Internalization of Clostridium botulinum C2 Toxin Is Regulated by Cathepsin B Released from Lysosomes.

Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.

Óleo essencial de Lippia gracilis: atividade antioxidante e antimicrobiana contra Clostridium botulinum / Essential oil of Lippia gracilis: antioxidant and antimicrobial activity against Clostridium botulinum

Apesar da utilização dos aditivos nos alimentos, alguns microrganismos, como o Clostridium botulinum ainda causam preocupação. Óleos essenciais são opções de aditivo para a indústria de alimentos, pois podem apresentar atividade antimicrobiana e antioxidante. Diante disso, o objetivo foi extrair o ó leo essencial de Lippia gracilis, caracterizar sua composição química por CG/ EM, avaliar a atividade antioxidante pela redução do radical DPPH e a concentração mínima esporicida (CME) sobreClostridium sporogenes. O óleo essencial é composto majoritariamente pelos monoterpenos carvacrol, p-cimeno e terpineno, e apresentou baixa capacidade de estabilização do radical DPPH, IC50 > 500 μg/ mL. Em relação à ação sobre os endósporos do Clostridium sporogenes, a concentração mínima esporicida foi 1,5% de óleo essencial.

[Toxin-infections and toxin-related diseases due to Clostridia other than Clostridium difficile]. / Toxi-infections et maladies toxiniques liées à des clostridies autres que Clostridium difficile.

Clostridia cause severe diseases. Tetanus is rare in Switzerland because of vaccine coverage and the application of guidelines for the management of contaminated wounds. Tetanus requires wound debridement and the administration of antibiotics and anti-tetanus immune. Besides gastroenteritis, infections due to C. perfringens most often require surgery, in addition to antibiotic treatment with penicillin and clindamycin. Botulism is a rare disease caused by a toxin produced by C. botulinum that causes flaccid paralysis. The clinical syndrome must be recognized early in order to administer the antitoxin and improve the prognosis. The other, rarer species of Clostridia require surgical and antibiotic management, but their prognosis remains poor.

Les clostridies causent des maladies graves. Le tétanos est rare en Suisse grâce à la vaccination et à l'application de directives pour la gestion des plaies souillées. Sa prise en charge nécessite un débridement de plaie, l'administration d'antibiotiques et d'immunoglobulines antitétaniques. En dehors des gastroentérites, les infections à C. perfringens nécessitent en règle générale une chirurgie, en sus d'une antibiothérapie par pénicilline et clindamycine. Le botulisme est une maladie rare due à une toxine produite par C. botulinum qui entraîne une paralysie flasque descendante. Le syndrome clinique doit être reconnu précocement afin d'administrer l'antitoxine et améliorer le pronostic. Les infections invasives dues à des espèces plus rares de clostridies nécessitent une prise en charge chirurgicale et l'administration d'antibiotiques, mais leur pronostic est défavorable.

Exploration of the Fecal Microbiota and Biomarker Discovery in Equine Grass Sickness.

Equine grass sickness (EGS) is a frequently fatal disease of horses, responsible for the death of 1 to 2% of the U.K. horse population annually. The etiology of this disease is currently uncharacterized, although there is evidence it is associated with Clostridium botulinum neurotoxin in the gut. Prevention is currently not possible, and ileal biopsy diagnosis is invasive. The aim of this study was to characterize the fecal microbiota and biofluid metabolic profiles of EGS horses, to further understand the mechanisms underlying this disease, and to identify metabolic biomarkers to aid in diagnosis. Urine, plasma, and feces were collected from horses with EGS, matched controls, and hospital controls. Sequencing the16S rRNA gene of the fecal bacterial population of the study horses found a severe dysbiosis in EGS horses, with an increase in Bacteroidetes and a decrease in Firmicutes bacteria. Metabolic profiling by 1H nuclear magnetic resonance spectroscopy found EGS to be associated with the lower urinary excretion of hippurate and 4-cresyl sulfate and higher excretion of O-acetyl carnitine and trimethylamine-N-oxide. The predictive ability of the complete urinary metabolic signature and using the four discriminatory urinary metabolites to classify horses by disease status was assessed using a second (test) set of horses. The urinary metabolome and a combination of the four candidate biomarkers showed promise in aiding the identification of horses with EGS. Characterization of the metabolic shifts associated with EGS offers the potential of a noninvasive test to aid premortem diagnosis.

