remains unclear. Prior studies have demonstrated that synovial fluid, in the joint cavity, promotes the development of bacterial aggregates with many biofilm-like properties, including antibiotic resistance. We anticipate these aggregates have an important role in biofilm formation and antibiotic tolerance during PJIs. Therefore, we sought to determine specifically how synovial fluid promotes aggregate formation and the impact of this process on surface attachment. Using flow cytometry and microscopy, we quantified the aggregation of various clinical S. aureus strains following exposure to purified synovial fluid components. We determined that fibrinogen and fibronectin promoted bacterial aggregation, while cell free DNA, serum albumin, and hyaluronic acid had minimal effect. To determine how synovial fluid mediated aggregation affects surface attachment, we utilized microscopy to measure bacterial attachment. Surprisingly, we found that synovial fluid significantly impeded bacterial surface attachment to a variety of materials. We conclude from this study that fibrinogen and fibronectin in synovial fluid have a crucial role in promoting bacterial aggregation and inhibiting surface adhesion during PJI. Collectively, we propose that synovial fluid may have conflicting protective roles for the host by preventing adhesion to surfaces, but by promoting bacterial aggregation is also contributing to the development of antibiotic tolerance.
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Project description:S. aureus is a pathogen that frequently causes severe morbidity and phage therapy is being discussed as an alternative to antibiotics for the treatment of S. aureus infections. In this in vitro and animal study, we demonstrated that the activity of anti-staphylococcal phages is severely impaired in 0.5% plasma or synovial fluid. Despite phage replication in these matrices, lysis of the bacteria was slower than phage propagation, and no reduction of the bacterial population was observed. The inhibition of the phages associated with a reduction in phage adsorption, quantified to 99% at 10% plasma. S. aureus is known to bind multiple coagulation factors, resulting in the formation of aggregates and blood clots that might protect the bacterium from the phages. Here, we show that purified fibrinogen at a sub-physiological concentration of 0.4 mg/ml is sufficient to impair phage activity. In contrast, dissolution of the clots by tissue plasminogen activator (tPA) partially restored phage activity. Consistent with these in vitro findings, phage treatment did not reduce bacterial burdens in a neutropenic mouse S. aureus thigh infection model. In summary, phage treatment of S. aureus infections inside the body may be fundamentally challenging, and more investigation is needed prior to proceeding to in-human trials.
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Project description:Spondyloarthritis (SpA) patients suffer from joint inflammation resulting in tissue damage, characterized by the presence of numerous neutrophils in the synovium and synovial fluid (SF). As it is yet unclear to what extent neutrophils contribute to the pathogenesis of SpA, we set out to study SF neutrophils in more detail. We analyzed the functionality of SF neutrophils of 20 SpA patients and 7 disease controls, determining ROS production and degranulation in response to various stimuli. In addition, the effect of SF on neutrophil function was determined. Surprisingly, our data show that SF neutrophils in SpA patients have an inactive phenotype, despite the presence of many neutrophil-activating stimuli such as GM-CSF and TNF in SF. This was not due to exhaustion as SF neutrophils readily responded to stimulation. Therefore, this finding suggests that one or more inhibitors of neutrophil activation may be present in SF. Indeed, when blood neutrophils from healthy donors were activated in the presence of increasing concentrations of SF from SpA patients, degranulation and ROS production were dose-dependently inhibited. This effect was independent of diagnosis, gender, age, and medication in the patients from which the SF was isolated. Treatment of SF with the enzyme hyaluronidase strongly reduced the inhibitory effect of SF on neutrophil activation, indicating that hyaluronic acid that is present in SF may be an important factor in preventing SF neutrophil activation. This finding provides novel insights into the role of soluble factors in SF regulating neutrophil function and may lead to the development of novel therapeutics targeting neutrophil activation via hyaluronic acid or associated pathways.
| S-EPMC9964069 | biostudies-literature
Project description:Health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) forms biofilm in vitro that is dependent on the surface-located fibronectin binding proteins A and B (FnBPA, FnBPB). Here we provide new insights into the requirements for FnBP-dependent biofilm formation by MRSA. We show that expression of FnBPs is sustained at high levels throughout the growth cycle in the HA-MRSA strain BH1CC in contrast to laboratory strain SH1000, where expression could be detected only in exponential phase. We found that FnBP-mediated biofilm accumulation required Zn(2+), while the removal of Zn(2+) had no effect on the ability of FnBPA to mediate bacterial adherence to fibrinogen. We also investigated the role of FnBPA expressed on the surface of S. aureus in promoting biofilm formation and bacterial adhesion to fibrinogen. The minimum part of FnBPA required for ligand binding has so far been defined only with recombinant proteins. Here we found that the N1 subdomain was not required for biofilm formation or for FnBPA to promote bacterial adherence to fibrinogen. Residues at the C terminus of subdomain N3 required for FnBPA to bind to ligands using the "dock, lock, and latch" mechanism were necessary for FnBPA to promote bacterial adherence to fibrinogen. However, these residues were not necessary to form biofilm, allowing us to localize the region of FnBPA required for biofilm accumulation to residues 166 to 498. Thus, FnBPA mediates biofilm formation and bacterial adhesion to fibrinogen using two distinct mechanisms. Finally, we identified a hitherto-unrecognized thrombin cleavage site close to the boundary between subdomains N1 and N2 of FnBPA.
| S-EPMC3676058 | biostudies-literature
Project description:Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (?sigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ?sigB mutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that an sspB scpA double mutant restored ?sigB biofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment of S. aureus biofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved across S. aureus strain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulating S. aureus biofilm architecture.
