scholarly journals A potential key role for alpha-haemolysin ofStaphylococcus aureusin mediating chondrocyte death in septic arthritis

2018 ◽  
Vol 7 (7) ◽  
pp. 457-467 ◽  
Author(s):  
I. D. M. Smith ◽  
K. M. Milto ◽  
C. J. Doherty ◽  
S. G. B. Amyes ◽  
A. H. R. W. Simpson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Septic Arthritis ◽  
Cartilage Damage ◽  
Laser Scanning Microscopy ◽  
Cartilage Explants ◽  
Confocal Laser ◽  
Dead Cell ◽  
Chondrocyte Viability ◽  
Chondrocyte Death ◽  
Bovine Cartilage

ObjectivesStaphylococcus aureus (S. aureus) is the most commonly implicated organism in septic arthritis, a condition that may be highly destructive to articular cartilage. Previous studies investigating laboratory and clinical strains of S. aureus have demonstrated that potent toxins induced significant chondrocyte death, although the precise toxin or toxins that were involved was unknown. In this study, we used isogenic S. aureus mutants to assess the influence of alpha (Hla)-, beta (Hlb)-, and gamma (Hlg)-haemolysins, toxins considered important for the destruction of host tissue, on in situ bovine chondrocyte viability.MethodsBovine cartilage explants were cultured with isogenic S. aureus mutants and/or their culture supernatants. Chondrocyte viability was then assessed within defined regions of interest in the axial and coronal plane following live- and dead-cell imaging using the fluorescent probes 5-chloromethylfluorescein diacetate and propidium iodide, respectively, and confocal laser-scanning microscopy.ResultsHla-producing mutants caused substantial chondrocyte death compared with the toxin-deficient control (Hla-Hlb-Hlg-), whilst mutants producing Hlb and Hlg in the absence of Hla induced minimal chondrocyte death. Coronal studies established that Hla-induced chondrocyte death started in the superficial zone of cartilage and spread to deeper layers, whereas Hlb and Hlg toxins were without significant effect.ConclusionThis study identified Hla as a highly potent S. aureus toxin that caused rapid chondrocyte death in bovine cartilage, with other toxins or metabolic products produced by the bacteria playing a minor role. The identification of Hla in mediating chondrocyte death may assist in the development of therapeutic strategies aimed at reducing the extent of cartilage damage during and after an episode of septic arthritis. Cite this article: I. D. M. Smith, K. M. Milto, C. J. Doherty, S. G. B. Amyes, A. H. R. W. Simpson, A. C. Hall. A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis. Bone Joint Res 2018;7:457–467. DOI: 10.1302/2046-3758.77.BJR-2017-0165.R1.

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

2022 ◽  
pp. 1-8
Author(s):  
Andrew J.T. Muir ◽  
Andrew J. Niehaus ◽  
Joseph W. Lozier ◽  
Sara L. Cole ◽  
Zarah A. Belacic ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Septic Arthritis ◽  
Metabolic Activity ◽  
Culture Medium ◽  
Platelet Rich Plasma ◽  
Cartilage Explants ◽  
Chondrocyte Death ◽  
Autologous Platelet Rich Plasma ◽  
Autologous Platelet

Abstract 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.

scholarly journals Rapid in situ chondrocyte death induced by Staphylococcus aureus toxins in a bovine cartilage explant model of septic arthritis

2013 ◽  
Vol 21 (11) ◽  
pp. 1755-1765 ◽  
Author(s):  
I.D.M. Smith ◽  
J.P. Winstanley ◽  
K.M. Milto ◽  
C.J. Doherty ◽  
E. Czarniak ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Septic Arthritis ◽  
Cartilage Explant ◽  
Chondrocyte Death ◽  
Bovine Cartilage

scholarly journals Quantitative Analysis of Surface Contouring with Pulsed Bipolar Radiofrequency on Thin Chondromalacic Cartilage

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Michaela Huber ◽  
Daniela Schlosser ◽  
Susanne Stenzel ◽  
Johannes Maier ◽  
Girish Pattappa ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Microscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chondrocyte Viability ◽  
Radio Frequency Energy ◽  
Bipolar Radiofrequency ◽  
Chondrocyte Death ◽  
Emery Paper ◽  
The Impact

