Hypervirulent R20291 Clostridioides difficile spores show disinfection resilience to sodium hypochlorite despite structural changes.

BACKGROUND: Clostridioides difficile is a spore forming bacterial species and the major causative agent of nosocomial gastrointestinal infections. C. difficile spores are highly resilient to disinfection methods and to prevent infection, common cleaning protocols use sodium hypochlorite solutions to decontaminate hospital surfaces and equipment. However, there is a balance between minimising the use of harmful chemicals to the environment and patients as well as the need to eliminate spores, which can have varying resistance properties between strains. In this work, we employ TEM imaging and Raman spectroscopy to analyse changes in spore physiology in response to sodium hypochlorite. We characterize different C. difficile clinical isolates and assess the chemical's impact on spores' biochemical composition. Changes in the biochemical composition can, in turn, change spores' vibrational spectroscopic fingerprints, which can impact the possibility of detecting spores in a hospital using Raman based methods. RESULTS: We found that the isolates show significantly different susceptibility to hypochlorite, with the R20291 strain, in particular, showing less than 1 log reduction in viability for a 0.5% hypochlorite treatment, far below typically reported values for C. difficile. While TEM and Raman spectra analysis of hypochlorite-treated spores revealed that some hypochlorite-exposed spores remained intact and not distinguishable from controls, most spores showed structural changes. These changes were prominent in B. thuringiensis spores than C. difficile spores. CONCLUSION: This study highlights the ability of certain C. difficile spores to survive practical disinfection exposure and the related changes in spore Raman spectra that can be seen after exposure. These findings are important to consider when designing practical disinfection protocols and vibrational-based detection methods to avoid a false-positive response when screening decontaminated areas.

The Effect of cytoskeleton inhibitors on coccolith morphology in Coccolithus braarudii and Scyphosphaera apsteinii.

The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was unclear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.

Retrospective Comparison of Patients ≥ 80 Years With Atrial Fibrillation Prescribed Either an FDA-Approved Reduced or Full Dose Direct-Acting Oral Anticoagulant.

Direct-acting oral anticoagulants (DOACs) represent the standard for preventing stroke and systemic embolization (SSE) in patients with atrial fibrillation (AF). There is limited information for patients ≥ 80 years. We report a retrospective analysis of AF patients ≥ 80 years prescribed either a US Food and Drug Administration (FDA)-approved reduced (n = 514) or full dose (n = 199) DOAC (Dabigatran, Rivaroxaban, or Apixaban) between January 1st, 2011 (first DOAC commercially available) and May 31st, 2017. The following multivariable differences in baseline characteristics were identified: patients prescribed a reduced dose DOAC were older (p < 0.001), had worse renal function (p = 0.001), were more often prescribed aspirin (p = 0.004) or aspirin and clopidogrel (p < 0.001), and more often had new-onset AF (p = 0.001). SSE and central nervous system (CNS) bleed rates were low and not different (1.02 vs 0 %/yr and 1.45 vs 0.44 %/yr) for the reduced and full dose groups, respectively. For non-CNS bleeds, rates were 10.89 vs 4.15 %/yr (p < 0.001, univariable) for the reduced and full doses, respectively. The mortality rate was 6.24 vs 1.75 %/yr (p = 0.001, univariable) for the reduced and full doses. Unlike the non-CNS bleed rate, mortality rate differences remained significant when adjusted for baseline characteristics. Thus, DOACs in patients ≥ 80 with AF effectively reduce SSE with a low risk of CNS bleeding, independent of DOAC dose. The higher non-CNS bleed rate and not the mortality rate is explained by the higher risk baseline characteristics in the reduced DOAC dose group. Further investigation of the etiology of non-CNS bleeds and mortality is warranted.

Comparison of Low-Dose Direct Acting Anticoagulant and Warfarin in patients Aged ≥80 years With Atrial Fibrillation.

