Aspergillus fumigatus is an increasingly prevalent opportunistic fungal pathogen of various immunocompromised individuals. It has the ability to form filaments within the lungs, producing dense intertwined mycelial balls, which are difficult to treat. The aim of this study was to develop a suitable model of A. fumigatus to examine the effects of antifungal challenge on these intertwined filamentous communities. A. fumigatus NCPF 7367 growth conditions were optimized on both Thermanox coverslips and on flat-bottomed microtitre plates to establish optimal conidial seeding densities. Isolates were treated with itraconazole, voriconazole, amphotericin B and caspofungin and their overall killing efficiency was measured using an XTT formazan metabolic dye assay. This was compared with the CLSI (formerly NCCLS) methodology of broth microdilution of moulds (standard M38-A). It was shown that 1¾10 5 conidia ml "1 in RPMI 1640 was the optimum concentration of spores for biofilm formation. Filamentous growth characteristics were not observed until 10 h incubation, followed by an exponential increase in the biofilm biomass (hyphae and extracellular material) and cellular activity (metabolism). When susceptibility testing of biofilms was compared with that of planktonic cells by CLSI broth microdilution testing, all antifungal drugs were at least 1000 times less effective at reducing the overall metabolic activity of 90 % of the cells. Overall, this study showed that A. fumigatus has the ability to form coherent multicellular biofilm structures that are resistant to the effects of antifungal drugs.

Highlights  Alopecia areata is a polygenic and multifactorial autoimmune disease characterised by non-scarring hair loss.  Autoreactive CD8 + , CD4 + , natural killer cells and plasmacytoid dendritic cells infiltrate around the hair follicles during the growth (anagen) phase.  Increased cytokine activity, particularly IFN-γ, results in disruption of the hair follicle immune privilege and premature termination of the anagen phase, followed by hair follicle atrophy and dystrophy in persistent disease.

In subjects with periodontitis, P. gingivalis infection may be responsible for inducing autoimmune responses that characterize rheumatoid arthritis.

BackgroundThe aim of this study was to characterise the microbiome of new and recurrent diabetic foot ulcers using 16S amplicon sequencing (16S AS), allowing the identification of a wider range of bacterial species that may be important in the development of chronicity in these debilitating wounds. Twenty patients not receiving antibiotics for the past three months were selected, with swabs taken from each individual for culture and 16S AS. DNA was isolated using a combination of bead beating and kit extraction. Samples were sequenced on the Illumina Hiseq 2500 platform.ResultsConventional laboratory culture showed positive growth from only 55 % of the patients, whereas 16S AS was positive for 75 % of the patients (41 unique genera, representing 82 different operational taxonomic units (OTU’s). S. aureus was isolated in 72 % of culture-positive samples, whereas the most commonly detected bacteria in all ulcers were Peptoniphilus spp., Anaerococcus spp. and Corynebacterium spp., with the addition of Staphylococcus spp. in new ulcers. The majority of OTU’s residing in both new and recurrent ulcers (over 67 %) were identified as facultative or strict anaerobic Gram-positive organisms. Principal component analysis (PCA) showed no difference in clustering between the two groups (new and recurrent ulcers).ConclusionsThe abundance of anaerobic bacteria has important implications for treatment as it suggests that the microbiome of each ulcer “starts afresh” and that, although diverse, are not distinctly different from one another with respect to new or recurrent ulcers. Therefore, when considering antibiotic therapy the duration of current ulceration may be a more important consideration than a history of healed ulcer.

Of 40 ready-to-eat salads, 3 (7.5%) were positive for Clostridium diffi cile by PCR. Two isolates were PCR ribotype 017 (toxin A-, B+), and 1 was PCR ribotype 001. Isolates were susceptible to vancomycin and metronidazole but variably resistant to other antimicrobial drugs. Ready-to-eat salads may be potential sources for virulent C. diffi cile.