A childhood-onset intestinal toxemia botulism during chemotherapy for relapsed acute leukemia.

BACKGROUND: Botulism is a potentially fatal infection characterized by progressive muscle weakness, bulbar paralysis, constipation and other autonomic dysfunctions. A recent report suggested that cancer chemotherapy might increase the risk for the intestinal toxemia botulism in both adults and children. CASE PRESENTATION: We report a 5-year-old boy, who developed general muscle weakness, constipation, ptosis and mydriasis during the third induction therapy for relapsed acute myeloid leukemia. He had recent histories of multiple antibiotic therapy for bacteremia and intake of well water at home. Repeated bacterial cultures identified Clostridium botulinum producing botulinum neurotoxin A. Botulinum toxin A was isolated from his stools at 17, 21, and 23 days after the onset. Symptoms were self-limiting, and were fully recovered without anti-botulinum toxin globulin therapy. CONCLUSION: This is the second report of a pediatric case with cancer chemotherapy-associated intestinal toxemia botulism. Our case provides further evidence that the immunocompromised status due to anti-cancer treatments increases the risk for the development of botulism at all ages in childhood.

The C-terminal heavy-chain domain of botulinum neurotoxin a is not the only site that binds neurons, as the N-terminal heavy-chain domain also plays a very active role in toxin-cell binding and interactions.

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin.

Structure-based drug discovery for botulinum neurotoxins.

Clostridium botulinum neurotoxin is the most poisonous substance known to humans. It is a potential biowarfare threat and a public health hazard. The only therapeutics available is antibody treatment which will not be effective for post-exposure therapy. There are no drugs available for post-intoxication treatment. Accordingly, it is imperative to develop effective drugs to counter botulism. Available structural information on botulinum neurotoxins both alone and in complex with their substrates offers an efficient method for designing structure-based drugs to treat botulism.

Sublingual immunization with adenovirus F protein-based vaccines stimulates protective immunity against botulinum neurotoxin A intoxication.

Sublingual (s.l.) vaccination is an efficient way to induce elevated levels of systemic and mucosal immune responses. To mediate mucosal uptake, ovalbumin (OVA) was genetically fused to adenovirus 2 fiber protein (OVA-Ad2F) to assess whether s.l. immunization was as effective as an alternative route of vaccination. Ad2F-delivered vaccines were efficiently taken up by dendritic cells and migrated mostly to submaxillary gland lymph nodes, which could readily stimulate OVA-specific CD4(+) T cells. OVA-Ad2F + cholera toxin (CT)-immunized mice elicited significantly higher OVA-specific serum IgG, IgA and mucosal IgA antibodies among the tested immunization groups. These were supported by elevated OVA-specific IgG and IgA antibody-forming cells. A mixed T(h)-cell response was induced as evident by the enhanced IL-4, IL-10, IFN-γ and TNF-α-specific cytokine-forming cells. To assess whether this approach can stimulate neutralizing antibodies, immunizations were performed with the protein encumbering the ß-trefoil domain of C-terminus heavy chain (Hcßtre) from botulinum neurotoxin A (BoNT/A) as well as when fused to Ad2F. Hcßtre-Ad2F + CT-dosed mice showed the greatest serum IgG, IgA and mucosal IgA titers among the immunization groups. Hcßtre-Ad2F alone also induced elevated antibody production in contrast to Hcßtre alone. Plasma from Hcßtre + CT- and Hcßtre-Ad2F + CT-immunized groups neutralized BoNT/A and protected mice from BoNT/A intoxication. Most importantly, Hcßtre-Ad2F + CT-immunized mice were protected from BoNT/A intoxication relative to Hcßtre + CT-immunized mice, which only showed ∼60% protection. This study shows that s.l. immunization with Ad2F-based vaccines is effective in conferring protective immunity.

