Effects of Antibiotics on α-Toxin Levels during <i>Staphylococcus aureus</i> Culture: Implications for the Protection of Chondrocytes in a Model of Septic Arthritis

Miller, R; Berlouis, Marie E; Simpson, A. H. R. W.; Smith, I.D.M.; Hall, Andrew C.

doi:10.1177/1947603519828433

Cited by 4 publications

(5 citation statements)

References 47 publications

(117 reference statements)

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“…27,28 As reported by Smith et al, 27,28 we utilized chondrocyte viability 24 hours following S aureus inoculation as our experimental endpoint to investigate the efficacy of PRP, AmpS, and PRP+AmpS treatments. The S aureus-derived toxin α-hemolysin is the key toxin responsible for rapid chondrocyte death with this model, 26,28 and both S aureus strains used in this study, ATCC 29213 and 43300, secrete α-hemolysin toxin. [36][37][38] The S aureus inoculum size used in this Figure 1-Box-and-whisker plots of metabolic activity (optical density) of bovine cartilage explants incubated with synovial fluid (SF) alone or incubated with S aureus-inoculated SF (SA) alone or with platelet-rich plasma (PRP; 25% culture medium volume; SA+PRP), ampicillin-sulbactam (AmpS; 2 mg/mL; SA+AmpS), or both PRP (25% culture medium volume) and AmpS (2 mg/ mL; SA+PRP+AmpS) for 24 hours.…”

Section: Discussionmentioning

confidence: 99%

“…Even though we reported the chondrocyte death-mitigating effects of PRP and AmpS, it must be noted that S aureus inoculation and PRP+AmpS treatments were initiated simultaneously in this study, and this does not mimic clinical scenarios. Inoculating explants with S aureus prior (ie, 12 to 14 hours) to adding treatments and subsequently quantifying chondroprotective indices relative to untreated controls as described by Miller et al 26 would have been more reflective of clinical septic arthritis. Platelet concentrations in PRP treatments between experiments from individual cattle were not standardized to control for intersubject variability of quantified outcome measures.…”

Section: Discussionmentioning

confidence: 99%

“…A previously validated S aureus-induced in vitro explant model of bovine septic arthritis was used. [26][27][28] We hypothesized that the chondroprotective effects of PRP and PRP+AmpS would not differ significantly from those of AmpS alone in an in vitro explant model of bovine S aureus-induced septic arthritis.…”

Section: Clinical Relevancementioning

confidence: 99%

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Autologous platelet-rich plasma effects on Staphylococcus aureus–induced chondrocyte death in an in vitro bovine septic arthritis model

Muir

Niehaus

Lozier

et al. 2022

ajvr

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OBJECTIVE To investigate the chondroprotective effects of autologous platelet-rich plasma (PRP), ampicillin-sulbactam (AmpS), or PRP combined with AmpS (PRP+AmpS) in an in vitro chondrocyte explant model of bovine Staphylococcus aureus–induced septic arthritis. SAMPLE Autologous PRP and cartilage explants obtained from 6 healthy, adult, nonlactating Jersey-crossbred cows. ProcedureS Autologous PRP was prepared prior to euthanasia using an optimized double centrifugation protocol. Cartilage explants collected from grossly normal stifle joints were incubated in synovial fluid (SF) alone, S aureus–inoculated SF (SA), or SA supplemented with PRP (25% culture medium volume), AmpS (2 mg/mL), or both PRP (25% culture medium volume) and AmpS (2 mg/mL; PRP+AmpS) for 24 hours. The metabolic activity, percentage of dead cells, and glycosaminoglycan content of cartilage explants were measured with a resazurin-based assay, live-dead cell staining, and dimethylmethylene blue assay, respectively. Treatment effects were assessed relative to the findings for cartilage explants incubated in SF alone. RESULTS Application of PRP, AmpS, and PRP+AmpS treatments significantly reduced S aureus–induced chondrocyte death (ie, increased metabolic activity and cell viability staining) in cartilage explants, compared with untreated controls. There were no significant differences in chondrocyte death among explants treated with PRP, AmpS, or PRP+AmpS. CLINICAL RELEVANCE In this in vitro explant model of S aureus–induced septic arthritis, PRP, AmpS, and PRP+AmpS treatments mitigated chondrocyte death. Additional work to confirm the efficacy of PRP with bacteria commonly associated with clinical septic arthritis in cattle as well as in vivo evaluation is warranted.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Autologous platelet-rich plasma effects on Staphylococcus aureus–induced chondrocyte death in an in vitro bovine septic arthritis model

Muir

Niehaus

Lozier

et al. 2022

ajvr

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“…[28][29][30] Binding of Hla to ADAM10 receptors on chondrocytes leads to cell membrane pore formation, and influx of Na + and water, causing cell lysis. 31 An example of Hlatargeted therapy lies in the choice of antibiotics as shown by Miller et al, 32 whereby a bacteriostatic antibiotic linezolid showed rapid curtailing of Hla-induced haemolysis in the rabbit erythrocyte assay. The action of linezolid was faster compared to the bacteriolytic antibiotic penicillin.…”

