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.

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.

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.

[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.

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.

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.

The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the ras-related protein Rho1p is essentially the only target for ADP-ribosylation by exoenzyme C3 of Clostridium botulinum. Using C3 to detect Rho1p in subcellular fractions, Rho1p was found primarily in the 10,000 g pellet (P2) containing large organelles; small amounts also were detected in the 100,000 g pellet (P3), and cytosol. When P2 organelles were separated in sucrose density gradients Rho1p comigrated with the Kex-2 activity, a late Golgi marker. Rho1p distribution was shifted from P2 to P3 in several mutants that accumulate post-Golgi vesicles. Rho1p comigrated with post-Golgi transport vesicles during fractionation of P3 organelles from wild-type or sec6 cells. Vesicles containing Rho1p were of the same size but different density than those bearing Sec4p, a ras-related protein located both on post-Golgi vesicles and the plasma membrane. Immunofluorescence microscopy detected Rho1p as a punctate pattern, with signal concentrated towards the cell periphery and in the bud. Thus, in S. cerevisiae Rho1p resides primarily in the Golgi apparatus, and also in vesicles that are likely to be early post-Golgi vesicles.

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.

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.

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.

Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.

Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot. Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein. When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function. NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins. When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins. Nevertheless, p21.bot was immunologically distinct from p21ras.

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.

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.

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.

Clostridium perfringens: toxinotype and genotype.

Clostridium perfringens is a ubiquitous pathogen that produces many toxins and hydrolytic enzymes. Because the toxin-encoding genes can be located on extrachromosomal elements or in variable regions of the chromosome, several pathovars have arisen, each of which is involved in a specific disease. Pathovar identification is required for a precise diagnosis of associated pathologies and to define vaccine requirements. For these purposes, toxin genotyping is more reliable than the classical toxinotyping.

Genotyping Clostridium botulinum toxinotype A isolates from patients using amplified rDNA restriction analysis.

In this study, the application of amplified rDNA restriction analysis (ARDRA) for characterizing Clostridium botulinum toxinotype A strains isolated from individuals with botulism was evaluated. Ten restriction enzymes were tested for their suitability in ARDRA as a typing method and HhaI was selected for the best outcome. Analysis of HhaI restriction profiles of the amplified products divided C. botulinum isolates into three clusters. Non-toxigenic Clostridium sporogenes strains showed an ARDRA restriction pattern that was distinct from those observed for C. botulinum. The successful use of ARDRA for subdivision of C. botulinum in this study confirmed that this technique is a powerful method for typing of C. botulinum toxinotype A clonal diversity. In addition, it is rapid, sensitive and simple.

Beta2 toxin, a novel toxin produced by Clostridium perfringens.

A novel toxin (Beta2) and its gene were characterized from a Clostridium perfringens strain isolated from a piglet with necrotic enteritis. At the amino-acid level, Beta2 toxin (27670 Da) has no significant homology with the previously identified Beta toxin (called Beta1) (34861 kDa) from C. perfringens type B NCTC8533 ( Hunter, S.E.C., Brown, J.E., Oyston, P.C.F., Sakurai, J., Titball, R.W., 1993. Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus. Infect. Immun. 61, 3958-3965). Both Beta1 and Beta2 toxins were lethal for mice and cytotoxic for the cell line 1407, inducing cell rounding and lysis without affecting the actin cytoskeleton. The genes encoding Beta1 and Beta2 toxins have been localized in unlinked loci in large plasmids of C. perfringens. In addition, Beta2 toxin-producing C. perfringens strains were found to be associated with animal diseases such as necrotic enteritis in piglets and enterocolitis in horses.