The clinical, radiological, microbiological, and molecular profile of the skin-penetration site of transfemoral amputees treated with bone-anchored prostheses.

The breach of the skin barrier is a critical issue associated with the treatment of individuals with transfemoral amputation (TFA) using osseointegrated, percutaneous titanium implants. Thirty TFA patients scheduled for abutment exchange or removal were consecutively enrolled. The aims were to determine the macroscopic skin signs, the presence of bacteria and the gene expression in abutment-adherent cells and to conduct correlative and comparative analyses between the different parameters. Redness and a granulation ring were present in 47% of the patients. Bacteria were detected in 27/30 patients, commonly in the bone canal. Staphylococcus aureus, coagulase-negative staphylococci, streptococci, and Enterococcus faecalis were the most common. A positive correlation was found between TNF-α expression and the detection of S. aureus. Staphylococcus aureus together with other bacterial species revealed a positive relationship with MMP-8 expression. A negative correlation was demonstrated between the length of the residual femur bone and the detection of a granulation ring and E. faecalis. A positive correlation was revealed between fixture loosening and pain and the radiological detection of endosteal bone resorption. Fixture loosening was also correlated with the reduced expression of interleukin-10 and osteocalcin. It is concluded that several relationships exist between clinical, radiological, microbiological, and molecular assessments of the percutaneous area of TFAs. Further long term studies on larger patient cohorts are required to determine the precise cause-effect relationships and unravel the role of host-bacteria interactions in the skin, bone canal and on the abutment for the longevity of percutaneous implants as treatment of TFA. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 578-589, 2017

Functional analysis of intergenic regulatory regions of genes encoding surface adhesins in Staphylococcus aureus isolates from periprosthetic joint infections

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI). Surface adhesins play an important role in the primary attachment to plasma proteins that coat the surface of prosthetic devices after implantation. Previous efforts to identify a genetic component of the bacterium that confers an enhanced capacity to cause PJI have focused on gene content, kmers, or single-nucleotide polymorphisms (SNPs) in coding sequences. Here, using a collection of S. aureus strains isolated from PJI and wounds, we investigated whether genetic variations in the regulatory region of genes encoding surface adhesins lead to differences in their expression levels and modulate the capacity of S. aureus to colonize implanted prosthetic devices. The data revealed that S. aureus isolates from the same clonal complex (CC) contain a specific pattern of SNPs in the regulatory region of genes encoding surface adhesins. As a consequence, each clonal lineage shows a specific profile of surface proteins expression. Co-infection experiments with representative isolates of the most prevalent CCs demonstrated that some lineages have a higher capacity to colonize implanted catheters in a murine infection model, which correlated with a greater ability to form a biofilm on coated surfaces with plasma proteins. Together, results indicate that differences in the expression level of surface adhesins may modulate the propensity of S. aureus strains to cause PJI. Given the high conservation of surface proteins among staphylococci, our work lays the framework for investigating how diversification at intergenic regulatory regions affects the capacity of S. aureus to colonize the surface of medical implants.This work was financially supported by the Spanish Ministry of Science and Innovation grant PID2020-113494RB-I00 to I.L. (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union), the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 754412 [MoRE2020 - Region Väs-tra Götaland], and the IngaBritt and Arne Lundberg Foundation (LU2021-0048). L.M.L was supported by the European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586 (IberusTalent)

Immunomodulatory effects exerted by extracellular vesicles from <i>Staphylococcus epidermidis</i> and <i>Staphylococcus aureus</i> isolated from bone-anchored prostheses