The purpose of this study was to evaluate the quality of surface contouring of chondromalacic cartilage by bipolar radio frequency energy using different treatment patterns in an animal model, as well as examining the impact of the treatment onto chondrocyte viability by two different methods. Our experiments were conducted on 36 fresh osteochondral sections from the tibia plateau of slaughtered 6-month-old pigs, where the thickness of the cartilage is similar to that of human wrist cartilage. An area of 1 cm2 was first treated with emery paper to simulate the chondromalacic cartilage. Then, the treatment with RFE followed in 6 different patterns. The osteochondral sections were assessed for cellular viability (live/dead assay, caspase (cell apoptosis marker) staining, and quantitative analysed images obtained by fluorescent microscopy). For a quantitative characterization of none or treated cartilage surfaces, various roughness parameters were measured using confocal laser scanning microscopy (Olympus LEXT OLS 4000 3D). To describe the roughness, the Root-Mean-Square parameter (Sq) was calculated. A smoothing effect of the cartilage surface was detectable upon each pattern of RFE treatment. The Sq for native cartilage was Sq=3.8±1.1 μm. The best smoothing pattern was seen for two RFE passes and a 2-second pulsed mode (B2p2) with an Sq=27.3±4.9 μm. However, with increased smoothing, an augmentation in chondrocyte death up to 95% was detected. Using bipolar RFE treatment in arthroscopy for small joints like the wrist or MCP joints should be used with caution. In the case of chondroplasty, there is a high chance to destroy the joint cartilage.

The effect of N-acetylcysteine in a combined antibiofilm treatment against antibiotic-resistant Staphylococcus aureus

2020 ◽  
Vol 75 (7) ◽  
pp. 1787-1798
Author(s):  
Arthika Manoharan ◽  
Theerthankar Das ◽  
Gregory S Whiteley ◽  
Trevor Glasbey ◽  
Frederik H Kriel ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Cleavage ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Combination Treatments ◽  
Resazurin Assay ◽  
Biofilm Architecture ◽  
Biofilm Disruption ◽  
The Uk

Abstract Background The WHO declared Staphylococcus aureus as a ‘pathogen of high importance’ in 2017. One-fifth of all bloodstream-related infections in Australia and 12 000 cases of bacteraemia in the UK (2017–18) were caused by the MRSA variant. To address the need for novel therapies, we investigated several permutations of an innovative combination therapy containing N-acetylcysteine (NAC), an antibiotic and an enzyme of choice in eradicating MRSA and MSSA biofilms. Methods Biofilm viability (resazurin assay) and colony count methods were used to investigate the effect of NAC, antibiotics and enzymes on S. aureus biofilm disruption and killing. The effects of NAC and enzymes on the polysaccharide content of biofilm matrices were analysed using the phenol/sulphuric acid method and the effect of NAC on DNA cleavage was determined using the Qubit fluorometer technique. Changes in biofilm architecture when subjected to NAC and enzymes were visualized using confocal laser scanning microscopy (CLSM). Results NAC alone displayed bacteriostatic effects when tested on planktonic bacterial growth. Combination treatments containing 30 mM NAC resulted in ≥90% disruption of biofilms across all MRSA and MSSA strains with a 2–3 log10 decrease in cfu/mL in treated biofilms. CLSM showed that NAC treatment drastically disrupted S. aureus biofilm architecture. There was also reduced polysaccharide production in MRSA biofilms in the presence of NAC. Conclusions Our results indicate that inclusion of NAC in a combination treatment is a promising strategy for S. aureus biofilm eradication. The intrinsic acidity of NAC was identified as key to maximum biofilm disruption and degradation of matrix components.

Lavage Solution Temperature Influences Depth of Chondrocyte Death and Surface Contouring during Thermal Chondroplasty with Temperature-Controlled Monopolar Radiofrequency Energy

2002 ◽  
Vol 30 (5) ◽  
pp. 667-673 ◽  
Author(s):  
Yan Lu ◽  
Ryland B. Edwards ◽  
Shane Nho ◽  
Brian J. Cole ◽  
Mark D. Markel
Keyword(s):  
Treatment Time ◽  
Solution Temperature ◽  
Confocal Laser ◽  
Radiofrequency Energy ◽  
Cartilage Surface ◽  
Chondrocyte Viability ◽  
Chondrocyte Death ◽  
Articular Cartilage Surface ◽  
Thermal Chondroplasty ◽  
Lavage Solution

