Viable Clostridium botulinum spores not detected in the household dust of major Canadian cities.

Clostridium botulinum causes infant botulism by colonising the intestines and producing botulinum neurotoxin in situ. Previous reports have linked infant botulism cases to C. botulinum spores in household dust, yet the baseline incidence of C. botulinum spores in residential households is currently unknown. Vacuum cleaner dust from 963 households in 13 major Canadian cities was tested for C. botulinum using a novel real-time PCR assay directed against all known subtypes of the botulinum neurotoxin gene. None of the samples tested positive for C. botulinum. Analysis of a random subset of samples by MALDI Biotyper revealed that the most common anaerobic bacterial isolates were of the genus Clostridium and the most common species recovered overall was Clostridium perfringens. Dust that was spiked with C. botulinum spores of each toxin type successfully produced positive real-time PCR reactions. These control experiments indicate that this is a viable method for the detection of C. botulinum spores in household dust. We make several recommendations for future work that may help discover a common environmental source of C. botulinum spores that could lead to effective preventative measures for this rare but deadly childhood disease.

Pathogenicity and virulence of Clostridium botulinum.

Clostridium botulinum, a polyphyletic Gram-positive taxon of bacteria, is classified purely by their ability to produce botulinum neurotoxin (BoNT). BoNT is the primary virulence factor and the causative agent of botulism. A potentially fatal disease, botulism is classically characterized by a symmetrical descending flaccid paralysis, which is left untreated can lead to respiratory failure and death. Botulism cases are classified into three main forms dependent on the nature of intoxication; foodborne, wound and infant. The BoNT, regarded as the most potent biological substance known, is a zinc metalloprotease that specifically cleaves SNARE proteins at neuromuscular junctions, preventing exocytosis of neurotransmitters, leading to muscle paralysis. The BoNT is now used to treat numerous medical conditions caused by overactive or spastic muscles and is extensively used in the cosmetic industry due to its high specificity and the exceedingly small doses needed to exert long-lasting pharmacological effects. Additionally, the ability to form endospores is critical to the pathogenicity of the bacteria. Disease transmission is often facilitated via the metabolically dormant spores that are highly resistant to environment stresses, allowing persistence in the environment in unfavourable conditions. Infant and wound botulism infections are initiated upon germination of the spores into neurotoxin producing vegetative cells, whereas foodborne botulism is attributed to ingestion of preformed BoNT. C. botulinum is a saprophytic bacterium, thought to have evolved its potent neurotoxin to establish a source of nutrients by killing its host.

Botulism in the Brazilian Amazon: a life-threatening disease in a neglected population.

BACKGROUND: Botulism is a rare and potentially fatal neuroparalytic syndrome caused by the gram-positive anaerobe spore-forming bacterium Clostridium botulinum. The microorganism produces a neurotoxin that inhibits the presynaptic release of acetylcholine at the neuromuscular junction, clinically leading to a myasthenic syndrome. OBJECTIVE: To describe the recent outbreak of botulism cases and its demographic, clinical, and laboratory characteristics. METHODS: We report 4 patients with botulism in the recent outbreak occurred between 2017 and 2019 in the state of Amazon. RESULTS: Out of four patients with botulism, three contracted it from eating contaminated food and one had wound botulism. We emphasize the excellent clinical outcome of the different disease presentations in our case series. CONCLUSION: The temporal proximity of these reports may suggest a new rise in the number of cases in the upcoming years. A possible hypothesis is that the rarity of the disease decreased the awareness regarding the primary prevention or even a diagnosis by an untrained physician.

