Infant botulism: Two case reports and electroneuromyogram findings.

Botulism is an uncommon severe neuromuscular disorder. We report two recent cases of confirmed infant botulism diagnosed in an 11-week and a 5-month-old infant along with electroneuromyogram (ENMG) findings. Then, we discuss the EMG features of infant botulism. In severe forms of infant botulism, presence of these features might help decide to use botulinum immune globulin. To our knowledge, case 1 is the first case reported in France based on confirmed dust contamination.

Development of a clinical prediction score for detection of suspected cases of equine grass sickness (dysautonomia) in France.

Equine grass sickness (EGS) (equine dysautonomia) is a neurodegenerative condition of grazing equines. Pre-mortem diagnosis of EGS is a challenge for practitioners as definitive diagnosis requires ileal/myenteric lymph node biopsies. This study aimed to develop a clinical score that could be used by practitioners to improve the detection of acute or subacute EGS cases in the field. Suspected EGS cases were declared by veterinary practitioners. A case was classified as confirmed positive if ileal or rectal biopsy samples showed neuronal degeneration typical of EGS. A semi-quantitative scoring system, including epidemiological and clinical data, was created to attempt to classify suspected EGS horses into confirmed positive or negative cases. Each variable was weighted based on a boosted regression trees model, while taking into account its clinical relevance. Twenty-eight EGS cases were confirmed by biopsy during the entire study period. The best cut-off value for the score to have a high sensitivity while maximizing specificity was 8, with a sensitivity of 100% and a specificity of 53%. In our dataset, 77% of animals would be correctly classified with this cut-off value of 8. Highest sensitivity was chosen in order to detect the highest number of potential cases. Our score represents an inexpensive and useful tool to aid in the identification of suspected EGS cases in the field and selection for further diagnostics procedures to confirm or rule out the disease. Application of the score to larger populations of animals would be required to further adapt and refine the score.

A large outbreak of bovine botulism possibly linked to a massive contamination of grass silage by type D/C Clostridium botulinum spores on a farm with dairy and poultry operations.

Type D bovine botulism outbreaks associated with poultry litter are increasingly reported in European countries, but the circumstances of exposure to Clostridium botulinum toxins remain unclear. In spring 2015, a large type D/C bovine botulism outbreak affected a farm with dairy and poultry operations. Epidemiological and laboratory investigations strongly suggest that the outbreak was caused by feeding cattle with insufficiently acidified grass silage that was contaminated by type D/C C. botulinum spores. The source of the spores remains unclear, but could have been a stack of poultry litter stored in the grass silage pasture before harvesting. The presence of putrefied poultry carcasses mixed in with the litter is relatively unlikely considering the careful daily removal of poultry carcasses. These findings reinforce the importance of proper ensiling of feed materials and highlight the need for safe disposal of poultry litter, even in the case of good management of poultry deadstock, in order to prevent bovine botulism.

A penicillin- and metronidazole-resistant Clostridium botulinum strain responsible for an infant botulism case.

The clinical course of a case of infant botulism was characterized by several relapses despite therapy with amoxicillin and metronidazole. Botulism was confirmed by identification of botulinum toxin and Clostridium botulinum in stools. A C. botulinum A2 strain resistant to penicillins and with heterogeneous resistance to metronidazole was isolated from stool samples up to 110 days after onset. Antibiotic susceptibility was tested by disc agar diffusion and MICs were determined by Etest. Whole genome sequencing allowed detection of a gene cluster composed of blaCBP for a novel penicillinase, blaI for a regulator, and blaR1 for a membrane-bound penicillin receptor in the chromosome of the C. botulinum isolate. The purified recombinant penicillinase was assayed. Resistance to ß-lactams was in agreement with the kinetic parameters of the enzyme. In addition, the ß-lactamase gene cluster was found in three C. botulinum genomes in databanks and in two of 62 genomes of our collection, all the strains belonging to group I C. botulinum. This is the first report of a C. botulinum isolate resistant to penicillins. This stresses the importance of antibiotic susceptibility testing for adequate therapy of botulism.

Pore-forming activity of clostridial binary toxins.

Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as Bacillus binary toxins, including Bacillus anthracis toxins consist of two independent proteins, one being the binding component which mediates the internalization into cell of the intracellularly active component. Clostridial binary toxins induce actin cytoskeleton disorganization through mono-ADP-ribosylation of globular actin and are responsible for enteric diseases. Clostridial and Bacillus binary toxins share structurally and functionally related binding components which recognize specific cell receptors, oligomerize, form pores in endocytic vesicle membrane, and mediate the transport of the enzymatic component into the cytosol. Binding components retain the global structure of pore-forming toxins (PFTs) from the cholesterol-dependent cytotoxin family such as perfringolysin. However, their pore-forming activity notably that of clostridial binding components is more related to that of heptameric PFT family including aerolysin and C. perfringens epsilon toxin. This review focuses upon pore-forming activity of clostridial binary toxins compared to other related PFTs. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

Cluster of two cases of botulism due to Clostridium baratii type F in France, November 2014.

The first two cases in France of botulism due to Clostridium baratii type F were identified in November 2014, in the same family. Both cases required prolonged respiratory assistance. One of the cases had extremely high toxin serum levels and remained paralysed for two weeks. Investigations strongly supported the hypothesis of a common exposure during a family meal with high level contamination of the source. However, all analyses of leftover food remained negative.

Lumbar discitis caused by Clostridium perfringens.

We report here a rare case of chronic lumbar discitis caused by Clostridium perfringens in an elderly patient that was treated with a combination of ß-lactams and clindamycin. Molecular analysis performed on the strain revealed an unusual toxin gene pattern.

Two outbreaks of botulism associated with consumption of green olive paste, France, September 2011.

Two family outbreaks of botulism (a total of nine cases) were identified in south-east and northern France in early September 2011. The source of infection was considered to be a ground green olive paste. Botulinum type A toxin was identified in seven cases and in the incriminated olive paste. Incorrect sterilisation techniques were observed at the artisanal producer's workshop. These episodes highlight the potential public health threat of Clostridium botulinum linked to inadequate sterilisation of food products.

Multifaceted interactions of bacterial toxins with the gastrointestinal mucosa.

The digestive tract is one of the ecosystems that harbors the largest number and greatest variety of bacteria. Among them, certain bacteria have developed various strategies, including the synthesis of virulence factors such as toxins, to interact with the intestinal mucosa, and are responsible for various pathologies. A large variety of bacterial toxins of different sizes, structures and modes of action are able to interact with the gastrointestinal mucosa. Some toxins, termed enterotoxins, directly stimulate fluid secretion in enterocytes or cause their death, whereas other toxins pass through the intestinal barrier and disseminate by the general circulation to remote organs or tissues, where they are active. After recognition of a membrane receptor on target cells, toxins can act at the cell membrane by transducing a signal across the membrane in a hormone-like manner, by pore formation or by damaging membrane compounds. Other toxins can enter the cells and modify an intracellular target leading to a disregulation of certain physiological processes or disorganization of some structural architectures and cell death. Toxins are fascinating molecules, which mimic or interfere with eukaryotic physiological processes. Thereby, they have permitted the identification and characterization of new natural hormones or regulatory pathways. Besides use as protective antigens in vaccines, toxins offer multiple possibilities in pharmacology, such as immune modulation or specific delivery of a protein of interest into target cells.

[Infant botulism in France, 1991-2009]. / Le botulisme infantile en France, 1991-2009.

Infant botulism is caused by the ingestion of spores of Clostridium botulinum and affects newborns and infants under 12 months of age. Ingested spores multiply and produce botulinum toxin in the digestive tract, which then induces clinical symptoms. A single French case was described in the literature prior to 1991. We describe the cases of infant botulism identified in France between 1991 and 2009. All clinical suspicions of botulism must be declared in France. Biological confirmation of the disease is provided by the National reference laboratory for anaerobic bacteria and botulism at the Pasteur Institute. During this period, 7 cases of infant botulism were identified, 1 per year from 2004 to 2008 and 2 in 2009. The median age of affected infants was 119 days and all were female. All infants presented with constipation and oculomotor symptoms. All were hospitalized and required mechanical ventilation. The infants recovered from their botulism. The diagnosis of infant botulism was biologically confirmed for all patients. One 4-month-old infant was treated with a single dose of the human-derived botulism antitoxin specific for infant botulism types A and B (BabyBIG®). The infants all had different feeding habits ranging from exclusive breast feeding to a mix of formula feeding and solid food consumption. The consumption of honey, the only documented risk food for this disease, was reported for 3 of the infants. The honey had been placed on the pacifier of 2 infants and directly in the mouth of the 3rd by the mother. Infant botulism, a form of botulism that was previously rarely recognized in France, has been reported more frequently during the last 6 years. This disease remains rare but nonetheless severe. In light of recent epidemiological data, efforts to raise awareness among parents of infants and health professionals on the danger of infant botulism and particularly, its association with honey consumption seems necessary.