Low dose direct acting oral anticoagulants (LDDOACS) were approved for elderly atrial Fibrillation (AF) patients with limited information. A retrospective analysis collecting baseline characteristics and outcomes in AF patients ≥ 80 prescribed LDDOAC or warfarin (W), from a multidisciplinary practice between 1/1/11 (First LDDOAC available) and 5/31/17 was conducted. From 9660 AF patients, 514 ≥ 80 received a LDDOAC and 422 W. A multivariable comparison found LDDOAC patients were older (p <0.001), had lower creatinine clearance (CrCl) (p = 0.006), used more anti-platelet drugs (p <0.001), and more often had new onset AF verses those prescribed W (p <0.001). There were no clinically significant differences among those patients receiving Dabigatran 75 mgs BID (D), Rivaroxaban 15mgs (R) or Apixaban 2.5mgs BID (A). Forty-eight and 50% of the patients remained on their LDDOAC or W for the observation period (p = 0.55). Stroke/systemic embolism (SSE) and CNS bleeds were 1.16 vs 2.22%/yr., (p = 0.143) and 1.46 vs 0.93%/yr., (p = 0.24). Mortality and major bleeds were 6.26 vs 1.67%/yr., and 12.3vs 3.77%/yr. (p <0.001). SSE were 1.1%/yr for D, R, and A (p = 0.94). CNS bleeds were 2.2 for D, 1.7 for R and 0.8%/yr. for A: p = 0.53. Major bleeding was: 14.3 for D, 14.1 for R and 9.1%/yr. for A, p = 0.048 (with A < R, p = 0.01). Mortality was 5.5 for D, 4.2 for R and 9.5% for A, p = 0.031. In conclusion, half the patients remained on their assigned anti-coagulant. SSE and intracranial bleed rates were similar and low. Major bleeds and deaths were different between groups emphasizing the need for prospective randomized trials in this growing population with AF.

Role of silicon in the development of complex crystal shapes in coccolithophores.

The development of calcification by the coccolithophores had a profound impact on ocean carbon cycling, but the evolutionary steps leading to the formation of these complex biomineralized structures are not clear. Heterococcoliths consisting of intricately shaped calcite crystals are formed intracellularly by the diploid life cycle phase. Holococcoliths consisting of simple rhombic crystals can be produced by the haploid life cycle stage but are thought to be formed extracellularly, representing an independent evolutionary origin of calcification. We use advanced microscopy techniques to determine the nature of coccolith formation and complex crystal formation in coccolithophore life cycle stages. We find that holococcoliths are formed in intracellular compartments in a similar manner to heterococcoliths. However, we show that silicon is not required for holococcolith formation and that the requirement for silicon in certain coccolithophore species relates specifically to the process of crystal morphogenesis in heterococcoliths. We therefore propose an evolutionary scheme in which the lower complexity holococcoliths represent an ancestral form of calcification in coccolithophores. The subsequent recruitment of a silicon-dependent mechanism for crystal morphogenesis in the diploid life cycle stage led to the emergence of the intricately shaped heterococcoliths, enabling the formation of the elaborate coccospheres that underpin the ecological success of coccolithophores.

Insights into growth kinetics and roles of enzymes of Krebs' cycle and sulfur oxidation during exochemolithoheterotrophic growth of Achromobacter aegrifaciens NCCB 38021 on succinate with thiosulfate as the auxiliary electron donor.

Achromobacter aegrifaciens NCCB 38021 was grown heterotrophically on succinate versus exochemolithoheterotrophically on succinate with thiosulfate as auxiliary electron donor. In batch culture, no significant differences in specific molar growth yield or specific growth rate were found for the two growth conditions, but in continuous culture in the succinate-limited chemostat, the maximum specific growth yield coefficient increased by 23.3% with thiosulfate present, consistent with previous studies of endo- and exochemolithoheterotrophs and thermodynamic predictions. Thiosulfate oxidation was coupled to respiration at cytochrome c551, and thiosulfate-dependent ATP biosynthesis occurred. Specific activities of cytochrome c-linked thiosulfate dehydrogenase (E.C. 1.8.2.2) and two other enzymes of sulfur metabolism were significantly higher in exochemolithoheterotrophically grown cell extracts, while those of succinyl-transferring 2-oxoglutarate dehydrogenase (E.C. 1.2.4.2), fumarate hydratase (E.C. 4.2.1.2) and malate dehydrogenase (NAD+, E.C. 1.1.1.37) were significantly lower-presumably owing to less need to generate reducing equivalents during Krebs' cycle, since they could be produced from thiosulfate oxidation.