Staphylococcus aureus and Staphylococcus epidermidis are the bacteria that most frequently cause osteomyelitis. This study aimed to determine whether staphylococci isolated from osteomyelitis associated with septic loosening of orthopedic prostheses release extracellular vesicles (EVs) and, if so, to determine tentative immunomodulatory effects on the human monocytic cell line THP-1. EVs were isolated from bacterial cultures using filtration and ultracentrifugation and characterized by scanning electron microscopy, nanoparticle tracking analysis and Western Blot. The cytotoxic effect of EVs was analyzed by NucleoCounter and lactate dehydrogenase (LDH) analyses. Confocal laser scanning microscopy was employed to visualize the uptake of EVs by THP-1 cells. Activation of the transcription factor nuclear factor-κB (NF-κB) was determined in THP1-Blue™ NF-κB cells, and the gene expression and secretion of cytokines were determined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. All investigated strains, irrespective of their biofilm formation ability, were able to secrete EVs in vitro. The S. aureus strains produced significantly more EVs than the S. epidermidis strains. Both S. aureus-derived EVs and S. epidermidis-derived EVs were internalized by THP-1 cells, upregulated Toll-like receptor 3 (TLR3) gene expression, activated NF-κB, and promoted the gene expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, matrix metallopeptidase (MMP)-9 and IL-10. Whereas EVs from both staphylococcal species upregulated the proapoptotic DNA damage-inducible transcript 4 (DDIT4) gene and downregulated the antiapoptotic B-cell lymphoma 2 (Bcl-2) gene, cytolysis was preferentially induced in S. aureus EV-stimulated cells, possibly related to the expression of cytolytic proteins predominantly in S. aureus EVs. In conclusion, staphylococcal EVs possess potent cytolytic and immunomodulatory properties

Role of sodium salicylate in Staphylococcus aureus quorum sensing, virulence, biofilm formation and antimicrobial susceptibility

The widespread threat of antibiotic resistance requires new treatment options. Disrupting bacterial communication, quorum sensing (QS), has the potential to reduce pathogenesis by decreasing bacterial virulence. The aim of this study was to investigate the influence of sodium salicylate (NaSa) on Staphylococcus aureus QS, virulence production and biofilm formation. In S. aureus ATCC 25923 (agr III), with or without serum, NaSa (10 mM) downregulated the agr QS system and decreased the secretion levels of alpha-hemolysin, staphopain A and delta-hemolysin. Inhibition of agr expression caused a downregulation of delta-hemolysin, decreasing biofilm dispersal and increasing biofilm formation on polystyrene and titanium under static conditions. In contrast, NaSa did not increase biofilm biomass under flow but caused one log10 reduction in biofilm viability on polystyrene pegs, resulting in biofilms being twice as susceptible to rifampicin. A concentrationdependent effect of NaSa was further observed, where high concentrations (10 mM) decreased agr expression, while low concentrations (≤0.1 mM) increased agr expression. In S. aureus 8325-4 (agr I), a high concentration of NaSa (10 mM) decreased hla expression, and a low concentration of NaSa (≤1 mM) increased rnaIII and hla expression. The activity of NaSa on biofilm formation was dependent on agr type and material surface. Eight clinical strains isolated from prosthetic joint infection (PJI) or wound infection belonging to each of the four agr types were evaluated. The four PJI S. aureus strains did not change their biofilm phenotype with NaSa on the clinically relevant titanium surface. Half of the wound strains (agr III and IV) did not change the biofilm phenotype in the 3D collagen wound model. In addition, compared to the control, ATCC 25923 biofilms formed with 10 mM NaSa in the collagen model were more susceptible to silver. It is concluded that NaSa can inhibit QS in S. aureus, decreasing the levels of toxin production with certain modulation of biofilm formation. The effect on biofilm formation was dependent on the strain and material surface. It is suggested that the observed NaSa inhibition of bacterial communication is a potential alternative or adjuvant to traditional antibiotics.This research was funded by the Swedish Foundation for Strategic Research (SSF; RMA15-0110 2016), Mölnlycke Health Care AB (Sweden), the European Commission within the H2020-MSCA grant agreement no. 861046 (BIOREMIA-ETN), the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement No: 754412 (MoRE2020— Region Västra Götaland), CARe—Centre for Antibiotic Resistance Research at University of Gothenburg; Swedish Research Council (2018–02891), the Swedish state under the agreement between the Swedish government and the county councils, the ALF agreement (ALFGBG-725641), the IngaBritt and Arne Lundberg Foundation (LU2021- 0048), the Hjalmar Svensson Foundation; the Doctor Felix Neuberghs Foundation, the Adlerbertska Foundation, and the Area of Advance Materials of Chalmers/GU Biomaterials within the Strategic Research Area initiative launched by the Swedish government

