Clostridium Botulinum Research Articles

Evaluation of long-term immune response in cattle to botulism using a recombinant E. coli bacterin formulated with Montanide™ ISA 50 and aluminum hydroxide adjuvants

Botulism is a severe disease caused by potent botulinum neurotoxins (BoNTs) produced by Clostridium botulinum. This disease is associated with high-lethality outbreaks in cattle, which have been linked to the ingestion of preformed BoNT serotypes C and D, emphasizing the need for effective vaccines. The potency of current commercial toxoids (formaldehyde-inactivated BoNTs) is assured through tests in guinea pigs according to government regulatory guidelines, but their short-term immunity raises concerns. Recombinant vaccines containing the receptor-binding domain have demonstrated potential for eliciting robust protective immunity. Previous studies have demonstrated the safety and effectiveness of recombinant E. coli bacterin, eliciting high titers of neutralizing antibodies against C. botulinum and C. perfringens in target animal species. In this study, neutralizing antibody titers in cattle and the long-term immune response against BoNT/C and D were used to assess the efficacy of the oil-based adjuvant compared with that of the aluminum hydroxide adjuvant in cattle. The vaccine formulation containing Montanide™ ISA 50 yielded significantly higher titers of neutralizing antibody against BoNT/C and D (8.64 IU/mL and 9.6 IU/mL, respectively) and induced an immune response that lasted longer than the response induced by aluminum, extending between 30 and 60 days. This approach represents a straightforward, cost-effective strategy for recombinant E. coli bacterin, enhancing both the magnitude and duration of the immune response to botulism.

Microbiological risk assessment and resistome analysis from shotgun metagenomics of bovine colostrum microbiome

Colostrum is known for its nutraceutical qualities, probiotic attributes, and health benefits. The aim of this study was to profile colostrum microbiome from bovine in rural sites of a developing country. The focus was on microbiological safety assessments and antimicrobial resistance, taking into account the risks linked with the consumption of raw colostrum. Shotgun sequencing was employed to analyze microbiome in raw buffalo and cow colostrum. Alpha and beta diversity analyses revealed increased inter and intra-variability within colostrum samples' microbiome from both livestock species. The colostrum microbiome was mainly comprised of bacteria, with over 90% abundance, whereas fungi and viruses were found in minor abundance. Known probiotic species, such as Leuconostoc mesenteroides, Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus paracasei, were found in the colostrum samples. A relatively higher number of pathogenic and opportunistic pathogenic bacteria were identified in colostrum from both animals, including clinically significant bacteria like Clostridium botulinum, Pseudomonas aeruginosa, Escherichia coli, and Listeria monocytogenes. Binning retrieved 11 high-quality metagenome-assembled genomes (MAGs), with three MAGs potentially representing novel species from the genera Psychrobacter and Pantoea. Notably, 175 antimicrobial resistance genes (ARGs) and variants were detected, with 55 of them common to both buffalo and cow colostrum metagenomes. These ARGs confer resistance against aminoglycoside, fluoroquinolone, tetracycline, sulfonamide, and peptide antibiotics. In conclusion, this study describes a thorough overview of microbial communities in buffalo and cow colostrum samples. It emphasizes the importance of hygienic processing and pasteurization in minimizing the potential transmission of harmful microorganisms linked to the consumption of colostrum.

Botulinum neurotoxin X lacks potency in mice and in human neurons.

The family of botulinum neurotoxins comprises the most potent toxins known to humankind. New members of this family of protein toxins as well as more distantly related homologs are being identified. The discovery of BoNT/X via bioinformatic screen in 2017 as a putative new BoNT serotype raised concern about its potential as a pathogenic agent with no available countermeasures. This study for the first time assessed both recombinantly produced and native purified BoNT/X for its vertebrate neurotoxicity.

From Toxin to Treatment: A Narrative Review on the Use of Botulinum Toxin for Autonomic Dysfunction.