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Project description:Biofilm formation in Staphylococcus aureus under in vitro growth conditions is generally promoted by high concentrations of sugar and/or salts. The addition of glucose to routinely used complex growth media triggered biofilm formation in S. aureus strain SA113. Deletion of ccpA, coding for the catabolite control protein A (CcpA), which regulates gene expression in response to the carbon source, abolished the capacity of SA113 to form a biofilm under static and flow conditions, while still allowing primary attachment to polystyrene surfaces. This suggested that CcpA mainly affects biofilm accumulation and intercellular aggregation. trans-Complementation of the mutant with the wild-type ccpA allele fully restored the biofilm formation. The biofilm produced by SA113 was susceptible to sodium metaperiodate, DNase I, and proteinase K treatment, indicating the presence of polysaccharide intercellular adhesin (PIA), protein factors, and extracellular DNA (eDNA). The investigation of several factors which were reported to influence biofilm formation in S. aureus (arlRS, mgrA, rbf, sarA, atl, ica, citZ, citB, and cidABC) showed that CcpA up-regulated the transcription of cidA, which was recently shown to contribute to eDNA production. Moreover, we showed that CcpA increased icaA expression and PIA production, presumably over the down-regulation of the tricarboxylic acid cycle genes citB and citZ.
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Project description:Hyaluronic acid (HA), a natural biomaterial present in healthy joints but depleted in osteoarthritis (OA), has been employed clinically to provide symptomatic relief of joint pain. Joint movement combined with a reduced joint lubrication in osteoarthritic knees can result in increased wear and tear, chondrocyte apoptosis, and inflammation, leading to cascading cartilage deterioration. Therefore, development of an appropriate cartilage model that can be evaluated for its friction properties with potential lubricants in different conditions is necessary, which can closely resemble a mechanically induced OA cartilage. Additionally, a comparison of different models with and without endogenous lubricating surface zone proteins, such as PRG4 promotes a well-rounded understanding of cartilage lubrication. In this study, we present our findings on the lubricating effects of HA on different articular cartilage model surfaces in comparison to synovial fluid, a physiological lubricating biomaterial. The mechanical testings data demonstrated that HA reduced average static and kinetic friction coefficient values of the cartilage samples by 75% and 70%, respectively. Furthermore, HA mimicked the friction characteristics of freshly harvested natural synovial fluid throughout all tested and modeled OA conditions with no statistically significant difference. These characteristics led us to exclusively identify HA as an effective boundary layer lubricant in the technology that we develop to treat OA (Singh et al., 2014).
| S-EPMC4526414 | biostudies-literature
Project description:BackgroundStaphylococcus aureus is a Gram-positive, pathogenic bacterium that causes a wide range of symptoms in humans and can form biofilm, which is a multicellular community of microorganisms that attaches to nonbiological and biological surfaces.MethodsHere, we aimed to isolate and characterize an S. aureus phage and examine the bactericidal activity alone and in conjunction with streptomycin treatment.ResultsWe isolated a virulent phage, WV, from a slaughterhouse in Jiangsu, China. This strain belonged to the family Myoviridae and presented a genome size of 141,342 bp. The optimal pH of the preservation buffer was 6-7, optimal growth temperature was 37°C, and optimal multiplicity of infection was 0.01. Phage WV can sterilize most clinical strains of S. aureus that had been isolated from clinical patients in the First People's Hospital of the Yunnan Province. Against low-concentration S. aureus culture, streptomycin demonstrated a greater antibiofilm effect than that of phage WV. By contrast, in high-concentration S. aureus culture, phage WV demonstrated greater antibiofilm effect than that of streptomycin. The use of phage WV and streptomycin together had a substantially greater overall antibiofilm effect than that achieved using either component alone.ConclusionThis study provides strong evidence for the effectiveness of phage application for the reduction of S. aureus biofilm growth and suggests that phages can be considered as a viable alternative to antibiotics in clinical settings.
| S-EPMC8619764 | biostudies-literature
Project description:Derivatives of the widely used laboratory strain Staphylococcus aureus NCTC8325, which are natural rsbU mutants, were shown to be unable to produce RsbU, a positive regulator of the alternative sigma factor sigma(B). The lack of RsbU prevented the heat-dependent production of sigma(B)-controlled transcripts and resulted in reduced H2O2 and UV tolerance, enhanced alpha-hemolysin activity, and the inability to produce the alkaline shock protein Asp23. After 48 h of growth, rsbU mutant strains failed to accumulate staphyloxanthin, the major stationary-phase carotenoid. Transcription of Asp23 was found to be exclusively controlled by sigma(B), making it an excellent target for the study of sigma(B) activity in S. aureus. Reporter gene experiments, using the firefly luciferase gene (luc+) fused to the sigma(B)-dependent promoter(s) of asp23, revealed that sigma(B) is almost inactive in 8325 derivatives. cis complementation of the 8325 derivative BB255 with the wild-type rsbU gene from strain COL produced the rsbU(+) derivative GP268, a strain possessing a sigma(B) activity profile comparable to that of the rsbU(+) wild-type strain Newman. In GP268, the heat inducibility of sigma(B)-dependent genes, Asp23 production, alpha-hemolysin activity, pigmentation, and susceptibility to H2O2 were restored to the levels observed in strain Newman, clearly demonstrating that RsbU is needed for activation of sigma(B) in S. aureus.
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