Genomics of Staphylococcus aureus and Staphylococcus epidermidis from periprosthetic joint infections and correlation to clinical outcome

The approach of sequencing or genotyping to characterize the pathogenic potential of staphylococci from orthopedic device-related infection (ODRI) has been applied in recent studies. These studies described the genomic carriage of virulence in clinical strains and compared it with those in commensal strains. Only a few studies have directly correlated genomic profiles to patient outcome and phenotypic virulence properties in periprosthetic joint infections (PJIs). We investigated the association between genomic variations and virulence-associated phenotypes (biofilm-forming ability and antimicrobial resistance) in 111 staphylococcal strains isolated from patients with PJI and the infection outcome (resolved/unresolved). The presence of a strong biofilm phenotype in Staphylococcus aureus and an antibiotic-resistant phenotype in Staphylococcus epidermidis were both associated with treatment failure of PJI. In S. epidermidis, multidrug resistance (MDR) and resistance to rifampicin were associated with unresolved infection. Sequence type 45 (ST45) and ST2 were particularly enriched in S. aureus and S. epidermidis, respectively. S. epidermidis ST2 caused the majority of relapses and was associated with MDR and strong biofilm production, whereas ST215 correlated with MDR and non/weak biofilm production. S. aureus agr II correlated with resolved infection, while S. epidermidis agr I was associated with strong biofilm production and agr III with non/weak production. Collectively, our results highlight the importance of careful genomic and phenotypic characterization to anticipate the probability of the strain causing treatment failure in PJI. Due to the high rate of resistant S. epidermidis strains identified, this study provides evidence that the current recommended treatment of rifampicin and a fluoroquinolone should not be administered without knowledge of the resistance pattern

Accelerated biodegradation of FeMn porous alloy coated with ZnO : Effect on cytocompatibility and antibiofilm properties

Altres ajuts: acords transformatius de la UABFe-based alloys are being studied as potential candidates for biodegradable implants; however, their degradation rates remain too slow. To accelerate biodegradation while simultaneously hindering biofilm formation, a ZnO coating was deposited onto porous equiatomic FeMn alloy discs by sol-gel method using dip coating. The effect of the ZnO coating on the microstructure, biodegradability, cytocompatibility, and antibacterial properties were investigated. Biodegradability experiments were performed by immersing the specimens in Hank's balanced salt solution and measuring ion release after up to 28 days of immersion. The experiments showed an increased degradation of the FeMn/ZnO sample due to Fe segregation towards the grain boundaries, formation of iron-manganese oxide, and limited formation of degradation products on ZnO. Further, indirect Saos-2 cell cytotoxicity testing in 24 h sample-conditioned media showed no significant cytotoxicity in concentrations equal to or below 50 %. In addition, the total biofilm biovolume formed by Staphylococcus aureus on the FeMn/ZnO surface was significantly reduced compared to the uncoated FeMn. Taken together, these results show that the ZnO coating on FeMn improves the degradation rate, maintains cytocompatibility, and reduces biofilm accumulation when compared to an uncoated FeMn alloy

Effect of minor gallium addition on corrosion, passivity, and antibacterial behaviour of novel β-type Ti–Nb alloys