Improving estimation of puma (Puma concolor) population density: clustered camera-trapping, telemetry data, and generalized spatial mark-resight models.

Obtaining reliable population density estimates for pumas (Puma concolor) and other cryptic, wide-ranging large carnivores is challenging. Recent advancements in spatially explicit capture-recapture models have facilitated development of novel survey approaches, such as clustered sampling designs, which can provide reliable density estimation for expansive areas with reduced effort. We applied clustered sampling to camera-traps to detect marked (collared) and unmarked pumas, and used generalized spatial mark-resight (SMR) models to estimate puma population density across 15,314 km2 in the southwestern USA. Generalized SMR models outperformed conventional SMR models. Integrating telemetry data from collars on marked pumas with detection data from camera-traps substantially improved density estimates by informing cryptic activity (home range) center transiency and improving estimation of the SMR home range parameter. Modeling sex of unmarked pumas as a partially identifying categorical covariate further improved estimates. Our density estimates (0.84-1.65 puma/100 km2) were generally more precise (CV = 0.24-0.31) than spatially explicit estimates produced from other puma sampling methods, including biopsy darting, scat detection dogs, and regular camera-trapping. This study provides an illustrative example of the effectiveness and flexibility of our combined sampling and analytical approach for reliably estimating density of pumas and other wildlife across geographically expansive areas.

Case Report of Rarely Described Polymorphous Low-Grade Neuroepithelial Tumor of the Young and Comparison with Oligodendroglioma.

BACKGROUND: Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a central nervous system tumor that shares many qualities with oligodendroglioma but is rarely and only very recently described as a distinct entity in the literature. CASE DESCRIPTION: A previously healthy, 19-year-old man presented with new onset of seizures. Imaging showed an intracranial mass, which was treated with surgical removal. Preoperative and postoperative magnetic resonance imaging, histopathologic examination, genetic testing, and immunohistochemical staining all supported a diagnosis of PLNTY. CONCLUSIONS: Diagnostic investigation of PLNTY shows many similarities with oligodendroglioma, and thus these entities can be mistaken for one another. Certain studies are needed to distinguish PLNTY and other dysembryoplastic neuroepithelial tumors, such as oligodendroglioma.

Vesicular glutamate release from central axons contributes to myelin damage.

The axon myelin sheath is prone to injury associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor activation but the source of glutamate in this context is unknown. Myelin damage results in permanent action potential loss and severe functional deficit in the white matter of the CNS, for example in ischemic stroke. Here, we show that in rats and mice, ischemic conditions trigger activation of myelinic NMDA receptors incorporating GluN2C/D subunits following release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath. Glial sources of glutamate such as reverse transport did not contribute significantly to this phenomenon. We demonstrate selective myelin uptake and retention of a GluN2C/D NMDA receptor negative allosteric modulator that shields myelin from ischemic injury. The findings potentially support a rational approach toward a low-impact prophylactic therapy to protect patients at risk of stroke and other forms of excitotoxic injury.

Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper.

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the "gut sac" method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner.

Phaeoviruses discovered in kelp (Laminariales).

Phaeoviruses are latent double-stranded DNA viruses that insert their genomes into those of their brown algal (Phaeophyceae) hosts. So far these viruses are known only from members of the Ectocarpales, which are small and short-lived macroalgae. Here we report molecular and morphological evidence for a new Phaeovirus cluster, referred to as sub-group C, infecting kelps (Laminariales) of the genera Laminaria and Saccharina, which are ecologically and commercially important seaweeds. Epifluorescence and TEM observations indicate that the Laminaria digitata Virus (LdigV), the type species of sub-group C, targets the host nucleus for its genome replication, followed by gradual degradation of the chloroplast and assembly of virions in the cytoplasm of both vegetative and reproductive cells. This study is the first to describe phaeoviruses in kelp. In the field, these viruses infected two thirds of their host populations; however, their biological impact remains unknown.