Nitric oxide (NO) can be generated endogenously via NO synthases or via the diet following the action of symbiotic nitrate-reducing bacteria in the oral cavity. Given the important role of NO in smooth muscle control there is an intriguing suggestion that cardiovascular homeostasis may be intertwined with the presence of these bacteria. Here, we measured the abundance of nitrate-reducing bacteria in the oral cavity of 25 healthy humans using 16S rRNA sequencing and observed, for 3.5 h, the physiological responses to dietary nitrate ingestion via measurement of blood pressure, and salivary and plasma NO metabolites. We identified 7 species of bacteria previously known to contribute to nitrate-reduction, the most prevalent of which were Prevotella melaninogenica and Veillonella dispar. Following dietary nitrate supplementation, blood pressure was reduced and salivary and plasma nitrate and nitrite increased substantially. We found that the abundance of nitrate-reducing bacteria was associated with the generation of salivary nitrite but not with any other measured variable. To examine the impact of bacterial abundance on pharmacokinetics we also categorised our participants into two groups; those with a higher abundance of nitrate reducing bacteria (> 50%), and those with a lower abundance (< 50%). Salivary nitrite production was lower in participants with lower abundance of bacteria and these individuals also exhibited slower salivary nitrite pharmacokinetics. We therefore show that the rate of nitrate to nitrite reduction in the oral cavity is associated with the abundance of nitrate-reducing bacteria. Nevertheless, higher abundance of these bacteria did not result in an exaggerated plasma nitrite response, the best known marker of NO bioavailability. These data from healthy young adults suggest that the abundance of oral nitrate-reducing bacteria does not influence the generation of NO through the diet, at least when the host has a functional minimum threshold of these microorganisms.

Epidemiological data indicate a link between rheumatoid arthritis (RA) and periodontal disease (PD). In vitro and in vivo studies have sought to dissect potential mechanisms by which PD may contribute to initiation and progression of RA. However, these are both multifactorial, chronic diseases, and their complex etiologies and pathogenesis themselves remain incompletely understood. Could there really be an etiological link or does this simply represent a statistical coincidence muddied by common risk factors? This review seeks to provide background on these two diseases in the context of recent discoveries suggesting that their pathogenesis may be related. In particular, the process of citrullination, a post-translational protein modification, has been highlighted as a process common to both diseases. The evidence for a relationship between the diseases is explored and its potential mechanisms discussed.

Cytokines mediate the balance between protective and destructive immunity in periodontitis. We sought to investigate the role of IL-33 in periodontitis. The expression of IL-33 in gingival tissue from healthy controls (n = 10) and patients with chronic periodontitis (n = 17) was investigated. Based on a murine model of periodontal disease, the function of IL-33 was determined first by administration of exogenous IL-33 and second by inhibition of IL-33 signaling using mice deficient in the IL-33 receptor ST2. Alveolar bone level, serum antibody, and lymphocyte responses were assessed in the murine model. Expression of IL-33 and ST2 was elevated in gingival tissues from patients with chronic periodontitis as compared with healthy tissues (P < 0.05). Similarly, Il33 expression was higher in periodontal tissues of Porphyromonas gingivalis-infected mice as compared with sham-infected controls (P < 0.05). IL-33 treatment of P. gingivalis-infected mice significantly exacerbated alveolar bone loss when compared with infection or IL-33 treatment alone (P < 0.001). Conversely, P. gingivalis infection-induced alveolar bone loss was attenuated in mice lacking ST2. The percentages of T and B lymphocytes expressing nuclear factor κB ligand (RANKL) in the gingival tissues and T lymphocytes expressing RANKL in the cervical draining lymph nodes were higher in IL-33-treated P. gingivalis-infected mice versus phosphate buffered saline-treated P. gingivalis-infected controls (all P < 0.001). Targeting the RANKL pathway by osteoprotegerin administration abrogated periodontal bone destruction in P. gingivalis-infected, IL-33-treated mice. These data demonstrate a previously unrecognized role for IL-33 in exacerbating bone loss in a RANKL-dependent manner in the context of bacterial infection and suggest that this pathway may be amenable to manipulation as a novel therapeutic target in periodontitis.