Diverse bacterial species contribute to antibiotic-associated diarrhoea and gastrointestinal damage

Objectives: Antibiotic-associated diarrhoea (AAD) caused by C. difficile is one of the most common nosocomial infections, however, little is known about infections related to antimicrobial use for pathogens other than C. difficile. We therefore aimed to provide insight into other bacterial causes of AAD, and how infection with these pathogens causes damage in the dysbiotic gut. Methods: Clinical isolates from C. difficile-negative AAD patients were whole genome sequenced for in silico analysis of potential virulence factors and antimicrobial resistance determinants. A mouse model of infection was developed to assess the capacity of these isolates to cause gastrointestinal damage, which was analysed by studying specific markers in the gastrointestinal mucosa of infected mice. Results: Several bacterial pathogens were isolated from patients with C. difficile-negative AAD. Each isolate showed the potential for virulence based on encoded virulence factors, as well as most showing antimicrobial resistance in vitro. Isolates of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were tested in the mouse model of infection, inducing damage primarily in the small intestine, affecting adherens junction integrity, cellular polarity, and cellular proliferation. Conclusions: Several pathogens of clinical importance other than C. difficile are able to cause gastrointestinal infection following antimicrobial-mediated dysbiosis. The virulence potential and multidrug resistance identified in these isolates illuminates the importance of further diagnostic screening in cases of C. difficile-negative AAD

Structural characterization of Clostridium sordellii spores of diverse human, animal, and environmental origin and comparison to Clostridium difficile spores

© 2017 Rabi et al. Clostridium sordellii is an often-lethal bacterium causing human and animal disease. Crucial to the infectious cycle of C. sordellii is its ability to produce spores, which can germinate into toxin-producing vegetative bacteria under favorable conditions. However, structural details of the C. sordellii spore are lacking. Here, we used a range of electron microscopy techniques together with superresolution optical microscopy to characterize the C. sordellii spore morphology with an emphasis on the exosporium. The C. sordellii spore is made up of multiple layers with the exosporium presenting as a smooth balloon-like structure that is open at the spore poles. Focusing on the outer spore layers, we compared the morphologies of C. sordellii spores derived from different strains and determined that there is some variation between the spores, most notably with spores of some strains having tubular appendages. Since Clostridium difficile is a close relative of C. sordellii, their spores were compared by electron microscopy and their exosporia were found to be distinctly different from each other. This study therefore provides new structural details of the C. sordellii spore and offers insights into the physical structure of the exosporium across clostridial species

Binaphthyl-1,2,3-triazole peptidomimetics with activity against Clostridium difficile and other pathogenic bacteria

Clostridium difficile (C. difficile) is a problematic Gram positive bacterial pathogen causing moderate to severe gastrointestinal infections. Based on a lead binaphthyl-tripeptide dicationic antimicrobial, novel mono-, di- and tri-peptidomimetic analogues targeting C. difficile were designed and synthesized incorporating one, two or three d-configured cationic amino acid residues, with a common 1,2,3-triazole ester isostere at the C-terminus. Copper- and ruthenium-click chemistry facilitated the generation of a 46 compound library for in vitro bioactivity assays, with structure-activity trends over the largest compound subset revealing a clear advantage to triazole-substitution with a linear or branched hydrophobic group. The most active compounds were dicationic-dipeptides where the triazole was substituted with a 4- or 5-cyclohexylmethyl or 4,5-diphenyl moiety, providing MICs of 4 μg mL-1 against three human isolates of C. difficile. Further biological screening revealed significant antimicrobial activity for several compounds against other common bacterial pathogens, both Gram positive and negative, including S. aureus (MICs ≥2 μg mL-1), S. pneumoniae (MICs ≥1 μg mL-1), E. coli (MICs ≥4 μg mL-1), A. baumannii (MICs ≥4 μg mL-1) and vancomycin-resistant E. faecalis (MICs ≥4 μg mL-1)

A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis.

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely supresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development

The effect of area and isolation on insular dwarf proboscideans

Aim We investigated the hypothesis that insular body size of fossil elephants is directly related to isolation and surface area of the focal islands. Location Palaeo-islands worldwide. Methods We assembled data on the geographical characteristics (area and isolation) of islands and body size evolution of palaeo-insular species for 22 insular species of fossil elephants across 17 islands. Results Our results support the generality of the island rule in the sense that all but one of the elephants experienced dwarfism on islands. The smallest islands generally harbour the smallest elephants. We found no support for the hypothesis that body size of elephants declines with island isolation. Body size is weakly and positively correlated with island area for proboscideans as a whole, but more strongly correlated for Stegodontidae when considered separately. Average body size decrease is much higher when competitors are present. Main conclusions Body size in insular elephants is not significantly correlated with the isolation of an island. Surface area, however, is a significant predictor of body size. The correlation is positive but relatively weak; c. 23% of the variation is explained by surface area. Body size variation seems most strongly influenced by ecological interactions with competitors, possibly followed by time in isolation. Elephants exhibited far more extreme cases of dwarfism than extant insular mammals, which is consistent with the substantially more extended period of deep geological time that the selective pressures could act on these insular populations

A series of three cases of severe Clostridium difficile infection in Australia associated with a binary toxin producing clade 2 ribotype 251 strain