Since its regulatory approval over a half-century ago, botulinum toxin has evolved from one of the most potent neurotoxins known to becoming routinely adopted in clinical practice. Botulinum toxin, a highly potent neurotoxin produced by Clostridium botulinum, can cause botulism illness, characterized by widespread muscle weakness due to inhibition of acetylcholine transmission at neuromuscular junctions. The observation of botulinum toxin's anticholinergic properties led to the investigation of its potential benefits for conditions with an underlying etiology of cholinergic transmission, including autonomic nervous system dysfunction. These conditions range from disorders of the integument to gastrointestinal and urinary systems. Several formulations of botulinum toxin have been developed and tested over time, significantly increasing the availability of this treatment for appropriate clinical use. Despite the accelerated and expanded use of botulinum toxin, there lacks an updated comprehensive review on its therapeutic use, particularly to treat autonomic dysfunction. This narrative review provides an overview of the effect of botulinum toxin in the treatment of autonomic dysfunction and summarizes the different formulations and dosages most widely studied, while highlighting reported outcomes and the occurrence of any adverse events.

Equine grass sickness is associated with major abnormalities in the ultrastructure of skeletal neuromuscular junctions.

Equine grass sickness (EGS) is a frequently fatal multisystem neuropathy of equids. The aetiology is unknown; proposed causes include toxicoinfection with Clostridium botulinum and a mycotoxicosis. The effect of EGS on the organisation and structural integrity of the skeletal neuromuscular junction (NMJ), the target of botulinum neurotoxins (BoNTs), is unknown. To compare the organisation and structural integrity of skeletal NMJs from EGS horses, control horses and one horse with a presumptive diagnosis of botulism. Blinded, retrospective case control. NMJs in samples of diaphragm or intercostal muscle from six EGS horses, three control horses and one equine botulism case were compared using electron microscopy, morphometry and confocal light microscopy. A significantly higher percentage of EGS NMJs had abnormal morphology (EGS 72.2%, 95% CI 55.6-84.4; Controls 6.9%, 1.7-23.8; OR 35.1, 8.47-244.8; p < 0.001). EGS NMJs had a significantly lower mean volume fraction occupied by synaptic vesicles (SVs) (EGS 18.7%, 12.6-28.0; Controls 36.3%, 20.8-63.4; p = 0.024). EGS NMJs had evidence of accelerated SV exocytosis and SV depletion, accumulation of neurofilament-like material in terminal boutons and/or bouton degeneration. NMJs from the botulism horse had dense packing of SVs towards the presynaptic membrane active zone, consistent with BoNT intoxication, but had absence of the abnormalities identified in EGS NMJs. Group sizes were limited by difficulties obtaining suitably processed samples. Ages of control and EGS horses differed. Botulism was diagnosed based on clinical and post mortem findings. EGS is associated with major changes in skeletal NMJ ultrastructure that are inconsistent with the effects of BoNTs. SV depletion may reflect increased exocytosis coupled with reduced repopulation of SVs via anterograde axonal transport and endocytosis, consistent with the action of an excitatory presynaptic toxin and/or neurotransmitter reuptake inhibitor. Skeletal NMJs represent a previously unrecognised target for the toxin that causes EGS.

Interaction of Fe2O3 and Fe3O4 Nanoparticle with Pathogenic Bacteria: A In-silico Molecular Mechanism Study

Background:: Magnetic materials like iron, nickel, and cobalt have been a subject of interest among the scientific and research community for centuries. Owing to their unique properties, they are prevalent in the mechanical and electronic industries. In recent times, magnetic materials have undeniable applications in biotechnology and nanomedicine. Bacteria like Salmonella enterica, Clostridium botulinum, Bacillus subtilis, etc, pose a hazard to human health and livestock. This ultimately leads to huge yields and economic losses on a global scale. Antimicrobial resistance has become a significant public health concern in recent years, with the increasing prevalence of drugresistant infections posing a significant threat to global health. Many coherent studies have successfully reported magnetic metal oxide nanoparticles to be highly selective, specific, and effective in neutralizing pathogens through various mechanisms like cell membrane disruption, direct contact-mediated killing, or by generating Reactive Oxygen Species (ROS) and numerous costimulatory and inflammatory cytokines. Therefore, we explored the inhibitory effects of iron oxide nanoparticles (NPs) on various pathogenic bacteria via an in-silico approach. This method helped us to understand the active sites where the iron oxide NPs bind with the bacterial proteins. Methods:: The 3D crystal structures of all the pathogenic proteins of Streptococcus pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica, Shigella flexneri, Clostridium botulinum and nanoparticles (Fe2O3 and Fe3O4) under study were downloaded from RCSB PDB and ChemSpider official websites respectively. It was followed by the in-silico molecular Docking using PyRx and AutoDock Vina and analyzed on LigPlot. Results:: This study interprets the efficacy of the Fe2O3 and Fe3O4 nanoparticles against all the test bacteria. At the same time, Fe2O3 and Fe3O4 formed the most stable complexes with cholera enterotoxin subunit B and lectin II (PA-IIL) mutant S23A of Pseudomonas aeruginosa, respectively. Conclusion:: As in this era of AMR, researchers have been exploring alternative strategies to combat bacterial infections, including using magnetic nanoparticles as a potential treatment. They possess unique physical and chemical properties that make them attractive candidates for antimicrobial therapy, including their ability to penetrate bacterial biofilms and selectively target pathogenic bacteria while leaving healthy cells unharmed. This study examined the inhibitory effects of iron oxide (magnetic) nanoparticles, namely Fe2O3 and Fe3O4, on various bacterial proteins involved in cell-to-cell interactions and pathogenesis.