[Mechanisms of action of botulinum toxins and neurotoxins]. / Mécanismes d'action des toxines et neurotoxines botuliques.

Several bacteria of the Clostridium genus (C. botulinum) produce 150 kDa di-chainal protein toxins referred as botulinum neurotoxins or BoNTs. They associate with non-toxic companion proteins and form a complex termed botulinum toxin. BoNTs specifically inhibit vesicular neurotransmitter release. The cellular action of BoNTs can be depicted according to a multi-step model : The toxin's heavy chain mediates binding to specific receptors comprised of a ganglioside moiety and a vesicular protein (SV2 for BoNT type A, synaptotagmin for BoNT type B), followed by endocytotic internalisation of the BoNT/receptor complex. Vesicle recycling induces BoNT internalisation. Upon acidification of vesicles, the light chain of the neurotoxin is translocated into the cytosol. Here, this zinc-endopeptidase cleaves one or two among three synaptic proteins (VAMP-synapto-brevin, SNAP25, and syntaxin). As the three protein targets of BoNT play major role in fusion of synaptic vesicles at the release sites, their cleavage is followed by blockade of neurotransmitter exocytosis. Importantly, as the BoNT receptors and intracellular targets are present in all nerve terminals, the BoNTs are not specific for cholinergic transmission. Duration of their inhibitory action is mainly determined by the the life-time of the toxin's light chain in the cytosol. Sprouting of new nerve-endings, which are retracted when the poisoned nerve terminals have recovered full functionality, may lead to anticipated recovery of the poisoned nerve terminals.

Inferring maps of forces inside cell membrane microdomains.

Mapping of the forces on biomolecules in cell membranes has spurred the development of effective labels, e.g., organic fluorophores and nanoparticles, to track trajectories of single biomolecules. Standard methods use particular statistics, namely the mean square displacement, to analyze the underlying dynamics. Here, we introduce general inference methods to fully exploit information in the experimental trajectories, providing sharp estimates of the forces and the diffusion coefficients in membrane microdomains. Rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. The method is then applied to infer forces and potentials acting on the receptor of the toxin labeled by lanthanide-ion nanoparticles. Our scheme is applicable to any labeled biomolecule and results show its general relevance for membrane compartmentation.

Generation of high-titer neutralizing antibodies against botulinum toxins A, B, and E by DNA electrotransfer.

Botulinum neurotoxins are known to be among the most toxic known substances. They produce severe paralysis by preventing the release of acetylcholine at the neuromuscular junction. Thus, new strategies for efficient production of safe and effective anti-botulinum neurotoxin antisera have been a high priority. Here we describe the use of DNA electrotransfer into the skeletal muscle to enhance antiserum titers against botulinum toxin serotypes A, B, and E in mice. We treated animals with codon-optimized plasmid DNA encoding the nontoxic but highly immunogenic C-terminal heavy chain fragment of the toxin. By employing both codon optimization and the electrotransfer procedure, the immune response and corresponding neutralizing antiserum titers were markedly increased. The cellular localization of the antigen and the immunization regimens were also shown to increase neutralizing titers to >100 IU/ml. This study demonstrates that DNA electrotransfer is an effective procedure for raising neutralizing antiserum titers to remarkably high levels.

Pore-forming epsilon toxin causes membrane permeabilization and rapid ATP depletion-mediated cell death in renal collecting duct cells.