Three patients with severe Clostridium difficile infection (CDI) caused by an unusual strain of C. difficile, PCR ribotype (RT) 251, were identified in New South Wales, Australia. All cases presented with severe diarrhoea, two had multiple recurrences and one died following a colectomy. C. difficile RT251 strains were isolated by toxigenic culture. Genetic characterisation was performed using techniques including toxin gene profiling, PCR ribotyping, whole genome sequencing (WGS), in-silico multi-locus-sequence-typing (MLST) and core-genome single nucleotide variant (SNV) analyses. Antimicrobial susceptibility was determined using an agar incorporation method. In vitro toxin production was confirmed by Vero cell cytotoxicity assay and pathogenicity was assessed in a murine model of CDI. All RT251 isolates contained toxin A (tcdA), toxin B (tcdB) and binary toxin (cdtA and cdtB) genes. Core-genome analyses revealed the RT251 strains were clonal, with 0–5 SNVs between isolates. WGS and MLST clustered RT251 in the same evolutionary clade (clade 2) as RT027. Despite comparatively lower levels of in vitro toxin production, in the murine model RT251 infection resembled RT027 infection. Mice showed marked weight loss, severe disease within 48 h post-infection and death. All isolates were susceptible to metronidazole and vancomycin. Our observations suggest C. difficile RT251 causes severe disease and emphasise the importance of ongoing surveillance for new and emerging strains of C. difficile with enhanced virulence

Susceptibility of hamsters to clostridium difficile isolates of differing toxinotype

Clostridium difficile is the most commonly associated cause of antibiotic associated disease (AAD), which caused ~21,000 cases of AAD in 2011 in the U.K. alone. The golden Syrian hamster model of CDI is an acute model displaying many of the clinical features of C. difficile disease. Using this model we characterised three clinical strains of C. difficile, all differing in toxinotype; CD1342 (PaLoc negative), M68 (toxinotype VIII) and BI-7 (toxinotype III). The naturally occurring non-toxic strain colonised all hamsters within 1-day post challenge (d.p.c.) with high-levels of spores being shed in the faeces of animals that appeared well throughout the entire experiment. However, some changes including increased neutrophil influx and unclotted red blood cells were observed at early time points despite the fact that the known C. difficile toxins (TcdA, TcdB and CDT) are absent from the genome. In contrast, hamsters challenged with strain M68 resulted in a 45% mortality rate, with those that survived challenge remaining highly colonised. It is currently unclear why some hamsters survive infection, as bacterial and toxin levels and histology scores were similar to those culled at a similar time-point. Hamsters challenged with strain BI-7 resulted in a rapid fatal infection in 100% of the hamsters approximately 26 hr post challenge. Severe caecal pathology, including transmural neutrophil infiltrates and extensive submucosal damage correlated with high levels of toxin measured in gut filtrates ex vivo. These data describes the infection kinetics and disease outcomes of 3 clinical C. difficile isolates differing in toxin carriage and provides additional insights to the role of each toxin in disease progression

Dwarf deer of Crete.

26 pages : illustrations (some color), map ; 26 cm.Age-graded fossils of Pleistocene endemic Cretan deer (Candiacervus spp.) reveal unexpectedly high juvenile mortality similar to that reported for extant mainland ruminants, despite the fact that these deer lived in a predator-free environment and became extinct before any plausible date for human arrival. Age profiles show that deer surviving past the fawn stage were relatively long-lived for ruminants, indicating that high juvenile mortality was not an expression of their living a "fast" life. Although the effects on survivorship of such variables as fatal accidents, starvation, and disease are difficult to gauge in extinct taxa, the presence of extreme morphological variability within nominal species/ecomorphs of Candiacervus is consistent with the view that high juvenile mortality can function as a key innovation permitting rapid adaptation in insular contexts

Real-world effectiveness of fremanezumab for the preventive treatment of migraine: Interim analysis of the pan-European, prospective, observational, phase 4 PEARL study.

BACKGROUND The ongoing Pan-European Real Life (PEARL) phase 4 study is evaluating fremanezumab effectiveness and safety for the prevention of episodic and chronic migraine. This interim analysis reports primary, secondary and exploratory endpoints from when 500 participants completed at least six months of treatment. METHODS Adults with episodic migraine or chronic migraine maintaining daily headache diaries were enrolled upon initiation of fremanezumab. Primary endpoint: proportion of participants with ≥50% reduction in monthly migraine days during the six-month period after fremanezumab initiation. Secondary endpoints: mean change from baseline across months 1-12 in monthly migraine days, acute migraine medication use, and headache-related disability. Exploratory endpoint: mean change in headache severity from baseline across months 1-12. Safety was assessed through adverse events reported. RESULTS Overall, 897 participants were enrolled and 574 included in the effectiveness analyses (episodic migraine, 25.8%; chronic migraine, 74.2%). Of participants with data available, 175/313 (55.9%) achieved ≥50% monthly migraine days reduction during the six-month period post-initiation. Across months 1-12, there were sustained reductions in mean monthly migraine days, acute medication use, disability scores, and headache severity. Few adverse events were reported. CONCLUSION PEARL interim results support the effectiveness and safety of fremanezumab for migraine prevention in a real-world population across several European countries.Trial registration: encepp.eu: EUPAS35111