ANTECEDENTES: O botulismo é uma síndrome neuroparalítica rara e potencialmente fatal causada pela bactéria anaeróbia gram-positiva formadora de esporos Clostridium botulinum. O microrganismo produz uma neurotoxina que inibe a liberação pré-sináptica de acetilcolina na junção neuromuscular, levando clinicamente a uma síndrome miastênica. OBJETIVO: Descrever o recente surto de casos de botulismo e suas características demográficas, clínicas e laboratoriais. MéTODOS: Relatamos quatro pacientes com botulismo em surto recente ocorrido entre 2017 e 2019 no estado do Amazonas. RESULTADOS: Dos quatro pacientes com botulismo, três o contraíram devido ao consumo de alimento contaminado e um por ferimento. Enfatizamos o excelente resultado clínico das diferentes apresentações da doença em nossa série de casos. CONCLUSãO: A proximidade temporal dessas notificações pode sugerir um novo aumento no número de casos nos próximos anos. Uma possível hipótese é que a raridade da doença diminuiu a conscientização quanto à prevenção primária ou mesmo ao diagnóstico por médico destreinado.

Tracing Foodborne Botulism Events Caused by Clostridium botulinum in Xinjiang Province, China, Using a Core Genome Sequence Typing Scheme.

Foodborne botulism is a rare but life-threatening illness resulting from the action of a potent toxin mainly produced by Clostridium botulinum. It grows in an oxygen-deficient environment and is extremely viable in meat and soy products, making it one of the most virulent bacteria. How to track foodborne botulism events quickly and accurately has become a key issue. Here, we investigated two foodborne botulism events that occurred in Xinjiang in 2019 based on whole-genome sequencing and also successfully traced the relationship between clinical and food C. botulinum isolates using whole-genome core gene markers. All 59 isolates were classified as group I strains. Of the strains isolated in this study, 44 were found to be botulinum toxin A(B), and 15 isolates contained only the toxin B locus. Both the toxin A and B gene segments were located on the chromosome and organized in an ha cluster. Antibiotic resistance and virulence factors were also investigated. A set of 329 universal core gene markers were established using C. botulinum strains from a public database. These core gene markers were applied to the published C. botulinum genomes, and three outbreaks were identified. This work demonstrates that universal core gene markers can be used to trace foodborne botulism events, and we hope that our work will facilitate this effort in future. IMPORTANCE In this study, we analyzed 59 foodborne botulism (FB)-related strains isolated in Xinjiang Province, China. Our findings not only reveal the group classification, neurotoxin locus organization, antibiotic resistance and virulence factors of these strains but also establish a set of core gene markers for tracing foodborne botulism events, which was verified using published genomes. These findings indicate that these gene markers might be used as a potential tracing tool for FB events caused by C. botulinum group I strains, which have relatively stable genomic components.

Regulatory Networks Controlling Neurotoxin Synthesis in Clostridium botulinum and Clostridium tetani.

Clostridium botulinum and Clostridium tetani are Gram-positive, spore-forming, and anaerobic bacteria that produce the most potent neurotoxins, botulinum toxin (BoNT) and tetanus toxin (TeNT), responsible for flaccid and spastic paralysis, respectively. The main habitat of these toxigenic bacteria is the environment (soil, sediments, cadavers, decayed plants, intestinal content of healthy carrier animals). C. botulinum can grow and produce BoNT in food, leading to food-borne botulism, and in some circumstances, C. botulinum can colonize the intestinal tract and induce infant botulism or adult intestinal toxemia botulism. More rarely, C. botulinum colonizes wounds, whereas tetanus is always a result of wound contamination by C. tetani. The synthesis of neurotoxins is strictly regulated by complex regulatory networks. The highest levels of neurotoxins are produced at the end of the exponential growth and in the early stationary growth phase. Both microorganisms, except C. botulinum E, share an alternative sigma factor, BotR and TetR, respectively, the genes of which are located upstream of the neurotoxin genes. These factors are essential for neurotoxin gene expression. C. botulinum and C. tetani share also a two-component system (TCS) that negatively regulates neurotoxin synthesis, but each microorganism uses additional distinct sets of TCSs. Neurotoxin synthesis is interlocked with the general metabolism, and CodY, a master regulator of metabolism in Gram-positive bacteria, is involved in both clostridial species. The environmental and nutritional factors controlling neurotoxin synthesis are still poorly understood. The transition from amino acid to peptide metabolism seems to be an important factor. Moreover, a small non-coding RNA in C. tetani, and quorum-sensing systems in C. botulinum and possibly in C. tetani, also control toxin synthesis. However, both species use also distinct regulatory pathways; this reflects the adaptation of C. botulinum and C. tetani to different ecological niches.