Survival of radio-marked mallards in relation to management of avian botulism.

Avian botulism outbreaks are frequently perpetuated by type C toxin produced by Clostridium botulinum proliferating in decomposing bird carcasses and consumption of toxic maggots from these carcasses by healthy birds. Therefore, removing bird carcasses has been advocated for disease management because availability of toxic maggots should be reduced, increasing duck survival. However, this management is expensive, and its effect on waterfowl mortality under field conditions is unknown. We radio-marked 419 molting mallards on 11 lakes in western Canada during July-August 1999-2001 and monitored them for 30 days, testing whether survival was higher on lakes with carcass removal. Botulism occurred on 10 lakes. On five carcass removal lakes, greater-than-normal effort was made to conduct early, thorough surveillance and immediately remove carcasses; on six nonremoval lakes, no carcasses were removed. In 1999, estimated 30-day survival probabilities ranged from 0.149 (95% CI=0.065-0.304) on one large lake with carcass removal to 0.466 (95% CI=0.270-0.674) and 0.618 (95% CI=0.443-0.767) on two nonremoval lakes. As a result, we conducted work on smaller wetlands thereafter, reasoning that any management benefit would be easier to detect. In 2000, estimated 30-day survival probabilities were 0.313 (95% CI=0.143-0.556) and 0.794 (95% CI=0.609-0.905) on two carcass removal lakes versus 0.525 (95% CI=0.362-0.682) and 0.743 (95% CI=0.564-0.866) on two nonremoval lakes. In 2001, botulism was detected on two nonremoval lakes where survival probabilities were 0.845 (95% CI=0.630-0.946) and 0.942 (95% CI=0.778-0.987), and on one removal lake where survival probability was 1.0 (95% CI=0.99-1.0), but not detected on the other removal lake where no marked birds died from botulism (1.0, 95% CI=0.99-1.0). Survival tended to be higher on lakes with lower carcass density, but when data were organized by carcass removal versus nonremoval, mallard survival was not consistently greater on lakes where carcasses were removed.

Wound botulism in drug users: a still underestimated diagnosis.

Wound botulism is a rare infectious disease that is becoming a frequent complication of parental drug use. Diagnosis is often difficult and based on clinical suspicion. We report the first Italian case of wound botulism due to intramuscular heroin injection in a 48-year-old man with an acute onset of slurred speech and dysphagia. The most considerable finding of electrophysiological study was the reduction in amplitude of compound muscle action potential which should be considered a useful initial electrodiagnostic sign in the clinical context of botulism. Alerting clinicians to botulism is crucial for a rapid diagnosis and appropriate treatment and thus decreasing mortality and complications.

Botulismo: estudo clínico / Botulism: clinical studies

O botulismo é uma doença infecciosa, neuroparalítica, envolvendo a placa motora, causada pela toxina produzida pelo Clostridium botulinum. Apresenta impacto significativo quando de sua ocorrência, com relação à gravidade dos quadros produzidos. Os autores revisam os aspectos mais relevantes da doença.

The bolulism is a neuroparalitic disease caused by toxin produced by Clostridium botulinum with important impact in public health. In this article is presented a review with the most relevant aspects of the botulism.

Determination of the median toxic dose of type C botulinum toxin in lactating dairy cows.

Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay [ELISA] for type C botulinum toxin). Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined. Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period. Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism. From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method. These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis. The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals.

Endogenous antibody production to botulinum toxin in an adult with intestinal colonization botulism and underlying Crohn's disease.