Pathogenic Escherichia coli (E. coli) food borne outbreak: Detection methods and controlling measures

Food-borne illnesses and diseases are major threats to human health. Common food borne pathogens include bacteria such as Escherichia coli Serogroup (O157), Bacillus cereus, Clostridium botulinum, Campylobacter sp., C. perfringens, some Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococcus aureus, and Vibrio spp. etc. Pathogenic E. coli infection usually causes severe diarrhea. Escherichia coli is the most common cause of acute urinary tract infections as well as urinary tract sepsis. Diarrheagenic E. coli pathotypes can be passed in the feces of humans and other animals. Safeguarding public health during mass gatherings is a big challenge. Despite the recent advances in food preservation techniques and food safety, significant disease outbreaks linked to food borne pathogens such as bacteria, fungi, and viruses still occur worldwide indicating that these pathogens still constitute significant risks to public health. However, at present the conventional method for E. coli detection requires several days, especially in cases where E. coli concentrations are low. Enrichment is a commonly used method for bacterial isolation to increase the cell counts of target bacteria. However, traditional culturing method is also a slow and laborious process requiring a series of steps and may require the use of adjunct methods (e.g. biochemical, serological, nucleic acid-based methods for conclusive identification) and can take up to a week for bacteria. The culture-based approaches coupled with other methods such as PCR, immunoassays, bacteriophages, NGS, Biosensors, and MALDI TOF MS are increasingly being used for the detection and identification of food borne pathogens. Nucleic acid-based methods such as Real-Time PCR and vPCR combined with sequencing approaches are more widely used than immunoassay and NGS-based approaches for pathogen detection. Nanobiotechnology is the latest approach for the detection of pathogens. Bacteriophages are one such unique biological entity that showed excellent host selectivity and have been actively used as recognition probes for pathogen detection. Antibiotics are efficient, powerful, and the most commonly used treatment against pathogenic E. coli. Other ways to treat or prevent E. coli contamination, such as probiotics, antimicrobial nanoparticles, and radiation treatment had been reported.

Simultaneous detection of multiple spoilage bacteria using a fluorogenic loop-mediated isothermal amplification-based multi-sample microfluidic chip

Spoilage bacteria, which ubiquitously contribute to food spoilage, pose a global public health risk. In this study, we developed a centrifugal microfluidic chip integrating fluorogenic loop-mediated isothermal amplification (LAMP) to detect six important spoilage bacteria: Bacillus cereus, Clostridium botulinum, Photobacterium phosphoreum, Pseudomonas putida, Streptococcus agalactiae, and Shewanella putrefaciens. The LAMP reaction components were pre-mixed (primers pre-embedded in each reaction chamber of the chip) and evenly pipetted into the two inlet holes, enabling the simultaneous analysis of six genetic targets from a single sample. This facilitates the automated detection of up to four samples simultaneously using a single chip. The LAMP reactions were conducted in a 5-μL volume at 65 °C for 60 min, achieving limits of detection ranging from 10−1 to 10−2 pg/μL of bacterial genomic DNA. Almost all coefficients of variation for time-to-positive values consistently remained under 10% when genomic DNA was used at concentrations within the limits of detection for the six bacteria. This demonstrated excellent reproducibility across replicates. The clinical sensitivity and specificity for field sample detection were 93.3% and 97.5%, respectively. This method offers ease of operation, rapid analysis, and minimal reagent usage and has great potential for the on-site detection of multiple spoilage bacteria.