Investigation and Identification of Food Poisoning Caused by Clostridium botulinum Type B1 in Shenzhen, China.

Clostridium botulinum produces botulinum neurotoxins (BoNTs), which cause people who ingest them to become seriously ill and sometimes die. In recent years, sporadic food poisoning cases associated with C. botulinum have occurred across the world. In 2016, two men were admitted to our hospital in Shenzhen, China, with foodborne botulism. In this study, we report on these two typical C. botulinum-related food poisoning incidents and the steps taken to identify and characterize the causative pathogen. We characterized the bacterial pathogen isolated from the first patient using cooked meat medium and egg yolk agar bacterial cultures under anaerobic conditions, and morphologically identified the isolate using Gram staining. The in vivo bioassay results in mice showed that the minimum lethal dose of the BoNTs produced by our isolate was 0.001-0.0001 mg/mL (LD50 of the culture was estimated to be 1.5812 mg/kg). Whole genome sequencing (WGS) results showed that the isolate was identified as C. botulinum B1 Okra. The causative strain was successfully isolated from the intestinal lavage fluid collected from the initial patient.

Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum.

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

Multi-dimensional nanoscale liquid chromatography and nano-electrospray ion-trap mass spectrometry for detection of Clostridium botulinum type C and the produced botulinum neurotoxin type C complex.

Botulinum neurotoxin types C, D and their mosaic forms C/D and D/C produced mainly by Clostridium botulinum types C and D cause botulism in animals and belong to the most toxic substances for poultry and fish. In addition to intoxications, also toxoinfections with C. botulinum types C and D play a role that should not be underestimated, especially in veterinary medicine. Contrary to other botulinum neurotoxin complexes (BT x), the biosynthesis of these types is phage-encoded. Currently, the gold standard for neurotoxin detection in cases of clinical botulism is the mouse bioassay. In the last few years, alternatives for replacing this mouse bioassay have become increasingly interesting for the detection and characterisation of botulinum neurotoxins. Therefore, immunological techniques based mainly on antibodies, PCR or mass spectral methods have been developed. In this context, the most promising development is that of different endopeptidase assays. In our study, we were able to show that the 2D-nano-LC-MS/MS method presented by Klaubert et al. 2009 especially for detecting BT x A, B, E and F in complex culture media can also be used for detecting BT x C. The focus was therefore on transferring this method to detecting BT x C and pointing out necessary modifications of this current method. For method development, we used different culture preparations and sample conditions. To find out whether BT x C is just as stable against acetic peptic pretreatment as other BT x, we used sample preparations with and without peptic pretreatment. The decisive difference to previous publications is the detection of produced BT x C directly from culture supernatant of different strains of C. botulinum type C. In addition, we present a new approach of detecting protein fragments from C3 and C2 toxin and some specific host cell proteins of the bacterium Clostridium spp. in order to specify the carrier bacterium, therefore verifying the presence of an intact neurotoxin-encoding phage also without directly detecting BT x C and thus the possibility to produce neurotoxin. Herein, we describe a new method to examine environmental samples or suspected feed samples in cases of toxoinfections as well as finding out the causes of clinical botulism. This new approach is particularly interesting for veterinary medicine, especially for diseases like chronic botulism in cows or equine grass sickness.

Botulism during SARS-CoV-2 pandemic: The importance of differential diagnoses.

Botulism is a neuroparalytic syndrome caused by a neurotoxin produced by Clostridium botulinum. We describe a patient with neurological symptoms associated with intoxication by Clostridium botulinum and infection by SARSCoV2. This report underlines that it is mandatory, even in case of SARS-CoV-2 positivity, to investigate all the causes of a clinical pattern.

Foodborne botulism presenting as small bowel obstruction: a case report.