A patient with obstruction of the terminal ileum from Crohn's disease developed complete paralysis in week 1 of hospitalization. Features initially suggested Guillain-Barre syndrome, but botulinum toxin was identified in serum and stool specimens from week 1 and type A toxin-producing Clostridium botulinum in stool specimens from weeks 3 to 19, confirming botulism due to intestinal colonization. In week 19, the inflamed small bowel was resected, and C. botulinum disappeared from the stool. In week 31, the patient was able to breath without assistance. Testing for an active immune response with neutralizing antibodies to C. botulinum at week 19 was positive; these antibodies remained at a protective level for >1 year. Intestinal colonization botulism, rare in adults, should be considered for patients with descending paralysis, especially those with a preceding alteration in small bowel function. An active immune response to botulinum toxin with production of protective antibodies has not been demonstrated previously in a patient with botulism and may have contributed to this patient's recovery.

Avaliaçäo da eficiência de diferentes tratamentos térmicos na estabilidade microbiológica do leite de coco / Evaluation of the effectiveness of different heating treatments on the microbiological stability of coconut milk

As indústrias de leite de coco no Brasil utilizam como método de conservaçäo deste produto a pasteurizaçäo acompanhada da adiçäo de conservadores químicos do tipo metabissulfito de sódio, sorbato de potássio e benzoato de sódio. Devido às características do produto (pH 6,0 e atividade de água maior que 0,85), esse tratamento näo é efetivo para garantir sua estabilidade microbiológica. Estudou-se entäo a estabilidade microbiológica do leite de coco, comercializado à temperatura ambiente, através da avaliaçäo da eficiência dos processos termoquímicos mais comumente empregados pelas indústrias, comparando-os ao processo de esterilizaçäo em autocalve rotativa. O leite de coco foi previamente formulado e acrescido de esporos de Clostridium sporogenes (PA 3679). Para cada processo utilizaram-se dois níveis de inoculaçäo, um de 2x dez ao quadrado e outro de 2x dez a sexta esporos/200ml. Os resultados obtidos demonstraram que existe a possibilidade do desenvolvimento de clostrídios anaeróbicos putrefativos, indicando inclusive o risco de desenvolvimento de Clostridium botulinum quando näo se utiliza a esterilizaçäo do leite de coco em autoclave rotativa. Portanto, o tratamento térmico à pressäo atmosférica praticado pela indústria implica em risco potencial à saúde pública

Boltulismo en una lactante / Boltulism in an infant

Se describe el caso de una paciente de 2 meses de edad, sin antecedentes mórbidos. Sus manifestaciones clínicas consistieron en constipación, rechazo alimentario e hipotonía agregándose luego dificultad respiratoria progresiva, diplejía facial y pupilas hiporreactivas. Se descartó una enfermedad metabólica e infecciosa. La punción lumbar fue normal y la TAC cerebral normal. La electromiografía fue sugerente de botulismo certificándose el diagnóstico con detección de toxina botulínica tipo A en deposiciones. Requirió ventilación mecánica por 37 días. Presenta mejoría espontánea dándose de alta a los 98 días de hospitalización con hipotonía leve

Diagnóstico bacteriológico de un caso de botulismo infantil y primer aislamiento de C. botulinum en Chile / Bacteriological diagnosis of an infant botulism case and first isolation of C. botulinum in Chile

En noviembre de 1994 se recibió, en el Laboratorio de Anaerobicos del Instituto de Salud Pública de Chile, una muestra de deposición de una lactante de dos meses de edad con el diagnóstico presuntivo de botulismo infantil. La muestra fus sembrada en los medios de cultivo correspondientes y en atmósfera anaerobia hasta obtener un cultivo puro. Este fue sometido a diferentes pruebas bioquímicas, las cuales fueron coincidentes con Clostridium botulinum grupo I. POsteriormente se procedió a confirmar el diagnóstico realizando el test de patogenicidad y neutralización en ratones CF-1. En base a los resultados observados se concluyó que la cepa aislada correspondía a clostridium botulinum grupo I tipo A, lo cual fue confirmado por el Centers for Disease Control de Atlanta. Este fue el primer aislamiento de C. botulinum a partir de una muestra clínica en Chile