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Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens , is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of β-PFTs (aβ-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double β-barrel, a common feature of the aβ-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications. Epsilon toxin (Etx) is a potent pore forming toxin (PFT) produced by Clostridium perfringens. Here authors show the cryo-EM structure of the Etx pore assembled on the membrane of susceptible cells and shed light on pore formation and mutant phenotypes.

Enterotoxaemia is a disease with a high associated mortality rate, affecting beef and veal calves worldwide, caused by C. perfringens alpha toxin and perfringolysin. A longitudinal study was conducted to determine the dynamics of antibodies against these toxins in 528 calves on 4 beef and 15 veal farms. The second study aimed to determine the effect of solid feed intake on the production of antibodies against alpha toxin and perfringolysin. The control group only received milk replacer, whereas in the test group solid feed was provided. Maternal antibodies for alpha toxin were present in 45% of the veal calves and 66% of the beef calves. In beef calves a fluent transition from maternal to active immunity was observed for alpha toxin, whereas almost no veal calves developed active immunity. Perfringolysin antibodies significantly declined both in veal and beef calves. In the second study all calves were seropositive for alpha toxin throughout the experiment and solid feed intake did not alter the dynamics of alpha and perfringolysin antibodies. In conclusion, the present study showed that veal calves on a traditional milk replacer diet had significantly lower alpha toxin antibodies compared to beef calves in the risk period for enterotoxaemia, whereas no differences were noticed for perfringolysin.

We performed epidemiologic studies at public freshwater bathing sites in Germany to provide a better scientific basis for the definition of recreational water quality standards. A total of 2,196 participants were recruited from the local population and randomized into bathers and non-bathers. Bathers were exposed for 10 min and had to immerse their head at least three times. Water samples for microbiological analysis were collected at 20-min intervals. Unbiased concentration-response effects with no-observed-adverse-effect levels (NOAELs) were demonstrated for three different definitions of gastroenteritis and four fecal indicator organisms. Relative risks for bathing in waters with levels above NOAELs compared with nonbathing ranged from 1.8 (95% CI, 1.2-2.6) to 4.6 (95% CI, 2.1-10.1), depending on the definition of gastroenteritis. The effect of swallowing water provided additional evidence for true dose-response relationships. Based on the NOAELs, the following guide values for water quality are suggested: 100 Escherichia coli, 25 intestinal enterococci, 10 somatic coliphages, or 10 Clostridium perfringens per 100 mL. Recreational water quality standards are intended to protect the health of those consumers who are not already immune or resistant to pathogens that may be associated with indicator organisms. In contrast to current World Health Organization recommendations, we concluded that standards should be based on rates of compliance with NOAELs rather than on attributable risks determined above NOAELs, because these risks depend mainly on the unpredictable susceptibility of the cohorts. Although in theory there is no threshold in real concentration-response relationships, we demonstrated that a NOAEL approach would be a more robust and practical solution to the complex problem of setting standards.

Clostridium perfringens, a rapid-growing pathogen known to secrete an arsenal of >20 virulent toxins, has been associated with intestinal diseases in both animals and humans throughout the past century. Recent advances in genomic analysis and experimental systems make it timely to re-visit this clinically and veterinary important pathogen. This Review will summarise our understanding of the genomics and virulence-linked factors, including antimicrobial potentials and secreted toxins of this gut pathogen, and then its up-to-date clinical epidemiology and biological role in the pathogenesis of several important human and animal-associated intestinal diseases, including pre-term necrotising enterocolitis. Finally, we highlight some of the important unresolved questions in relation to C. perfringens-mediated infections, and implications for future research directions.

Simple, effective and affordable methods are needed to treat and safely store non-piped, gathered household water. This study evaluated point-of-use chlorination and storage in special plastic containers of gathered household water for improving microbial quality and reducing diarrhoeal illness of consumers living under conditions of poor sanitation and hygiene. Community families were recruited and randomly divided into intervention (household water chlorination and storage in a special container) and control (no intervention) households. Microbes in stored household water were extensively inactivated by 1-5-mg/L doses of hypochlorite. Escherichia coli levels in stored household waters were <1/100 mL in most intervention households but readily detectable at high levels in control households. Stored water of intervention households was also lower in Clostridium perfringens and heterotrophic plate count bacteria than in control households. The intervention reduced household diarrhoeal illness. In Bolivia, monthly episodes of household diarrhoeal illness were 1.25 and 2.2 in intervention and control families, respectively (P = <0.002) indicating that 43% of community diarrhoea was preventable by using the intervention. In Bangladesh, mean episodes of child diarrhoea/1,000 d were 19.6 and 24.8 in intervention and control groups respectively (P = <0.03) indicating that about 24% of observed diarrhoea was preventable by using the intervention. Chlorine disinfection and storage in an appropriate container significantly improved the microbiological quality of non-piped household drinking water and reduced community diarrhoeal disease. Widespread use of this simple treatment and storage system for non-piped domestic water has the potential to dramatically reduce the global burden of waterborne diarrhoeal disease.

The impact of probiotics on dogs with acute hemorrhagic diarrhea syndrome (AHDS) has not been evaluated so far. The study aim was to assess the effect of probiotic treatment on the clinical course, intestinal microbiome, and toxigenic Clostridium perfringens in dogs with AHDS in a prospective, placebo-controlled, blinded trial. Twenty-five dogs with AHDS with no signs of sepsis were randomly divided into a probiotic (PRO; Visbiome, ExeGi Pharma) and placebo group (PLAC). Treatment was administered for 21 days without antibiotics. Clinical signs were evaluated daily from day 0 to day 8. Key bacterial taxa, C. perfringens encoding NetF toxin and enterotoxin were assessed on days 0, 7, 21. Both groups showed a rapid clinical improvement. In PRO a significant clinical recovery was observed on day 3 (p = 0.008), while in PLAC it was observed on day 4 (p = 0.002) compared to day 0. Abundance of Blautia (p<0.001) and Faecalibacterium (p = 0.035) was significantly higher in PRO on day 7 compared to day 0, while in PLAC the abundance of Faecalibacterium was not significantly higher on any study day and Blautia (p = 0.016) was only significantly higher on day 21 compared to day 0. Abundance of C. perfringens was significantly lower on day 7 (p = 0.011) compared to day 0 in PRO but not in PLAC. Enterotoxin genes were significantly lower in PRO on day 21 (p = 0.028) compared to PLAC. Fecal samples of 57% of all dogs were positive for netF toxin genes on day 0 and the abundance was significantly lower on day 7 compared to day 0 in PRO (p = 0.016) and PLAC (p = 0.031). The probiotic treatment was associated with an accelerated normalization of the intestinal microbiome. Dogs with aseptic AHDS showed a rapid decrease of netF toxin genes and fast clinical recovery in both groups under symptomatic treatment without antibiotics.

Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases.

Background Clostridium perfringens , a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. Results The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. Conclusion Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Enteritis necroticans (EN) in humans caused by infection with Clostridium perfringens type C, once thought limited to the highlands of Papua New Guinea has been identified sporadically worldwide. Outbreaks still occur among children in low-income countries and isolated cases occur among children and adults in other countries. Here the disease seems to be associated with diabetes mellitus and other risk factors. C. perfringens type C is also an important cause of necrotizing enteritis among animals, particularly pigs. Research into the pathogenesis of this disease has confirmed the central role of beta toxin and its target, the endothelial cell. Unlike most bacterial enteric infections, the primary anatomic location of EN is the proximal small intestine, reasons for which are not completely understood. Ongoing surveillance for C. perfringens type C infection is warranted as well as public health measures of prevention in locations where environmental and food hygiene is poor.

CPE targets a subpopulation of colonocytes, thereby inducing focal barrier leaks. CPE-mediated damage is restricted by an intact tight junctional barrier. Toxicity and barrier disruption by CPE are mediated by apically dislocated nonjunctional claudins that serve as CPE receptors. Abstract Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (eg, claudin-4) as receptors to form Ca2+-permeable pores in the membrane, damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+ depletion, proinflammatory cytokine tumor necrosis factor α, or dedifferentiation. Microscopy, Ca2+ monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis—if intestinal barrier dysfunction, eg, during inflammation facilitates claudin accessibility.