Metastable Ti–Nb alloys are promising bone-implant materials due to improved mechanical biofunctionality and biocompatibility. To overcome increasing bacterial infection risk, alloying with antibacterial elements is a promising strategy. This study investigates the effect of minor gallium (Ga) additions (4, 8 wt% Ga) to as-cast and solution-treated β-type Ti–45Nb-based alloy (96(Ti–45Nb)-4Ga, 92(Ti–45Nb)-8Ga (wt.%)) on corrosion and passive film properties, as well as cytocompatibility and antibacterial activity. The electrochemical properties were evaluated by potentiodynamic polarization, electrochemical impedance spectroscopy (EIS), and Mott-Schottky analyses in phosphate-buffered saline (PBS). X-ray photoelectron spectroscopy (XPS) was performed to analyze the chemical composition of passive films. Early adhesion and viability of macrophages and Staphylococcus aureus were assessed by nucleocounting and colony-forming unit counting, respectively. The results showed that high corrosion resistance and passive film properties of Ti–45Nb are retained and even slightly improved with Ga. EIS results revealed that Ga addition improves the passive film resistance. XPS measurements of 92(Ti–45Nb)-8Ga show that the passive film contains Ti-, Nb- and Ga-based oxides, implying the formation of mixed (Ti–Nb-Ga) oxides. In addition, marginal Ga ion release rate was detected under free corrosion conditions. Therefore, it can be assumed that Ga species may contribute to passive film formation on Ga-containing alloys. The 92(Ti–45Nb)-8Ga elicited an antibacterial effect against S. aureus compared to cp-Ti at 4 h. Moreover, Ga-containing alloys showed good cytocompatibility with THP-1 macrophages at 24 h. In conclusion, it was demonstrated that Ga additions to Ti–45Nb are beneficial to corrosion resistance and showed promising initial host and bacterial interactions

Microbiome on the Bone-Anchored Hearing System: A Prospective Study

The bone-anchored hearing system (BAHS) has evolved to a common treatment option for various types of hearing revalidation. The BAHS consists of an implant in the skull that breeches the skin. Soft tissue reactions are a common complication associated with BAHS and are generally poorly understood. This study aims to investigate the influence of BAHS and associated skin reactions around the implant. A total of 45 patients were prospectively followed from implantation up to at least 1 year. Swabs were obtained at baseline, 12 weeks follow-up and during cases of inflammation (Holgers score ≥2). The microbiota was assessed using IS-proTM, a bacterial profiling method based on the interspace region between the 16S–23S rRNA genes. Detection of operational taxonomic units, the Shannon Diversity Index, sample similarity analyses and Partial Least Squares Discriminant Analysis (PLS-DA) were employed. Staphylococcus epidermidis, Streptococcus pneumoniae/mitis, Propionibacterium acnes, Staphylococcus capitis, Staphylococcus hominis, Bifidobacterium longum, Haemophilus parainfluenzae, Lactobacillus rhamnosus, Bordetella spp., Streptococcus sanguinis, Peptostreptococcus anaerobius, Staphylococcus aureus, Lactococcus lactis, Enterobacter cloacae, and Citrobacter koseri were the most commonly found bacterial species. S. pneumoniae/mitis was significantly more often observed after implantation, whereas P. acnes was significantly less observed after implantation compared with baseline. The relative abundance of S. epidermidis (17%) and S. aureus (19.4%) was the highest for the group of patients with inflammation. The Shannon Diversity Index was significantly increased after implantation compared with pre-surgical swabs for Firmicutes, Actinobacteria, Fusobacteria, Verrucomicrobia (FAFV), but not for other phyla. When combining all phyla, there was no significant increase in the Shannon Diversity Index. The diversity index was similar post-surgically for patients experiencing inflammation and for patients without inflammation. With a supervised classifier (PLS-DA), patients prone to inflammation could be identified at baseline with an accuracy of 91.7%. In addition, PLS-DA could classify post-surgical abutments as non-inflamed or inflamed with an accuracy of 97.7%. This study shows the potential of using IS-proTM to describe and quantify the microbiota associated with the percutaneous BAHS. Furthermore, the results indicate the possibility of an early identification of patients susceptible to adverse skin reaction following implantation. Both S. aureus and S. epidermidis should be considered as relevant bacteria for BAHS-associated inflammation