Background: Although radiofrequency energy can smooth and contour cartilage surface, it has deleterious effects on chondrocyte viability. Hypothesis: Monopolar thermal chondroplasty in a 37°C lavage solution, as compared with a 22° lavage solution, will reduce chondrocyte death and result in greater smoothing of the articular cartilage surface. Study Design: Controlled laboratory study. Methods: Sixteen chondromalacic samples from patients undergoing total knee arthroplasty were divided into two groups: 22°C and 37°C lavage solution. Each sample was divided into two equal parts and half of each group was treated for 10 seconds and the other half for 15 seconds. Results: Confocal laser microscopy demonstrated that the depth of chondrocyte death in the 37°C lavage solution group was significantly less (range, 200 to 340 μm) than that in the 22°C solution group for both 10- and 15-second treatment times. Scanning electron microscopy demonstrated that the cartilage surface in the 37°C lavage solution group was smoother than that in the 22°C solution group for the 10-second treatment time. Energy delivery power in the 37°C lavage solution group was significantly lower than in the 22°C solution group for both treatment times. Conclusions: Thermal chondroplasty with 37°C lavage solution resulted in less depth of chondrocyte death and produced smoother surfaces than with 22°C solution for 10 seconds of treatment. Clinical Relevance: Less chondrocyte death would permit increased use of thermal chondroplasty.

scholarly journals Antibiofilm Properties of Temporin-L on Pseudomonas fluorescens in Static and In-Flow Conditions

2020 ◽  
Vol 21 (22) ◽  
pp. 8526
Author(s):  
Angela Di Somma ◽  
Federica Recupido ◽  
Arianna Cirillo ◽  
Alessia Romano ◽  
Alessandra Romanelli ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Biofilm Formation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Antibiofilm Activity ◽  
Dead Cell ◽  
Flow Conditions ◽  
Dynamic Conditions ◽  
Ability To Act ◽  
Static Conditions

Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation of this structure represents the most important adaptive mechanism that leads to antibacterial resistance, and therefore, closely connected to pathogenicity. Antimicrobial peptides (AMPs) could represent attractive candidates for the design of new antibiotics because of their specific characteristics. AMPs show a broad activity spectrum, a relative selectivity towards their targets (microbial membranes), the ability to act on both proliferative and quiescent cells, a rapid mechanism of action, and above all, a low propensity for developing resistance. This article investigates the effect at subMIC concentrations of Temporin-L (TL) on biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, showing that TL displays antibiofilm properties. Biofilm formation in static conditions was analyzed by the Crystal Violet assay. Investigation of biofilms in dynamic conditions was performed in a commercial microfluidic device consisting of a microflow chamber to simulate real flow conditions in the human body. Biofilm morphology was examined using Confocal Laser Scanning Microscopy and quantified via image analysis. The investigation of TL effects on P. fluorescens showed that when subMIC concentrations of this peptide were added during bacterial growth, TL exerted antibiofilm activity, impairing biofilm formation both in static and dynamic conditions. Moreover, TL also affects mature biofilm as confocal microscopy analyses showed that a large portion of preformed biofilm architecture was clearly perturbed by the peptide addition with a significative decrease of all the biofilm surface properties and the overall biomass. Finally, in these conditions, TL did not affect bacterial cells as the live/dead cell ratio remained unchanged without any increase in damaged cells, confirming an actual antibiofilm activity of the peptide.

Influence of Stress Magnitude on Water Loss and Chondrocyte Viability in Impacted Articular Cartilage

2003 ◽  
Vol 125 (5) ◽  
pp. 594-601 ◽  
Author(s):  
Dejan Milentijevic ◽  
David L. Helfet ◽  
Peter A. Torzilli
Keyword(s):  
Articular Cartilage ◽  
Water Loss ◽  
Articular Surface ◽  
Axial Strain ◽  
Biological Response ◽  
Peak Stress ◽  
Cartilage Explants ◽  
Axial Deformation ◽  
Chondrocyte Viability ◽  
Chondrocyte Death

