Investigating best practice for specimen preparation for biological testing of root canal sealers

INTRODUCTION: Biological characterization of root canal sealers is important as it assesses the ability of the root canal sealer to exert antimicrobial properties thus avoiding treatment failures caused by microbial challenge and also assess the cytotoxic effect on the periapical tissues. Assessment of the biological testing of root canal sealers necessitates the sterilisation of the materials prior to evaluation. This study aims to analyse the influence of various sterilisation techniques conducted prior to biological testing on the microstructure and surface properties of endodontic sealers. Assessment of the initial microbial contamination on the material was also undertaken.METHODS: Four commercial sealers were investigated. The sealers were either prepared in a laminar flow cabinet or on a laboratory bench top under ambient conditions. Each group was further divided into 5 groups (n = 3) based on the sterilization technique:1) ethanol-10 mins, 2) ultraviolet-1 h, 3) ethanol-10 mins + ultraviolet-1 h, 4) autoclave, and 5) no sterilisation (control). Microbial levels in the materials were assessed by plate streaking technique. The materials were characterized by scanning electron microscopy and energy dispersive spectroscopy, and Fourier transform infrared spectroscopy, before and after sterilisation, to assess any changes in microstructure and chemical composition.RESULTS: All the materials did not exhibit contamination when prepared in laminar flow chamber in sterile conditions compared with sealers prepared on the bench top. Three of the commercial materials showed changes in microstructure while one (TotalFill) was not affected by the sterilisation. AH Plus and BioRoot RCS exhibited alterations in water and alcohol peaks in FT-IR while the single syringe sealers (TotalFill and BioRoot Flow) showed no changes.CONCLUSIONS: Sterilisation methods cause physical and chemical alterations to sealers. Material preparation should be performed in a laminar flow cabinet and a test for sterility should be performed prior to any biological testing being undertaken. If the materials are not sterile, assessment of the effects of the sterilization methods is recommended.</p

Noise reduction strategies in metagenomic chromosome confirmation capture to link antibiotic resistance genes to microbial hosts

The gut microbiota is a reservoir for antimicrobial resistance genes (ARGs). With current sequencing methods, it is difficult to assign ARGs to their microbial hosts, particularly if these ARGs are located on plasmids. Metagenomic chromosome conformation capture approaches (meta3C and Hi-C) have recently been developed to link bacterial genes to phylogenetic markers, thus potentially allowing the assignment of ARGs to their hosts on a microbiome-wide scale. Here, we generated a meta3C dataset of a human stool sample and used previously published meta3C and Hi-C datasets to investigate bacterial hosts of ARGs in the human gut microbiome. Sequence reads mapping to repetitive elements were found to cause problematic noise in, and may importantly skew interpretation of, meta3C and Hi-C data. We provide a strategy to improve the signal-to-noise ratio by discarding reads that map to insertion sequence elements and to the end of contigs. We also show the importance of using spike-in controls to quantify whether the cross-linking step in meta3C and Hi-C protocols has been successful. After filtering to remove artefactual links, 87 ARGs were assigned to their bacterial hosts across all datasets, including 27 ARGs in the meta3C dataset we generated. We show that commensal gut bacteria are an important reservoir for ARGs, with genes coding for aminoglycoside and tetracycline resistance being widespread in anaerobic commensals of the human gut

Comparison of culture based methods for the isolation of Clostridium difficile from stool samples in a research setting

AbstractEffective isolation of Clostridium difficile from stool samples is important in the research setting, especially where low numbers of spores/vegetative cells may be present within a sample. In this study, three protocols for stool culture were investigated to find a sensitive, cost effective and timely method of C. difficile isolation. For the initial enrichment step, the effectiveness of two different rich media, cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared. For the comparison of four different, selective solid media; Cycloserine-cefoxitin fructose agar (CCFA), Cycloserine-cefoxitin egg yolk agar (CCEY), ChromID C. difficile and tryptone soy agar (TSA) with 5% sheep's blood with and without preceding broth enrichment were used. As a means to enable differentiation between C. difficile and other fecal flora, the effectiveness of the inclusion of a pH indictor (1% Neutral Red), was also evaluated. The data derived indicated that CCFB is more sensitive than CCMB-TAL, however, the latter had an improved recovery rate. A broth enrichment step had a reduced sensitivity over direct plating. ChromID C. difficile showed the best recovery rate whereas CCEY egg yolk agar was the most sensitive of the four. The addition of 1% Neutral Red did not show sufficient colour change when added to CCEY egg yolk agar to be used as a differential medium. For a low cost, timely and sensitive method of isolating C. difficile from stool samples we recommend direct plating onto CCEY egg yolk agar after heat shock

Clostridium difficile modulates host innate immunity via toxin-independent and dependent mechanism(s)