BACKGROUND: Small bowel obstruction is one of the leading reasons for accessing to the Emergency Department. Food poisoning from Clostridium botulinum has emerged as a very rare potential cause of small bowel obstruction. The relevance of this case report regards the subtle onset of pathognomonic neurological symptoms, which can delay diagnosis and subsequent life-saving treatment. CASE PRESENTATION: A 24-year-old man came to our Emergency Department complaining of abdominal pain, fever and sporadic self-limiting episodes of diplopia, starting 4 days earlier. Clinical presentation and radiological imaging suggested a case of small bowel obstruction. Non-operative management was adopted, which was followed by worsening of neurological signs. On specifically questioning the patient, we discovered that his parents had experienced similar, but milder symptoms. The patient also recalled eating home-made preserves some days earlier. A clinical diagnosis of foodborne botulism was established and antitoxin was promptly administered with rapid clinical resolution. CONCLUSIONS: Though very rare, botulism can mimic small bowel obstruction, and could be associated with a rapid clinical deterioration if misdiagnosed. An accurate family history, frequent clinical reassessments and involvement of different specialists can guide to identify this unexpected diagnosis.

Foodborne Botulism in Ukraine from 1955 to 2018.

We aim to identify possible biological, social, and economic factors that could influence the prevalence of foodborne botulism (FB). The objective of this article is to assess epidemiological peculiarities of FB in Ukraine from 1955 to 2018 using national epidemiological surveillance data. This article presents an epidemiological descriptive population-based study of the epidemiology of FB using correlation analysis. From 1955 to 2018, 8614 cases of botulism were recorded in Ukraine causing 659 deaths. The distribution of types of botulism toxins is represented by type A (7.97%), B (59.64%), suspected as C (0.56%), E (25.47%), others (5.33%), and unidentified (1.04%). From 1990 to 2015, the rate correlation between Human Development Index (HDI) and incidence of botulism was -0.75 ± 0.20. Homemade canned meat and fish continue to be the leading causes of botulism in Ukraine. Cases related to commercial food were rare or absent, but in recent years (2017-2018), their percentage has increased to 32.56%. The HDI and botulism have an inverse mathematical correlation and predictable logical relationship: with an HDI increase, the incidence of FB decreased. In general, food botulism in Ukraine is related to traditional socioeconomic factors related to cultural food habits. In the face of declining living standards and uncertainty that food products will be physically or economically available, homemade preservation increases. Home food preservation is a major cause of botulism in Ukraine. The elimination of FB is possible in Ukraine only with the complete cessation of home canning and state control over the manufacture and sale of commercial canned products.

First confirmed case of infant botulism in Africa, caused by a dual-toxin-producing Clostridium botulinum strain.

Botulism, a rare life-threatening toxemia, is probably underdiagnosed in all of its forms in Africa. This study reports the first laboratory-supported case of infant botulism on the African continent. A 10-week-old, previously well infant presented with progressive global weakness, feeding difficulty, and aspiration pneumonia. During a lengthy hospitalization, a rare bivalent Clostridium botulinum strain, producing subtype B3 and F8 toxins and with a new multilocus sequence type, was isolated from stool. The infant was successfully treated with a heptavalent botulinum antitoxin infusion and pyridostigmine. Despite the relative rarity of infant botulism, this case illustrates the importance of maintaining a high level of clinical suspicion when assessing hypotonic infants. The value of modern diagnostic modalities in identifying and characterizing this under-recognized condition is also demonstrated.

Production and characterization of a neutralizing antibody against botulinum neurotoxin A.