Clostridium perfringens epsilon toxin (ET) is a potent pore-forming cytotoxin causing fatal enterotoxemia in livestock. ET accumulates in brain and kidney, particularly in the renal distal-collecting ducts. ET binds and oligomerizes in detergent-resistant membranes (DRMs) microdomains and causes cell death. However, the causal linkage between membrane permeabilization and cell death is not clear. Here, we show that ET binds and forms 220-kDa insoluble complexes in plasma membrane DRMs of renal mpkCCD(cl4) collecting duct cells. Phosphatidylinositol-specific phospholipase C did not impair binding or the formation of ET complexes, suggesting that the receptor for ET is not GPI anchored. ET induced a dose-dependent fall in the transepithelial resistance and potential in confluent cells grown on filters, transiently stimulated Na+ absorption, and induced an inward ionic current and a sustained rise in [Ca2+]i. ET also induced rapid depletion of cellular ATP, and stimulated the AMP-activated protein kinase, a metabolic-sensing Ser/Thr kinase. ET also induced mitochondrial membrane permeabilization and mitochondrial-nuclear translocation of apoptosis-inducing factor, a potent caspase-independent cell death effector. Finally, ET induced cell necrosis characterized by a marked reduction in nucleus size without DNA fragmentation. DRM disruption by methyl-beta-cyclodextrin impaired ET oligomerization, and significantly reduced the influx of Na+ and [Ca2+]i, but did not impair ATP depletion and cell death caused by the toxin. These findings indicate that ET causes rapid necrosis of renal collecting duct cells and establish that ATP depletion-mediated cell death is not strictly correlated with the plasma membrane permeabilization and ion diffusion caused by the toxin.

Positive modulation by Ras of interleukin-1beta-mediated nitric oxide generation in insulin-secreting clonal beta (HIT-T15) cells.

In the present study, we have shown that exposure of insulin-secreting clonal beta (HIT-T15) cells to interleukin-1beta (IL-1beta) results in a time- and concentration-dependent increase in nitric oxide (NO) release. These effects by IL-1beta on NO release were mediated by induction of inducible nitric oxide synthase (iNOS) from the cells. Preincubation of HIT cells with Clostridium sordellii lethal toxin-82, which irreversibly glucosylates and inactivates small G-proteins, such as Ras, Rap, Ral, and Rac, but not Cdc42, completely abolished IL-1beta-induced NO release. Pre-exposure of HIT cells to C. sordellii lethal toxin-9048, which monoglucosylates and inhibits Ras, Cdc42, Rac, and Rap, but not Ral, also attenuated IL-1beta-mediated NO release. These data indicate that activation of Ras and/or Rac may be necessary for IL-1beta-mediated NO release. Preincubation of HIT cells with C. difficile toxin-B, which monoglucosylates Rac, Cdc42, and Rho, had no demonstrable effects on IL-mediated NO release, ruling out the possibility that Rac may be involved in this signaling step. Further, two structurally dissimilar inhibitors of Ras function, namely manumycin A and damnacanthal, inhibited, in a concentration-dependent manner, the IL-1beta-mediated NO release from these cells. Together, our data provide evidence, for the first time, that Ras activation is an obligatory step in IL-1beta-mediated NO release and, presumably, the subsequent dysfunction of the pancreatic beta cell. Our data also provide a basis for future investigations to understand the mechanism of cytokine-induced beta cell death leading to the onset of insulin-dependent diabetes mellitus.

[Mechanism of action and therapeutic uses of botulinum and tetanus neurotoxins]. / Mécanismes d'action et utilisations thérapeutiques des neurotoxines botuliques et tétanique.

Botulinum neurotoxins are produced by anaerobic spore forming bacteria, Clostridiumbotulinum. They are synthesized as a single chain protein (150kDa) which is not or weakly active. The active form results from proteolysis that cleaves the precursor into a light chain (about 50kDa) and a heavy chain (about 100kDa) which are linked by a disulfide bridge. The heavy chain is involved in the recognition of a specific neurone surface receptor and mediates the internalization of the light chain into the cytosol. The light chain is responsible for the intracellular activity. It catalyzes the proteolysis of SNARE proteins which are involved in the exocytosis of synaptic vesicles containing acetylcholine. Hence, the release of acetylcholine at the neuromuscular junction is blocked leading to a flaccid paralysis. The tetanus neurotoxin shares with botulinum neurotoxins a common structure and mechanism of action. Tetanus neurotoxin blocks the release of neurotransmitters in the inhibitory interneurons leading to spastic paralysis. The paralytic properties of the botulinum neurotoxins are used to treat certain myoclonies such as blepharospasm, torticolis, hemifacial paralysis. Botulinum neurotoxins are thus efficient therapeutic agents helpful in avoiding surgery.

Clostridium perfringens iota-toxin: mapping of receptor binding and Ia docking domains on Ib.

Clostridium perfringens iota-toxin is a binary toxin consisting of iota a (Ia), an ADP-ribosyltransferase that modifies actin, and iota b (Ib), which binds to a cell surface protein and translocates Ia into a target cell. Fusion proteins of recombinant Ib and truncated variants were tested for binding to Vero cells and docking with Ia via fluorescence-activated cytometry and cytotoxicity experiments. C-terminal residues (656 to 665) of Ib were critical for cell surface binding, and truncated Ib variants containing > or = 200 amino acids of the C terminus were effective Ib competitors and prevented iota cytotoxicity. The N-terminal domain (residues 1 to 106) of Ib was important for Ia docking, yet this region was not an effective competitor of iota cytotoxicity. Further studies showed that Ib lacking just the N-terminal 27 residues did not facilitate Ia entry into a target cell and subsequent cytotoxicity. Five monoclonal antibodies against Ib were also tested with each truncated Ib variant for epitope and structural mapping by surface plasmon resonance and an enzyme-linked immunosorbent assay. Each antibody bound to a linear epitope within the N terminus (residues 28 to 66) or the C terminus (residues 632 to 655). Antibodies that target the C terminus neutralized in vitro cytotoxicity and delayed the lethal effects of iota-toxin in mice.

Clostridium perfringens epsilon toxin induces a rapid change of cell membrane permeability to ions and forms channels in artificial lipid bilayers.

Epsilon toxin is a potent toxin produced by Clostridium perfringens types B and D, which are responsible for a rapidly fatal enterotoxemia in animals. One of the main properties of epsilon toxin is the production of edema. We have previously found that epsilon toxin causes a rapid swelling of Madin-Darby canine kidney cells and that the toxin does not enter the cytosol and remains associated with the cell membrane by forming a large complex (Petit, L., Gibert, M., Gillet, D., Laurent-Winter, C., Boquet, P., and Popoff, M. R. (1997) J. Bacteriol. 179, 6480-6487). Here, we report that epsilon toxin induced in Madin-Darby canine kidney cells a rapid decrease of intracellular K(+), and an increase of Cl(-) and Na(+), whereas the increase of Ca(2+) occurred later. The entry of propidium iodide that was correlated with the loss of cell viability monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test indicates that epsilon toxin formed large pores. In artificial lipid bilayers, epsilon toxin caused current steps with a single-channel conductance of 60 pS in 100 mm KCl, which represented general diffusion pores. The channels were slightly selective for anions, but cations could also penetrate. Epsilon toxin formed wide and water-filled channels permeable to hydrophilic solutes up to a molecular mass of at least 1 kDa, which probably represents the basic mechanism of toxin action on target cells.

Evidence for Rho protein regulation of renal tubular epithelial cell function.

BACKGROUND: Rho proteins are small guanine 5'-triphosphate (GTP)-binding proteins felt to be important regulators of several aspects of cell function, including the organization of the actin cytoskeleton. The effects of Rho proteins on the regulation of renal tubular epithelial cell function are not known. METHODS: Selected bacterial toxins that inhibit Rho protein function were used to examine the effect of Rho in cultured renal tubular epithelial cells. RESULTS: Clostridium difficile toxin A significantly and dose dependently inhibited LLC-PK(1) cell (3)H-thymidine uptake and healing of small wounds made in confluent monolayers, and it induced apoptosis. A second Clostridium difficile toxin (toxin B) that acted via a different receptor also impaired LLC-PK(1) thymidine uptake and wound healing, and it induced apoptosis. A third bacterial toxin, C3 toxin from Clostridium botulinum, also impaired LLC-PK(1) thymidine uptake and stimulated apoptosis in LLC-PK(1) cells. Since Rho inhibition disrupted organization of the actin cytoskeleton, we examined the effects of another agent that disrupted the actin cytoskeleton (cytochalasin D) and found significant dose-dependent effects that impaired LLC-PK1 thymidine uptake and wound healing and that induced apoptosis. The effects of toxin A and cytochalasin D to induce apoptosis were not associated with significant changes in expression of Bcl-2, BAD, or BAK proteins and were significantly attenuated by a pancaspase inhibitor. CONCLUSIONS: Our results suggest that Rho proteins are important endogenous regulators of several aspects of renal tubular epithelial cell function, including proliferation, migration, and apoptosis. Further studies are needed to clarify the cellular mechanisms of Rho regulation of renal epithelial cell function.