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.

[Seniority of neurobladder and effectiveness of a first intradetrusor injection of botulinum toxin]. / Ancienneté de la neurovessie et efficacité d'une première injection de toxine botulique intradétrusorienne.

UNLABELLED: Intradetrusor injection of botulinum toxin is one of the second-line therapy of neurologenic detrusor overactivity. GOAL OF THE STUDY: In 26% to 66% of the cases, intradetrusor injection of botulinum toxin is inefficient in order to reduce overactive bladder symptoms and/or overactive detrusor. The objective of this study is to determine whether it exists a link between the efficacy of the first IDBT and the length of neurological detrusor overactivity symptoms. METHODS: Retrospective study on 79 patients which have a first intradetrusor injection of botulinum toxin between January 2001 and December 2013. Inclusion criteria were patients older than 18 and having neurological detrusor overactivity. RESULTS: There is no significant difference of intradetrusor injection of botulinum toxin efficacy according to duration of urinary symptoms in the general neurologigal population (multiple sclerosis, spinal cord injury, spinal cord compression, ischemic pathology, infectious pathology) with the mean age being 46 years. On the contrary, the length of evolution of neurological detrusor overactivity symptoms before the intradetrusor botox injection therapy and the efficiency of the first intradetrusor injection of botulinum toxin seem to be correlated with negative results in patients with multiple sclerosis. CONCLUSIONS: The duration of urinary symptoms is a predictive factor of primary failure of intradetrusor injection of botulinum toxin in multiple sclerosis patients, in univariate analysis.

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.

Structure of a C. perfringens enterotoxin mutant in complex with a modified Claudin-2 extracellular loop 2.

CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells.

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.

Botulism and hot-smoked whitefish: a family cluster of type E botulism in France, September 2009.

A family cluster of three cases of type E botulism were identified in south-east France in September 2009. The suspected food source of infection was a vacuum packed hot-smoked whitefish of Canadian origin purchased by the family during a visit to Finland and consumed several weeks later in France on the day prior to symptom onset. No leftover fish was available to confirm this hypothesis. Vacuum packed hot-smoked whitefish has previously been associated with cases of type E botulism in multiple countries, including Finland, Germany, the United States and Israel.

[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.

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum.

AIMS: To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism. METHODS AND RESULTS: Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin (bont) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A, bont/B, bont/E, bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg-1000 fg of total DNA in the PCR tube (25-250 genome equivalents) which correspond to 10(3) to 10(4) cells ml(-1). After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak. CONCLUSION: These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism. SIGNIFICANCE AND IMPACT OF THE STUDY: Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.

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.

Alternative vaccination against equine botulism (BoNT/C).

REASON FOR PERFORMING STUDY: In Europe the incidence of botulism in horses has increased in the last decade due to the growing popularity of haylage feeding. Recombinant vaccines are safer and less expensive to produce and are generally better tolerated than toxoids. OBJECTIVES: To investigate whether the recombinant C-terminal half of the heavy chain of the botulinum neurotoxin C (Hc BoNT/C) in combination with an immunstimulatory adjuvant is an appropriate vaccine candidate for horses by testing its efficacy to induce neutralising antibodies and by comparing its immunogenic properties and adverse reactions to a commercial toxoid vaccine. Formation of oedema and local pain reactions were assessed. ELISA and Western blot assay against Hc BoNT/C and testing of neutralising antibody induction in a mouse protection assay were used to evaluate the immune response. RESULTS: With the recombinant vaccine, only minor local swelling with full recovery after 5 days was noted after brisket injections. The toxoid vaccine produced local, painful reactions with longer recovery periods of up to 2 weeks. Horses vaccinated with either vaccine induced neutralising antibodies after the second booster vaccination, while seroconversion on ELISA and Western blot to Hc BoNT/C was apparent after the first recombinant vaccination, and at various time points in the vaccination schedule in horses that received commercial toxoid vaccine. CONCLUSION: The recombinant vaccine showed fewer adverse reactions compared to the only commercially available vaccine but induced similar concentrations of neutralising antibodies. There was no correlation between the serological response to Hc BoNT/C and the neutralising capacity of serum. POTENTIAL RELEVANCE: Recombinant Hc BoNT/C is an appropriate vaccine candidate to stimulate production of neutralising antibodies against botulinum neurotoxin C in horses and creates only minor local reactions at the injection site.

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.

Botulism in patients who inhale cocaine: the first cases in France.

We describe 2 cases of mild botulism in patients who inhaled cocaine. Botulism, though rare, is increasing in incidence among illicit drug users. To our knowledge, these are the first cases of botulism in illicit drug users in France. Clinicians should be aware of this phenomenon; botulism should be considered in illicit drug users with neurological symptoms.