Section: Discussionmentioning

confidence: 99%

Septic arthritis in an in vivo murine model induced by Staphylococcus aureus

Hall

Wong

Howie

et al. 2022

Bone & Joint Research

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Aims Staphylococcus aureus is a major cause of septic arthritis, and in vitro studies suggest α haemolysin (Hla) is responsible for chondrocyte death. We used an in vivo murine joint model to compare inoculation with wild type S. aureus 8325-4 with a Hla-deficient strain DU1090 on chondrocyte viability, tissue histology, and joint biomechanics. The aim was to compare the actions of S. aureus Hla alone with those of the animal’s immune response to infection. Methods Adult male C57Bl/6 mice (n = 75) were randomized into three groups to receive 1.0 to 1.4 × 107 colony-forming units (CFUs)/ml of 8325-4, DU1090, or saline into the right stifle joint. Chondrocyte death was assessed by confocal microscopy. Histological changes to inoculated joints were graded for inflammatory responses along with gait, weight changes, and limb swelling. Results Chondrocyte death was greater with 8325-4 (96.2% (SD 5.5%); p < 0.001) than DU1090 (28.9% (SD 16.0%); p = 0.009) and both were higher than controls (3.8% (SD 1.2%)). Histology revealed cartilage/bone damage with 8325-4 or DU1090 compared to controls (p = 0.010). Both infected groups lost weight (p = 0.006 for both) and experienced limb swelling (p = 0.043 and p = 0.018, respectively). Joints inoculated with bacteria showed significant alterations in gait cycle with a decreased stance phase, increased swing phase, and a corresponding decrease in swing speed. Conclusion Murine joints inoculated with Hla-producing 8325-4 experienced significantly more chondrocyte death than those with DU1090, which lack the toxin. This was despite similar immune responses, indicating that Hla was the major cause of chondrocyte death. Hla-deficient DU1090 also elevated chondrocyte death compared to controls, suggesting a smaller additional deleterious role of the immune system on cartilage. Cite this article: Bone Joint Res 2022;11(9):669–678.

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“…A hemolysis assay was performed as previously described (Miller et al, 2021). Approximately 10 ml of whole blood was collected from the central auricular artery of a New Zealand male rabbit, which was purchased from the Laboratory Animal Center of the Third Military Medical University, and added to 1 ml heparin sodium solution, followed by three times of wash with PBS with centrifugation at 1,000 × g for 5 min at 4 • C. A concentration of 2% (v/v) rabbit red blood cells (RBCs) was prepared with PBS.…”

Section: Hemolysis Assaymentioning

confidence: 99%

Loaded delta-hemolysin shapes the properties of Staphylococcus aureus membrane vesicles

Chen,

Lv,

Shang

et al. 2023

Front. Microbiol.

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BackgroundMembrane vesicles (MVs) are nanoscale vesicular structures produced by bacteria during their growth in vitro and in vivo. Some bacterial components can be loaded in bacterial MVs, but the roles of the loaded MV molecules are unclear.MethodsMVs of Staphylococcus aureus RN4220 and its derivatives were prepared. Dynamic light scattering analysis was used to evaluate the size distribution, and 4D-label-free liquid chromatography–tandem mass spectrometry analysis was performed to detect protein composition in the MVs. The site-mutation S. aureus RN4220-Δhld and agrA deletion mutant RN4220-ΔagrA were generated via allelic replacement strategies. A hemolysis assay was performed with rabbit red blood cells. CCK-8 and lactate dehydrogenase release assays were used to determine the cytotoxicity of S. aureus MVs against RAW264.7 macrophages. The serum levels of inflammatory factors such as IL-6, IL-1β, and TNFα in mice treated with S. aureus MVs were detected with an enzyme-linked immunosorbent assay kit.ResultsDelta-hemolysin (Hld) was identified as a major loaded factor in S. aureus MVs. Further study showed that Hld could promote the production of staphylococcal MVs with smaller sizes. Loaded Hld affected the diversity of loaded proteins in MVs of S. aureus RN4220. Hld resulted in decreased protein diversity in MVs of S. aureus. Site-mutation (RN4220-Δhld) and agrA deletion (RN4220-ΔagrA) mutants produced MVs (ΔhldMVs and ΔagrAMVs) with a greater number of bacterial proteins than those derived from wild-type RN4220 (wtMVs). Moreover, Hld contributed to the hemolytic activity of wtMVs. Hld-loaded wtMVs were cytotoxic to macrophage RAW264.7 cells and could stimulate the production of inflammatory factor IL-6 in vivo.ConclusionThis study presented that Hld was a major loaded factor in S. aureus MVs, and the loaded Hld played vital roles in the MV-property modification.

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Effects of Antibiotics on α-Toxin Levels during Staphylococcus aureus Culture: Implications for the Protection of Chondrocytes in a Model of Septic Arthritis

Abstract: toxin levels during Staphylococcus aureus culture: implications for the protection of chondrocytes in a model of septic arthritis.', Cartilage.

Cited by 4 publications

References 47 publications

Autologous platelet-rich plasma effects on Staphylococcus aureus–induced chondrocyte death in an in vitro bovine septic arthritis model

Autologous platelet-rich plasma effects on Staphylococcus aureus–induced chondrocyte death in an in vitro bovine septic arthritis model

Septic arthritis in an in vivo murine model induced by Staphylococcus aureus

Loaded delta-hemolysin shapes the properties of Staphylococcus aureus membrane vesicles

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