Clostridium difficile infection (CDI) is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea and currently represents a significant health burden. Although the role and contribution of C. difficile toxins to disease pathogenesis is being increasingly understood, at present other facets of C. difficile-host interactions, in particular, bacterial-driven effects on host immunity remain less studied. Using an ex-vivo model of infection, we report that the human gastrointestinal mucosa elicits a rapid and significant cytokine response to C. difficile. Marked increase in IFN-γ with modest increase in IL-22 and IL-17A was noted. Significant increase in IL-8 suggested potential for neutrophil influx while presence of IL-12, IL-23, IL-1β and IL-6 was indicative of a cytokine milieu that may modulate subsequent T cell immunity. Majority of C. difficile-driven effects on murine bone-marrow-derived dendritic cell (BMDC) activation were toxin-independent; the toxins were however responsible for BMDC inflammasome activation. In contrast, human monocyte-derived DCs (mDCs) released IL-1β even in the absence of toxins suggesting host-specific mediation. Infected DC-T cell crosstalk revealed the ability of R20291 and 630 WT strains to elicit a differential DC IL-12 family cytokine milieu which culminated in significantly greater Th1 immunity in response to R20291. Interestingly, both strains induced a similar Th17 response. Elicitation of mucosal IFN-γ/IL-17A and Th1/Th17 immunity to C. difficile indicates a central role for this dual cytokine axis in establishing antimicrobial immunity to CDI

Coinfection and Emergence of Rifamycin Resistance during a Recurrent Clostridium difficile Infection

Clostridium difficile (Peptoclostridium difficile) is a common health care-associated infection with a disproportionately high incidence in elderly patients. Disease symptoms range from mild diarrhea to life-threatening pseudomembranous colitis. Around 20% of patients may suffer recurrent disease, which often requires rehospitalization of patients. C. difficile was isolated from stool samples from a patient with two recurrent C. difficile infections. PCR ribotyping, whole-genome sequencing, and phenotypic assays were used to characterize these isolates. Genotypic and phenotypic screening of C. difficile isolates revealed multiple PCR ribotypes present and the emergence of rifamycin resistance during the infection cycle. Understanding both the clinical and bacterial factors that contribute to the course of recurrent infection could inform strategies to reduce recurrence. (This study has been registered at ClinicalTrials.gov under registration no. NCT01670149.

Clostridioides difficile binary toxin binding component (cdtb) increases virulence in a hamster model

Background Clostridioides difficile is the leading cause of hospital-acquired gastrointestinal infection, in part due to the existence of binary toxin (CDT)-expressing hypervirulent strains. Although the effects of the CDT holotoxin on disease pathogenesis have been previously studied, we sought to investigate the role of the individual components of CDT during in vivo infection. Methods To determine the contribution of the separate components of CDT during infection, we developed strains of C difficile expressing either CDTa or CDTb individually. We then infected both mice and hamsters with these novel mutant strains and monitored them for development of severe illness. Results Although expression of CDTb without CDTa did not induce significant disease in a mouse model of C difficile infection, we found that complementation of a CDT-deficient C difficile strain with CDTb alone restored virulence in a hamster model of C difficile infection. Conclusions Overall, this study demonstrates that the binding component of C difficile binary toxin, CDTb, contributes to virulence in a hamster model of infection

What's a SNP between friends: The lineage of Clostridioides difficile R20291 can effect research outcomes

Clostridioides difficile R20291 is the most studied PCR-Ribotype 027 isolate. The two predominant lineages of this hypervirulent strain, however, exhibit substantive phenotypic differences and possess genomes that differ by a small number of nucleotide changes. It is important that the source of R20291 is taken into account in research outcomes

Conjugated Polyimidazole Nanoparticles as Biodegradable Electrode Materials for Organic Batteries

Conjugated polymers are promising active materials for batteries. Batteries not only need to have high energy density but should also combine safe handling with recyclability or biodegradability after reaching their end-of-life. Here, π-conjugated polyimidazole particles are developed, which are prepared using atom economic direct arylation adapted to a dispersion polymerization protocol. The synthesis yields polyimidazole nanoparticles of tunable size and narrow dispersity. In addition, the degree of crosslinking of the polymer particles can be controlled. It is demonstrated that the polyimidazole nanoparticles can be processed together with carbon black and biodegradable carboxymethyl cellulose binder as an active material for organic battery electrodes. Electrochemical characterization shows that a higher degree of crosslinking significantly improves the electrochemical performance and leads to clearer oxidation and reduction signals of the polymer. Polyimidazole as part of the composite electrode shows complete degradation by exposure to composting bacteria over the course of 72 h

The glucosyltransferase activity of C. difficile toxin b is required for disease pathogenesis

© 2020 Bilverstone et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition