Background Graves’ disease (GD) is an autoimmune condition in which autoantibodies to the thyrotropin receptor (TSHR) cause hyperthyroidism. About 50% of GD patients also have Graves’ orbitopathy (GO), an intractable disease in which expansion of the orbital contents causes diplopia, proptosis and even blindness. Murine models of GD/GO, developed in different centres, demonstrated significant variation in gut microbiota composition which correlated with TSHR-induced disease heterogeneity. To investigate whether correlation indicates causation, we modified the gut microbiota to determine whether it has a role in thyroid autoimmunity. Female BALB/c mice were treated with either vancomycin, probiotic bacteria, human fecal material transfer (hFMT) from patients with severe GO or ddH2O from birth to immunization with TSHR-A subunit or beta-galactosidase (βgal; age ~ 6 weeks). Incidence and severity of GD (TSHR autoantibodies, thyroid histology, thyroxine level) and GO (orbital fat and muscle histology), lymphocyte phenotype, cytokine profile and gut microbiota were analysed at sacrifice (~ 22 weeks). Results In ddH2O-TSHR mice, 84% had pathological autoantibodies, 67% elevated thyroxine, 77% hyperplastic thyroids and 70% orbital pathology. Firmicutes were increased, and Bacteroidetes reduced relative to ddH2O-βgal; CCL5 was increased. The random forest algorithm at the genus level predicted vancomycin treatment with 100% accuracy but 74% and 70% for hFMT and probiotic, respectively. Vancomycin significantly reduced gut microbiota richness and diversity compared with all other groups; the incidence and severity of both GD and GO also decreased; reduced orbital pathology correlated positively with Akkermansia spp. whilst IL-4 levels increased. Mice receiving hFMT initially inherited their GO donors’ microbiota, and the severity of induced GD increased, as did the orbital brown adipose tissue volume in TSHR mice. Furthermore, genus Bacteroides, which is reduced in GD patients, was significantly increased by vancomycin but reduced in hFMT-treated mice. Probiotic treatment significantly increased CD25+ Treg cells in orbital draining lymph nodes but exacerbated induced autoimmune hyperthyroidism and GO. Conclusions These results strongly support a role for the gut microbiota in TSHR-induced disease. Whilst changes to the gut microbiota have a profound effect on quantifiable GD endocrine and immune factors, the impact on GO cellular changes is more nuanced. The findings have translational potential for novel, improved treatments.

Experimental models of hyperthyroid Graves' disease (GD) and Graves' orbitopathy (GO) are efficiently developed by genetic immunisation by electroporation with human thyrotropin hormone receptor (hTSHR) A-subunit plasmid in female BALB/c (H-2d) mice. We investigated susceptibility in C57BL/6 J (H-2b) animals to allow studies on disease mechanisms in transgenic and immune response gene knock-out mice. Higher numbers of female C57BL/6 J were positive for pathogenic thyroid stimulating antibodies, but induced hyperthyroidism remained at a low frequency compared to BALB/c animals. Assessment of hTSHR specific T cells showed reduced proliferation in C57BL/6 J animals accompanied with anti-inflammatory IL-10, with less pro-inflammatory IFN-γ compared to BALB/c. Whilst the orbital tissue from immune BALB/c mice showed inflammation and adipogenesis, in contrast C57BL/6 J animals showed normal pathology. We characterised the gut microbiota using 16 S ribosomal RNA gene sequencing to explore its possible pathogenic role in the model. Despite being housed under identical conditions, we observed significantly different organisation of the microbiota (beta-diversity) in the two strains. Taxonomic differences were also noted, with C57BL/6 J showing an enrichment of Operational Taxonomic Units (OTUs) belonging to the and, followed by , and genera. A higher number of genera significantly correlating with clinical features was observed in C57BL/6 J compared to BALB/c; for example, OTUs correlated negatively with thyroid-stimulating antibodies in C57BL/6 J mice. Thus, our data suggest gut microbiota may play a pivotal immunomodulatory role that differentiates the thyroid function and orbital pathology outcome in these two inbred strains undergoing experimental GO.

Summary Background The number of invasive Candida infections has significantly increased in recent decades. For the successful treatment of fungal infections, rapid identification at the species level, particularly in polyfungal infections, is a key factor. In this study, four commercially available chromogenic media, CandiSelect™ 4 (CS4), chromID™ Candida Agar (CCA), BBL™ CHROMagar™ Candida Medium (BBL) and Brilliance™ Candida Agar (BCA) were evaluated for Candida identification. Material/Methods Overall, 181 bronchial secretion samples from intensive care patients were analysed prospectively. In addition, 18 primarily sterile materials, previously tested positive for Candida, were investigated retrospectively. All samples were cultured as recommended by the manufacturer and visually inspected after 24 and 48 hours by three independent investigators. As a control, colonies were identified by MALDI‐TOF MS. Specificity and sensitivity were determined for C albicans identification prospectively. Results CS4 and BCA showed the best overall consensus with the identification results reached by MALDI‐TOF MS for Candida albicans and species. A clear differentiation between the species could be ascertained via easily identifiable, species‐specific coloration in contrast to BBL and CCA. Sensitivity for C albicans (n = 73) identification varied between 32% (BCA) and 69% (CS4 and CCA) after 24 hours and 68% (BBL) and 82% (BCA) after 48 hours incubation, while specificity ranged between 62% (BBL) and 81% (CCA) after 24 hours and 82% (BBL) and 85% (CS4) after 48 hours. Conclusion CS4 and BCA are recommended for routine identification of Candida species in human samples.

Periprosthetic infection (PPI) is a devastating complication in joint replacement surgery. On the background of an aging population, the number of joint replacements and associated complications is expected to increase. The capability for biofilm formation and the increasing resistance of different microbes to antibiotics have complicated the treatment of PPI, requiring the need for the development of alternative treatment options. The bactericidal effect of the naturally occurring amino alcohol sphingosine has already been reported. In our study, we demonstrate the antimicrobial efficacy of sphingosine on three different strains of biofilm producing Staphylococcus epidermidis, representing one of the most frequent microbes involved in PPI. In an in vitro analysis, sphingosine's capability for prevention and treatment of biofilm-contamination on different common orthopedic implant surfaces was tested. Coating titanium implant samples with sphingosine not only prevented implant contamination but also revealed a significant reduction of biofilm formation on the implant surfaces by 99.942%. When testing the antimicrobial efficacy of sphingosine on sessile biofilm-grown Staphylococcus epidermidis, sphingosine solution was capable to eliminate 99.999% of the bacteria on the different implant surfaces, i.e., titanium, steel, and polymethylmethacrylate. This study provides evidence on the antimicrobial efficacy of sphingosine for both planktonic and sessile biofilm-grown Staphylococcus epidermidis on contaminated orthopedic implants. Sphingosine may provide an effective and cheap treatment option for prevention and reduction of infections in joint replacement surgery. Key messages • Here we established a novel technology for prevention of implant colonization by sphingosine-coating of orthopedic implant materials. • Sphingosine-coating of orthopedic implants prevented bacterial colonization and significantly reduced biofilm formation on implant surfaces by 99.942%. • Moreover, sphingosine solution was capable to eliminate 99.999% of sessile biofilm-grown Staphylococcus epidermidis on different orthopedic implant surfaces.