Commercial farmed swine harbour a variety of pathogenic bacteria and antimicrobial resistance genes.

Introduction. The northern region of Thailand serves as a crucial area for swine production, contributing to the Thai community food supply. Previous studies have highlighted the presence of foodborne bacterial pathogens originating from swine farms in this region, posing a threat to both human and animal health.Gap statement. Multiple swine bacterial pathogens have been studied at a species level, but the distribution and co-occurrence of bacterial pathogens in agricultural swine has not been well established.Aim. Our study employed the intestinal scraping technique to directly examine the bacterial micro-organisms interacting with the swine host.Methodology. We used shotgun metagenomic sequencing to analyse the bacterial pathogens inhabiting the caecal microbiome of swine from five commercial farms in northern Thailand.Results. A variety of pathogenic and opportunistic bacteria were identified, including Escherichia coli, Clostridium botulinum, Staphylococcus aureus and the Corynebacterium genus. From a One Health perspective, these species are important foodborne and opportunistic pathogens in both humans and agricultural animals, making swine a critical pathogen reservoir that can cause illness in humans, especially farm workers. Additionally, the swine caecal microbiome contains commensal bacteria such as Bifidobacterium, Lactobacillus and Faecalibacterium, which are associated with normal physiology and feed utilization in healthy swine. Antimicrobial resistance genes were also detected in all samples, specifically conferring resistance to tetracycline and aminoglycosides, which have historically been used extensively in swine farming.Conclusion. The findings further support the need for improved sanitation standards in swine farms, and additional monitoring of agricultural animals and farm workers to reduce contamination and improved produce safety for human consumption.

The Latent Threat in Wild Birds: Clostridium botulinum.

Avian botulism caused by Clostridium botulinum emerged in 1910, affecting birds across North America, leading to severe outbreaks exacerbated by climate change, decreasing water levels, and inadequate wastewater management. While deadly for birds, its epidemiological impact on humans and other animals remains limited. Despite its significance, understanding and controlling the disease remain challenging. This review delves into the pathogen's epidemiology in wild bird populations, exploring the transmission, pathogenicity, clinical symptoms, diagnosis and treatment. The disease's growing concern in wild birds relates to the bacterium's adaptability and expansive spread, evident through genetic similarities among strains across countries. Outbreaks are influenced by environmental factors such as temperature and soil characteristics. Wild birds inadvertently transmit the bacterium, perpetuating the cycle through carcasses and flies. Some species suffer severely, while others, like scavengers, show resistance. Understanding disease mechanisms, involving potential toxin ingestion or internal production, remains ongoing. Clinical signs vary, affecting diverse bird orders. Diagnostic methods evolve, with treatment success varying among affected populations. Prevention and surveillance take precedence due to treatment challenges, emphasising population-based strategies and preventive measures to manage the widespread presence of C. botulinum.

Botulism is a Rare but Serious Disease

Botulism is a rare but serious disease caused by the anaerobic bacterium Clostridium botulinum, which produces spores and a toxin. It is very widespread in nature, in the ground and in the digestion of mice, rats and cats. The toxin is often created in rotten food, and the most common sources of infection for humans are canned goods and dried meat products. It is also possible to get the disease if an injured spot on the skin comes into contact with the causative agent of the disease. The disease is not contagious, but occurs suddenly and attacks the nerves.

Botulism in Two Juvenile Wild Red Foxes (Vulpes vulpes) in Somerset, England, UK.

Two juvenile red foxes (Vulpes vulpes) were euthanased because of severe nervous signs and paralysis. Detailed postmortem examinations were carried out with bacteriology, histology, and Clostridium botulinum toxin screening, which confirmed botulism as the cause of the clinical signs.

Closed genome sequence of Clostridium botulinum type B1 strain isolated from an infant botulism case in the United States.

We present the closed genome sequence of the Clostridium botulinum BT-22100019 strain isolated from the stool specimen of an infant diagnosed with botulism. With 4.33-Mb genome size and 28.0% G + C content, the bont/B1 gene encoded for botulinum neurotoxin serotype B was found on a 262 kb plasmid arranged in a ha+ orfx - cluster.

Complete genomes of Clostridium botulinum type B(F) isolates associated with a 1995 foodborne botulism outbreak from commercial pâté reveals a recombination event disrupting the ntnh gene.

Foodborne botulism is a neuroparalytic disease caused by ingestion of foods contaminated with botulinum neurotoxin (BoNT), produced by Clostridium botulinum. In 1995 a husband and wife from Québec, Canada, were hospitalized for several months with prolonged muscle paralysis after ingesting a commercial pâté de campagne. Examination of faecal samples from both patients and the pâté produced viable Group I (proteolytic) C. botulinum type B from each of the three samples. Whole genome sequencing revealed that all three isolates contain identical bont/B5 and bont/F2 genes encoded on a plasmid. Both faecal isolate genomes were identical in chromosome and plasmid length, as well as gene content. The genome of the pâté isolate was nearly identical to that of the faecal isolates with the notable difference of a missing 13-gene insertion on the bont/B5 cluster disrupting the ntnh gene. Examination of the insertion revealed several mobile genetic elements that participate in recombination.

Investigation of a Clostridium botulinum mass poisoning and lessons learned, Ivory Coast, 2023

Investigation of a Clostridium botulinum mass poisoning and lessons learned, Ivory Coast, 2023

Development and characterization of a novel neutralizing scFv vectored immunoprophylaxis against botulinum toxin type A

Botulinum toxin is a protein toxin secreted by Clostridium botulinum that is strongly neurotoxic. Due to its characteristics of being super toxic, quick acting, and difficult to prevent, the currently reported antiviral studies focusing on monoclonal antibodies have limited effectiveness. Therefore, for the sake of effectively prevention and treatment of botulism and to maintain country biosecurity as well as the health of the population, in this study, we intend to establish a single chain antibody (scFv) targeting the carboxyl terminal binding functional domain of the botulinum neurotoxin heavy chain (BONT/AHc) of botulinum neurotoxin type A, and explore the value of a new passive immune method in antiviral research which based on adeno-associated virus (AAV) mediated vector immunoprophylaxis (VIP) strategy. The scFv small-molecular single-chain antibody sequenced, designed, constructed, expressed and purified by hybridoma has high neutralising activity and affinity level, which can lay a good foundation for the modification and development of antibody engineering drugs. In vivo experiments, AAV-mediated scFv engineering drug has good anti-BONT/A toxin neutralisation ability, has advantages of simple operation, stable expression and good efficacy, and may be one of the effective treatment strategies for long-term prevention and protection of BONT/A botulinum neurotoxin.

That head lag is impressive! Infantile botulism in the NICU: a case report

BackgroundInfantile botulism (IB) is a devastating and potentially life-threatening neuromuscular disorder resulting from intestinal colonization by Clostridium botulinum and the resultant toxin production. It can present with constipation, descending paralysis, and, potentially, respiratory failure. Botulism is a diagnosis that is more commonly seen in the pediatric intensive care unit (PICU) or on the general pediatric wards and would not typically be managed in the neonatal intensive care unit (NICU), and therefore requires high clinical suspicion to ensure prompt diagnosis and treatment.Case presentationWe discuss a case where an infant from central Pennsylvania presented to a Level IV NICU rather than to the PICU for an evaluation for sepsis and was uniquely diagnosed with IB. The infant presented with poor oral feeding and reduced oral intake, hypothermia, and lethargy. His symptoms progressed into hypoxia and acute respiratory failure. Interestingly, this infant had no known exposure to honey or any other identifiable sources of botulism contact. The infant’s twin brother and the other infants who attended the mother’s in-home daycare remained asymptomatic. This infant was initially evaluated and managed for a potential infectious etiology. However, a diagnosis of IB was suspected, and was later confirmed through the detection of botulinum toxin in the infant’s stools. A high level of suspicion allowed for timely treatment with Botulism Immune Globulin neutralizing antibodies (BabyBIG), even prior to confirmatory testing. We describe the process of obtaining BabyBIG, as well as the natural course of illness after treatment in our patient who ultimately made a complete recovery.ConclusionsThis case highlights the importance of considering infantile botulism as a diagnostic possibility even in the absence of risk factors, and the need for vigilance in diagnosing and treating this rare but potentially life-threatening condition. With timely recognition, subsequent treatment with BabyBIG, and supportive care, infants with infantile botulism can be expected to recover completely. This information is particularly important for neonatologists providing care for infants outside the neonatal period, especially during times of high patient census and resulting overflow of pediatric admissions in the NICU.

Dissecting and tracing the gut microbiota of infants with botulism: a cross sectional and longitudinal study.

Infant botulism is caused by botulinum neurotoxin (BoNT), which is mainly produced by Clostridium botulinum. However, there is a lack of longitudinal cohort studies on infant botulism. Herein, we have constructed a cross-sectional and longitudinal cohort of infants infected with C. botulinum. Our goal was to reveal the differences in the intestinal microbiota of botulism-infected and healthy infants as well as the dynamic changes over time through multi-omics analysis. We performed 16S rRNA sequencing of 20 infants' stools over a period of 3 months and conducted whole genome sequencing of isolated C. botulinum strains from these laboratory-confirmed cases of infant botulism. Through bioinformatics analysis, we focused on the changes in the infants' intestinal microbiota as well as function over time series. We found that Enterococcus was significantly enriched in the infected group and declined over time, whereas Bifidobacterium was significantly enriched in the healthy group and gradually increased over time. 18/20 isolates carried the type B 2 botulinum toxin gene with identical sequences. In silico Multilocus sequence typing found that 20°C. botulinum isolates from the patients were typed into ST31 and ST32. Differences in intestinal microbiota and functions in infants were found with botulism through cross-sectional and longitudinal studies and Bifidobacterium may play a role in the recovery of infected infants.

Microbial Spoilage Organisms in Seafood Products: Pathogens and Quality Control

Consuming contaminated seafood can lead to various bacterial illnesses, either from contamination at the source or during processing and distribution. These illnesses can result from bacterial infection or the ingestion of toxins formed in the food before consumption. However, the distinction between the two causes is not always straightforward. For instance, toxins from Clostridium botulinum and Staphylococcus aureus are preformed in food during bacterial growth, while the toxin from Clostridium perfringens is usually formed when the bacteria sporulate in the intestinal tract. Moreover, the toxin from Vibrio cholerae O1 (and O139) is generated as the bacteria multiply in the intestinal tract. Scombrotoxin, a common illness linked to seafood consumption, is caused by the production of histamine and potentially related substances by certain types of Gram-negative bacteria on histidine-rich fish tissue. This topic is examined in greater detail in another chapter of this book, and therefore will not be extensively discussed here. This paper will explore the various types of bacteria that can lead to illness in humans after consuming seafood, the reported incidence of such infections in specific countries with available data. It will also examine both conventional and molecular methods for detecting, identifying, and categorizing these bacteria. There is growing interest in vibrios as the cause of bacterial illnesses linked to the consumption of bivalve molluscan shellfish and other seafood varieties, with a specific focus on this bacterial group.

The analgesic effects of botulinum neurotoxin by modulating pain-related receptors; A literature review.

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, have been used for the treatment of various central and peripheral neurological conditions. Recent studies have suggested that BoNTs may also have a beneficial effect on pain conditions. It has been hypothesized that one of the mechanisms underlying BoNTs' analgesic effects is the inhibition of pain-related receptors' transmission to the neuronal cell membrane. BoNT application disrupts the integration of synaptic vesicles with the cellular membrane, which is responsible for transporting various receptors, including pain receptors such as TRP channels, calcium channels, sodium channels, purinergic receptors, neurokinin-1 receptors, and glutamate receptors. BoNT also modulates the opioidergic system and the GABAergic system, both of which are involved in the pain process. Understanding the cellular and molecular mechanisms underlying these effects can provide valuable insights for the development of novel therapeutic approaches for pain management. This review aims to summarize the experimental evidence of the analgesic functions of BoNTs and discuss the cellular and molecular mechanisms by which they can act on pain conditions by inhibiting the transmission of pain-related receptors.