Ingestion of metal-nanoparticle contaminated food disrupts endogenous microbiota in zebrafish (Danio rerio).

Nanoparticles (NPs) can be ingested by organisms, and NPs with antimicrobial properties may disrupt beneficial endogenous microbial communities and affect organism health. Zebrafish were fed diets containing Cu-NPs or Ag-NPs (500 mg kg(-1) food), or an appropriate control for 14 d. Intestinal epithelium integrity was examined by transmission electron microscopy, and microbial community structure within the intestine was assessed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA. No lesions were observed in intestinal epithelia; however, presence of NPs in diets changed intestinal microbial community structure. In particular, some beneficial bacterial strains (e.g., Cetobacterium somerae) were suppressed to non-detectable levels by Cu-NP exposure, and two unidentified bacterial clones from the Firmicutes phylum were sensitive (not detected) to Cu, but were present in Ag and control fish. Unique changes in zebrafish microbiome caused by exposure to Ag-NP and Cu-NP indicate that NP ingestion could affect digestive system function and organism health.

Effect of dietary alginic acid on juvenile tilapia (Oreochromis niloticus) intestinal microbial balance, intestinal histology and growth performance.

The aim of the present study was to assess the effect of a commercial alginic acid source (Ergosan) on tilapia Oreochromis niloticus intestinal microbial balance, intestinal morphology, and growth parameters. Fish were fed a basal control diet or the basal diet plus a source of alginic acid (5 g kg(-1) Ergosan; Schering-Plough Aquaculture, UK) for 9 weeks. At the end of the trial, light and electron microscopy demonstrated that the morphology of the intestinal tract at the gross and ultra-structural level was not affected by dietary alginic acid inclusion. Both groups of fish displayed healthy, normal morphology with no signs of disease, cell or tissue damage. Intestinal epithelial leucocyte infiltration was not affected by dietary alginic acid. Molecular bacterial profiles derived from PCR-DGGE illustrated highly similar microbial communities (both within the lumen and associated with the intestinal mucosa) in the respective treatment groups. Microbial ecological parameters (e.g. species diversity and richness) also remained unaffected. Although not significant, trends towards elevated survival and body protein content were observed in the alginic acid-fed fish. These results are suggestive that alginic acid does not adversely impact the indigenous gastrointestinal microbial balance and subsequently does not impact upon the epithelial brush border integrity. Validation of non-detrimental impacts of immunostimulatory products on gastric microbiota and epithelial integrity should be pursued in future studies as maintaining microbial balance and epithelial integrity is essential for proper gut functionality.

A unicellular algal virus, Emiliania huxleyi virus 86, exploits an animal-like infection strategy.

Emiliania huxleyi virus 86 (EhV-86) belongs to the family Phycodnaviridae, a group of viruses that infect a wide range of freshwater and marine eukaryotic algae. Phycodnaviridae is one of the five families that belong to a large and phylogenetically diverse group of viruses known as nucleocytoplasmic large dsDNA viruses (NCLDVs). To date, our understanding of algal NCLDV entry is based on the entry mechanisms of members of the genera Chlorovirus and Phaeovirus, both of which consist of non-enveloped viruses that 'inject' their genome into their host via a viral inner-membrane host plasma membrane fusion mechanism, leaving an extracellular viral capsid. Using a combination of confocal and electron microscopy, this study demonstrated for the first time that EhV-86 differs from its algal virus counterparts in two fundamental areas. Firstly, its capsid is enveloped by a lipid membrane, and secondly, EhV-86 enters its host via either an endocytotic or an envelope fusion mechanism in which an intact nucleoprotein core still encapsulated by its capsid is seen in the host cytoplasm. Real-time fluorescence microscopy showed that viral internalization and virion breakdown took place within the host on a timescale of seconds. At around 4.5 h post-infection, virus progeny were released via a budding mechanism during which EhV-86 virions became enveloped with host plasma membrane. EhV-86 therefore appears to have an infection mechanism different from that employed by other algal NCLDVs, with entry and exit strategies showing a greater analogy to animal-like NCLDVs.