The objective of this study was to assess mechano-biological response of articular cartilage when subjected to a single impact stress. Mature bovine cartilage explants were impacted with peak stresses ranging from 10 to 60 MPa at a stress rate of 350 MPa/s. Water loss, matrix axial deformation, dynamic impact modulus (DIM), and cell viability were measured immediately after impaction. The water loss through the articular surface (AS) was small and ranged from 1% to 6% with increasing peak stress. The corresponding axial strains ranged from 2.5% to 25%, respectively, while the DIM was 455.9±111.9 MPa. Chondrocyte death started at the articular surface and increased in depth to a maximum of 6% (70 μm) of the cartilage thickness at the highest stress. We found that the volumetric (axial) strain was more than twice the amount of water loss at the highest peak stress. Furthermore, specimens impacted such that the interstitial water was forced through the deep zone (DZ) had less water loss, a higher DIM, and no cell death. These findings appear to be due to matrix compaction in the superficial region causing higher compressive strains to occur at the surface rather than in the deeper zones.

scholarly journals Staphylococcus aureus Interferes with Streptococci Spatial Distribution and with Protein Expression of Species within a Polymicrobial Oral Biofilm

Antibiotics ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 116
Author(s):  
Etyene Schnurr ◽  
Pune N. Paqué ◽  
Thomas Attin ◽  
Paolo Nanni ◽  
Jonas Grossmann ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Laser Scanning ◽  
Bacterial Species ◽  
Fusobacterium Nucleatum ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Oral Diseases ◽  
Streptococcus Oralis ◽  
Oral Environment ◽  
Associated Species

We asked whether transient Staphylococcus aureus in the oral environment synergistically interacts with orally associated bacterial species such as Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans, and Veillonella dispar (six-species control biofilm 6S). For this purpose, four modified biofilms with seven species that contain either the wild type strain of the S. aureus genotype (USA300-MRSA WT), its isogenic mutant with MSCRAMM deficiency (USA300-MRSA ΔMSCRAMM), a methicillin-sensitive S. aureus (ST72-MSSA-) or a methicillin-resistant S. aureus (USA800-MRSA) grown on hydroxyapatite disks were examined. Culture analyses, confocal-laser-scanning microscopy and proteome analyses were performed. S. aureus strains affected the amount of supragingival biofilm-associated species differently. The deletion of MSCRAMM genes disrupted the growth of S. aureus and the distribution of S. mutans and S. oralis within the biofilms. In addition, S. aureus caused shifts in the number of detectable proteins of other species in the 6S biofilm. S. aureus (USA300-MRSA WT), aggregated together with early colonizers such as Actinomyces and streptococci, influenced the number of secondary colonizers such as Fusobacterium nucleatum and was involved in structuring the biofilm architecture that triggered the change from a homeostatic biofilm to a dysbiotic biofilm to the development of oral diseases.

scholarly journals Subinhibitory Concentrations of Mupirocin Stimulate Staphylococcus aureus Biofilm Formation by Upregulating cidA

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Ye Jin ◽  
Yinjuan Guo ◽  
Qing Zhan ◽  
Yongpeng Shang ◽  
Di Qu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Multidrug Resistant ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Content Type ◽  
Subinhibitory Concentrations ◽  
Biofilm Development

ABSTRACT Previous studies have shown that the administration of antibiotics at subinhibitory concentrations stimulates biofilm formation by the majority of multidrug-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated the effect of subinhibitory concentrations of mupirocin on biofilm formation by the community-associated (CA) mupirocin-sensitive MRSA strain USA300 and the highly mupirocin-resistant clinical S. aureus SA01 to SA05 isolates. We found that mupirocin increased the ability of MRSA cells to attach to surfaces and form biofilms. Confocal laser scanning microscopy (CLSM) demonstrated that mupirocin treatment promoted thicker biofilm formation, which also correlated with the production of extracellular DNA (eDNA). Furthermore, quantitative real-time PCR (RT-qPCR) results revealed that this effect was largely due to the involvement of holin-like and antiholin-like proteins (encoded by the cidA gene), which are responsible for modulating cell death and lysis during biofilm development. We found that cidA expression levels significantly increased by 6.05- to 35.52-fold (P < 0.01) after mupirocin administration. We generated a cidA-deficient mutant of the USA300 S. aureus strain. Exposure of the ΔcidA mutant to mupirocin did not result in thicker biofilm formation than that in the parent strain. We therefore hypothesize that the mupirocin-induced stimulation of S. aureus biofilm formation may involve the upregulation of cidA.

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