As a category A toxic, the botulinum toxin(BoNT) is responsible for human botulism with an estimated lethal dose of 1 ng/kg which greatly increases the potential risk of use as bioweapons. Therefore, the development of anti-BoNT antibodies is urgent. In this paper, the HC domain of BoNT/A was purified and immunized with Balb/c mice. Monoclonal antibodies were screened against BoNT/A from 55 stable positive hybridoma cell lines, and one with the strongest neutralizing activity, designated as ML06, was subcloned, sequenced, and classified as IgG1(κ) subclass. The mouse protection assays showed that ML06 can neutralize the toxin of BoNT/A effectively both in vitro and in vivo, in a dose-dependent manner. The therapeutic assays showed that only 20% of mice injected with 4 LD50 BoNT/A can survive another injection of ML06 after 4 h. The prophylaxis assays showed the residual ML06 from mice injected with ML06 two weeks ago can protect mice against 4 LD50 BoNT/A challenge completely. Collectively, our results indicated that ML06 served as a good candidate for further development of immune therapeutics for BoNT/A.

Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function.

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Diversity of the Genomes and Neurotoxins of Strains of Clostridium botulinum Group I and Clostridium sporogenes Associated with Foodborne, Infant and Wound Botulism.

Clostridium botulinum Group I and Clostridium sporogenes are closely related bacteria responsible for foodborne, infant and wound botulism. A comparative genomic study with 556 highly diverse strains of C. botulinum Group I and C. sporogenes (including 417 newly sequenced strains) has been carried out to characterise the genetic diversity and spread of these bacteria and their neurotoxin genes. Core genome single-nucleotide polymorphism (SNP) analysis revealed two major lineages; C. botulinum Group I (most strains possessed botulinum neurotoxin gene(s) of types A, B and/or F) and C. sporogenes (some strains possessed a type B botulinum neurotoxin gene). Both lineages contained strains responsible for foodborne, infant and wound botulism. A new C. sporogenes cluster was identified that included five strains with a gene encoding botulinum neurotoxin sub-type B1. There was significant evidence of horizontal transfer of botulinum neurotoxin genes between distantly related bacteria. Population structure/diversity have been characterised, and novel associations discovered between whole genome lineage, botulinum neurotoxin sub-type variant, epidemiological links to foodborne, infant and wound botulism, and geographic origin. The impact of genomic and physiological variability on the botulism risk has been assessed. The genome sequences are a valuable resource for future research (e.g., pathogen biology, evolution of C. botulinum and its neurotoxin genes, improved pathogen detection and discrimination), and support enhanced risk assessments and the prevention of botulism.

Assessment of the risk of botulism from chilled, vacuum/modified atmosphere packed fresh beef, lamb and pork held at 3 °C-8 °C.

The safety of current UK industry practice (including shelf-life) for chilled, vacuum/modified atmosphere-packed fresh red meat (beef, lamb and pork) held at 3°C-8°C has been evaluated with respect to non-proteolytic Clostridium botulinum. UK industry typically applies a retail pack shelf-life at 3°C-8°C to 13 days for fresh red meat, with a maximum of 23 days for beef, 27 days for lamb, and 18 days for pork. An exposure assessment established that current commercial practice for fresh red meat provided strong protection with more than 1010 person servings marketed in the UK without association with foodborne botulism. A challenge test demonstrated that spores of non-proteolytic C. botulinum inoculated on chilled vacuum-packed fresh red meat did not lead to detectable neurotoxin at day 50 for beef, day 35 for lamb, or day 25 for pork (i.e. <40 pg type B toxin and type E toxin g-1 of meat). The products were visually spoiled many days before these end points. The exposure assessment and challenge test demonstrated the safety of current UK industry practices for the shelf-life of fresh, vacuum-packed beef, lamb and pork held at 3°C-8°C with respect to C. botulinum, and that botulinum neurotoxin was not detected within their organoleptic shelf-life.

Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment.

The neurotoxin formed by Clostridium botulinum Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of C. botulinum Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse C. botulinum Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages-one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of C. botulinum and its neurotoxin genes, dissecting key aspects of C. botulinum Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.

Fully Human Monoclonal Antibodies Effectively Neutralizing Botulinum Neurotoxin Serotype B.

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine-human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately 10 i.p. LD50/100 µg of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of two HuMAbs, M2 (1.25 µg) and M4 (1.25 µg), was able to completely neutralize BoNT/B1 (80 i.p. LD50) with a potency greater than 80 i.p. LD50/2.5 µg of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication.