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Many bacteria act pathogenic by the release of AB-type protein toxins that efficiently enter human or animal cells and act as enzymes in their cytosol. This leads to disturbed cell functions and the clinical symptoms characteristic for the individual toxin. Therefore, molecules that directly target and neutralize these toxins provide promising novel therapeutic options. Here, we found that the FDA-approved drug disulfiram (DSF), used for decades to treat alcohol abuse, protects cells from intoxication with diphtheria toxin (DT) from Corynebacterium diphtheria, the causative agent of diphtheria, lethal toxin (LT) from Bacillus anthracis, which contributes to anthrax, and C2 enterotoxin from Clostridium botulinum when applied in concentrations lower than those found in plasma of patients receiving standard DSF treatment for alcoholism (up to 20 µM). Moreover, this inhibitory effect is increased by copper, a known enhancer of DSF activity. LT and C2 are binary toxins, consisting of two non-linked proteins, an enzyme (A) and a separate binding/transport (B) subunit. To act cytotoxic, their proteolytically activated B subunits PA63 and C2IIa, respectively, form barrelshaped heptamers that bind to their cellular receptors and form complexes with their respective A subunits LF and C2I. The toxin complexes are internalized via receptor-mediated endocytosis and in acidified endosomes, PA63 and C2IIa form pores in endosomal membranes, which facilitate translocation of LF and C2I into the cytosol, where they act cytotoxic. In DT, A and B subunits are located within one protein, but DT also forms pores in endosomes that facilitate translocation of the A subunit. If cell binding, membrane translocation, or substrate modification is inhibited, cells are protected from intoxication. Our results implicate that DSF neither affects cellular binding nor the catalytic activity of the investigated toxins to a relevant extend, but interferes with the toxin pore-mediated translocation of the A subunits of DT, LT and C2 toxin, as demonstrated by membranetranslocation assays and toxin pore conductivity experiments in the presence or absence of DSF. Since toxin translocation across intracellular membranes represents a central step during cellular uptake of many bacterial toxins, DSF might neutralize a broad spectrum of medically relevant toxins. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is the most toxic protein known, capable of causing severe paralysis and posing a significant bioterrorism threat due to its extreme lethality even in minute quantities. Despite this, there are currently no FDA-approved vaccines for widespread public use. To address this urgent need, we have developed an innovative vaccine platform by fusing the neuronal binding domain of BoNT/E (Hc/E) with core-streptavidin (CS), resulting in a stable CS-Hc/E vaccine. Mice vaccinated with CS-Hc/E exhibited superior antibody titers compared to those receiving Hc/E alone. To develop a trivalent vaccine against BoNT/A, BoNT/B, and BoNT/E--key contributors to the vast majority of human botulism--we conjugated CS-Hc/E with a biotinylated atoxic chimeric protein incorporating neutralizing epitopes from BoNT/A and BoNT/B. This chimeric protein includes the binding domain of BoNT/A, along with the protease-inactive light chain and translocation domains of BoNT/B. The interaction between CS and biotin formed a stable tetrameric antigen, EBA. Vaccination with EBA in mice elicited robust antibody responses and provided complete protection against lethal doses of BoNT/A, BoNT/B, and BoNT/E. Our findings highlight EBA's potential as a stable and effective broadspectrum vaccine against BoNT. Moreover, our technology offers a versatile platform for developing multivalent, stable vaccines targeting various biological threats by substituting the BoNT domain(s) with neutralizing epitopes from other life-threatening pathogens, thereby enhancing public health preparedness and biodefense strategies. [ABSTRACT FROM AUTHOR]

BACKGROUND Botulinum neurotoxins produced by Clostridium botulinum consist of a complex of a core neurotoxin protein and one or more nontoxin accessory proteins. The accessory proteins are generally thought to protect the neurotoxin from the gastric environment in botulism poisoning, dissociating away upon absorption. Other than their questionable immunogenicity, they are rarely mentioned in botulinum toxin therapy. OBJECTIVE To review evidence that accessory proteins potentially play a role in neurotoxin activity. RESULTS Evidence suggests that the accessory proteins do not dissociate from the neurotoxin complex and enhance neurotoxin activity. Complexed type A botulinum toxin has dramatically higher endopeptidase activity than noncomplexed neurotoxin. A primary accessory protein, hemagglutinin-33, exhibits this same effect on both type A and type E core neurotoxin proteins, the latter not natively having this accessory protein. A clinical study using an objective computer assessment assay has shown a correlation between type A complex size and glabellar strain reduction, which reflects increasing clinical efficacy. Finally, a systematic review found no correlation between type A complex size and neutralizing antibody formation. CONCLUSION Accessory proteins may play a role in the efficacy of botulinum toxin and could remain complexed to the neurotoxin for longer than previously reported. [ABSTRACT FROM AUTHOR]

Tilapia stands out as one of the most extensively farmed and consumed fish species globally, valued for its ease of preparation and relative affordability. Although tilapia is a valuable protein source, it can also function as a vector for foodborne pathogens. This literature review reveals that tilapia could carry a variety of contamination with various foodborne pathogens, including Plesiomonas shigelloides, diarrheagenic Escherichia coli, Vibrio parahaemolyticus, Salmonella Weltevreden, Salmonella enterica, Shigella, Staphylococcus aureus, Campylobacter jejuni, Clostridium botulinum, and Listeria monocytogenes. Although guidelines from entities, such as the Global Seafood Alliance, Aquaculture Stewardship Council, and International Organization for Standardization, have been established to ensure the microbiological safety of tilapia, the unique challenges posed by pathogens in tilapia farming call for a more nuanced and targeted approach. Recognizing that contaminants could emerge at various stages of the tilapia supply chain, there is a crucial need for enhanced detection and monitoring of pathogens associated with this fish and its culturing environment. Additionally, it is essential to acknowledge the potential impact of climate change on the safety of tilapia, which may elevate the prevalence and contamination levels of pathogens in this fish. Proactive measures are essential to understand and mitigate the effects of climate change on tilapia production, ensuring the sustainability and safety of this seafood product for both present and future generations. [ABSTRACT FROM AUTHOR]

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO). Recombinant protein engineering experiments with cognate mini-flagellin constructs and chimeras and non-cognate flagellins highlight the potential of Clostridium botulinum Maf glycosyltransferase for future glycoengineering applications. [ABSTRACT FROM AUTHOR]

Simple Summary: In all continents, except in Antarctica, avian botulism outbreaks occur in wild waterbirds with different recurrences and a high severity. This feared disease is due to the ingestion of botulinum neurotoxins (BoNT) mainly produced by Clostridium botulinum, also able to produce persistent spores representing an efficient form of environmental resistance while waiting for suitable conditions enabling bacterial vegetative growth. In addition, concomitant ecological and environmental changes (e.g., the presence of decaying organic material of plant and animal origins) represent a suitable substrate for the replication of Clostridium spp. strains producing BoNT in conditions of high temperatures and the absence of oxygen. This study describes the occurrence, evolution, and management of a severe outbreak of botulism that occurred in a protected wetland area of northeastern Italy, where over 2000 waterbirds (mostly carcasses, and only to a lesser extent sick birds) were recovered in 2019. We also describe the activities underlying the avian botulism monitoring and management performed in this area in 2020, 2021, and 2022. According to an ecological approach, anthropogenic changes that may trigger the occurrence of avian botulism in wetland habitats should be carefully assessed, and habitat management actions should be adaptively planned to protect the biodiversity of these vulnerable ecosystems, which are increasingly affected by ongoing global change. Avian botulism is a paralytic disease due to the ingestion of botulinum neurotoxins (BoNT) produced by anaerobic, sporigenic bacteria (notably, Clostridium botulinum). Wild waterbirds worldwide are affected with variable recurrence and severity, and organic material decaying in wetland habitats may constitute a suitable substrate for the replication of clostridia strains producing BoNT in conditions of high temperatures and the absence of oxygen. Here, we describe a large outbreak of avian botulism that occurred in the Valle Mandriole protected area of northeastern Italy (VM). After the recovery in late summer of a few duck carcasses that molecularly tested positive for BoNT-producing clostridia, in October 2019, the avian botulism escalation led to a total of 2367 birds being recovered (2158 carcasses and 209 sick birds). Among these, 2365/2367 were waterbirds, with ducks accounting for 91.8% of the total (2173/2367) and green-winged teals representing 93.5% of the ducks. After the quick collection of dead and sick birds (from 4 to 11 October 2019) and the flooding of the VM wetland (from 5 to 12 October 2019), the 2019 botulism emergency apparently ended. Following two water inputs in May and July 2020, only one pooled sample obtained from 16 bird carcasses found that year in VM tested positive for clostridia type C by real-time PCR, whereas, after to the implementation of measures deterring the bird's presence, new avian botulism cases—due to clostridia type C and C/D, according to molecular and animal-model tests of confirmation—led to the collection of 176 waterbirds (82 carcasses and 94 sick ducks) and 16 waterbirds (9 carcasses and 7 sick ducks) in the summers 2021 and 2022, respectively. In conclusion, the prevention, management, and control of the disease rely on habitat management, the quick and careful collection/removal of animal carcasses, and the regular monitoring and surveillance of live and dead birds. [ABSTRACT FROM AUTHOR]

Clostridium bacteria were already known to Hippocrates many years before Christ. The name of the Clostridium species is owed to the Polish microbiologist, Adam Prażmowski. It is now known that these Clostridium bacteria are widespread in the natural environment, and their presence in food products is a threat to human health and life. According to European Food Safety Authority (EFSA) reports, every year, there are poisonings or deaths due to ingestion of bacterial toxins, including those of the Clostridium spp. The strengthening of consumer health awareness has increased interest in consuming products with minimal processing in recent years, which has led to a need to develop new techniques to ensure the safety of microbiological food, including elimination of bacteria from the Clostridium genera. On the other hand, the high biochemical activity of Clostridium bacteria allows them to be used in the chemical, pharmaceutical, and medical industries. Awareness of microbiological food safety is very important for our health. Unfortunately, in 2022, an increase in infections with Clostridium bacteria found in food was recorded. Knowledge about food contamination should thus be widely disseminated. [ABSTRACT FROM AUTHOR]

A mortadella is a cooked sausage made from raw pork, beef, chicken or lamb. It is popular in Jordan and neighboring countries for its taste, texture, nutritional value and ease of inclusion in sandwiches. The product is sold in plastic packaging, in circular rolls or vacuum packed. Although it poses no serious threat to human health, the heat can be dangerous if used improperly. A HACCP system is used to manage the production process, with a focus on quality control (CCP) throughout the manufacturing phase. Safety risks are identified at all stages of production, from raw materials to storage and distribution. Foodborne illnesses are primarily caused by pathogenic microorganisms, and these raw materials include pathogenic plant cells, Bacillus cereus, Clostridium botulinum and Clostridium perfringens that survive after cooking and contamination detected after monitoring. Post-manufacturing controls are performed through Good Manufacturing Practices (GMPs). The objectives of the study were to identify potential hazards associated with the production of beef mortadella, determine the CCPs to be implemented, develop a monitoring plan and establish corrective action on a case-by-case basis. The HACCP plan was developed in collaboration with local stakeholders and follows a process flow diagram to identify potential hazards and preventive measures. The system includes the steps for obtaining and storing the ingredients, preparing the meat, adding non-meat ingredients, and transferring the raw mixture to an incubator. The meat is weighed and filled with the mixture to improve cleanliness and avoid excess nitrite. The storage time between incubation and cooking is controlled to allow bacteria to multiply. To kill plant pathogens and reduce the total number of microorganisms, the product is cooked in a steam oven at 75°C for 5 minutes. The aim of the cooking process is to reduce microbial growth before lethal temperatures have been reached and uniform heating of the product has been ensured. Cooking suitability is checked by thermographic records and visual inspection of the cooked product. The plan also includes personnel in charge of every stage of production, ensuring a clean and hygienic environment. [ABSTRACT FROM AUTHOR]

Infant botulism in a 4-month-old boy in China who continued to excrete toxins for over a month despite antitoxin therapy was further treated with fecal microbiota transplantation. After treatment, we noted increased gut microbial diversity and altered fecal metabolites, which may help reduce intestinal pH and enhance anti-inflammatory capabilities.

Botulism, caused by Clostridium botulinum, continues to pose a significant threat to public health. This review explores the historical context and contemporary relevance of botulism, emphasizing its potential lethality and evolving medical applications. In recent years, the use of botulinum toxin in medical procedures, particularly in cosmetic and therapeutic applications, has increased the risk of iatrogenic botulism. The rise in iatrogenic cases underscores the importance of vigilance among health-care providers, especially those in emergency departments, where prompt diagnosis and intervention are critical. This review underscores the necessity for health-care professionals to consider botulism in the differential diagnosis of patients presenting with relevant symptoms, given the potential severity of the condition.

Background Aims Methods Results Conclusions Botulinum Toxin (BoNTA) is the most used nonsurgical aesthetic procedure to treat facial expression lines.This study compared the efficacy of Prabotulinum toxin‐A, a novel BoNTA that originates from Clostridium botulinum Hall‐A, with onabotulinum toxin‐A in treating facial expression lines using the Facial Wrinkle Scale (FWS) and FACE‐Q questionnaires.This was an experimental, comparative, longitudinal, open‐label, and prospective study. Patients aged between 25 and 40 years with upper‐third facial expression lines were included. Follow‐ups were made at three, seven, 30, and 120 days.A total of 26 patients were included: 20 female, and six males, with a mean age of 28.26 years. An average of 31.00 IU and 31.38 IU were administered to the onabotulinum and prabotulinum groups, respectively. The prabotulinum group demonstrated superiority in FWS and Face‐Q scores between the first and third days (p ≤ 0.001, p < 0.001, respectively), which continued on day 7. By day 30, there were no differences in the scores of the two questionnaires.Prabotulinum toxin‐A is a safe and effective treatment for upper‐third facial wrinkles. On day three and seven, the results suggest that prabotulinum toxin‐A has a quicker onset of action than onabotulinum toxin‐A. On days 30 and 120, both groups showed similar FWS and Face‐Q scores. [ABSTRACT FROM AUTHOR]

Introduction: Botulinum neurotoxins (BoNTs) cause botulism and are the most potent natural toxins known. Immunotherapy with neutralizing monoclonal antibodies (MAbs) is considered to be the most effective immediate response to BoNT exposure. Hybridoma technology remains the preferred method for producing MAbs with naturally paired immunoglobulin genes and with preserved innate functions of immune cells. The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against BoNTs. In an effort to develop novel BoNT type A (BoNT/A) immunotherapeutics, sorted by flow cytometry plasmablasts and activated memory B cells from a donor repeatedly injected with BoNT/A for aesthetic botulinum therapy could be used due to obtain hybridomas producing native antibodies. Methods: Plasmablasts and activated memory B-cells were isolated from whole blood collected 7 days after BoNT/A injection and sorted by flow cytometry. The sorted cells were then electrofused with the K6H6/B5 cell line, resulting in a producer of native human monoclonal antibodies (huMAbs). The 3 antibodies obtained were then purified by affinity chromatography, analyzed for binding by Western blot assay and neutralization by FRET assay. Results: We have succeeded in creating 3 hybridomas that secrete huMAbs specific to native BoNT/A and the proteolytic domain (LC) of BoNT/A. The 1B9 antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusion: The use activated plasmablasts and memory B-cells isolated at the peak of the immune response (at day 7 of immunogenesis) that have not yet completed the terminal stage of differentiation but have undergone somatic hypermutation for hybridization allows us to obtain specific huMAbs even when the immune response of the donor is weak (with low levels of specific antibodies and specific B-cells in blood). A BoNT/A LC-specific antibody is capable of effectively inhibiting BoNT/A by mechanisms not previously associated with antibodies that neutralize BoNT. Antibodies specific to BoNT LC can be valuable components of a mixture of antibodies against BoNT exposure. [ABSTRACT FROM AUTHOR]

This investigation was applied to isolate and identify bacteria in three different restaurants order to predict their effects on people. Power plate method for isolation of bacteria the samples were inoculated into nutrient agar media by streak plate technique to obtain well isolated colonies. The results show the identity and the total colony forming units (CFU) for bacteria. The most frequently isolated bacteria were the bacterial species: Bacillus cereus, Clostridium botulinum, Escherichia coli, Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus and Lactobacillus subtilus (11.12, 8.24, 20.11,17.32, 17.01, 10.31 and 8.27) % respectively. According to the bacteria the higher numbers of bacteria were isolated on menu and containers. [ABSTRACT FROM AUTHOR]

This study investigates the application of microwave combined induction heating (MCIH) to steam ready-to-eat pork with rice powder, emphasizing the advantages of rapid and uniform heating. The experimental setup included a mixture of 180 g pork strips, 30 g rice powder, and 10 g water in a CPET tray using MCIH with 1080 W microwave (MW) and 130 °C induction heating (IH) for 150 s. The results showed a quick temperature increase rate of 0.56 °C/s that achieved pasteurization against a variety of pathogenic bacteria, such as Listeria monocytogenes, but not Clostridium botulinum, by lethality calculation. Compared to typical electric cooker steaming, MCIH significantly shortened cooking time (8.6 times faster). To address rice starch gelatinization, two-stage heating techniques to steam pork with rice powder were MCIH: 150 s, and then IH: 60 s (MW1), and MCIH: 180 s, and then IH: 30 s (MW2), with no significant differences seen in color or the nine-point taste scale between treatment groups. MCIH groups had smaller shear forces than control. After MCIH cooking, no microbial counts were detected in the MW1 and MW2 groups initially, and the pork with rice powder had a shelf life of 14 days at 4 °C based on aerobic plate count assay. [ABSTRACT FROM AUTHOR]

Given the growing global demand for seafood, it is imperative to conduct a comprehensive study on the prevalence and persistence patterns of pathogenic bacteria and viruses associated with specific seafood varieties. This assessment thoroughly examines the safety of seafood products, considering the diverse processing methods employed in the industry. The importance of understanding the behavior of foodborne pathogens, such as Salmonella typhimurium, Vibrio parahaemolyticus, Clostridium botulinum, Listeria monocytogenes, human norovirus, and hepatitis A virus, is emphasized by recent cases of gastroenteritis outbreaks linked to contaminated seafood. This analysis examines outbreaks linked to seafood in the United States and globally, with a particular emphasis on the health concerns posed by pathogenic bacteria and viruses to consumers. Ensuring the safety of seafood is crucial since it directly relates to consumer preferences on sustainability, food safety, provenance, and availability. The review focuses on assessing the frequency, growth, and durability of infections that arise during the processing of seafood. It utilizes next‐generation sequencing to identify the bacteria responsible for these illnesses. Additionally, it analyzes methods for preventing and intervening of infections while also considering the forthcoming challenges in ensuring the microbiological safety of seafood products. This evaluation emphasizes the significance of the seafood processing industry in promptly responding to evolving consumer preferences by offering current information on seafood hazards and future consumption patterns. To ensure the continuous safety and sustainable future of seafood products, it is crucial to identify and address possible threats. [ABSTRACT FROM AUTHOR]

The role of Real-Time PCR assays for surveillance and rapid screening for pathogens is garnering more and more attention because of its versatility and ease of adoption. The goal of this study was to design, test, and evaluate Real-Time TaqMan PCR assays for the detection of botulinum neurotoxin (bont/A-G) genes from currently recognized BoNT subtypes. Assays were computationally designed and then laboratory tested for sensitivity and specificity using DNA preparations containing bont genes from 82 target toxin subtypes, including nine bivalent toxin types; 31 strains representing other clostridial species; and an extensive panel that consisted of DNA from a diverse set of prokaryotic (bacterial) and eukaryotic (fungal, protozoan, plant, and animal) species. In addition to laboratory testing, the assays were computationally evaluated using in silico analysis for their ability to detect bont gene sequences from recently identified toxin subtypes. Seventeen specific assays (two for each of the bont/C, bont/D, bont/E, and bont/G subtypes and three for each of the bont/A, bont/B, and bont/F subtypes) were designed and evaluated for their ability to detect bont genes encoding multiple subtypes from all seven serotypes. These assays could provide an additional tool for the detection of botulinum neurotoxins in clinical, environmental and food samples that can complement other existing methods used in clinical diagnostics, regulatory, public health, and research laboratories. [ABSTRACT FROM AUTHOR]

Background: Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is one of the most potent known toxins. Moreover, BoNT is classified as one of the most important biological warfare agents that threatens the biosafety of the world. Currently, the approved treatment for botulism in humans is the use of polyvalent horse serum antitoxins. However, they are greatly limited because of insufficient supply and adverse reactions. Thus, treatment of human botulism requires the development of effective toxin-neutralizing antibodies. Considering their advantages, neutralizing nanobodies will play an increasing role as BoNTs therapeutics. Methods: Herein, neutralizing nanobodies binding to the heavy chain (Hc) domain of BoNT/B (BHc) were screened from a phage display library. Then, BoNT/B-specific clones were identified and fused with the human Fc fragment (hFc) to form chimeric heavy chain antibodies. Finally, the affinity, specificity, and neutralizing activity of antibodies against BoNT/B in vivo were evaluated. Results: The B5-hFc, B9-hFc and B12-hFc antibodies demonstrated high affinity for BHc in the nanomolar range. The three antibodies were proven to have potent neutralizing activity against BoNT/B in vivo. Conclusion: The results demonstrate that inhibiting toxin binding to the host receptor is an efficient strategy and the three antibodies could be used as candidates for the further development of drugs to prevent and treat botulism. [ABSTRACT FROM AUTHOR]

Published information about fish botulism is scant. We review here the current literature on fish botulism. Freshwater fish are susceptible to botulism. Only anecdotal evidence exists about possible botulism cases in saltwater fish. With only a few exceptions, the etiology of all cases of fish botulism reported is Clostridium botulinum type E, although fish are sensitive to, and may carry, various C. botulinum types. Clinical signs of botulism in fish include loss of equilibrium and motion, abducted opercula, open mouths, dark pigmentation, and head up/tail down orientation in which attempts to swim result in breaching the surface of the water. Dark pigmentation is thought to be associated with acetylcholine imbalance in botulinum neurotoxin (BoNT)-affected fish. Rarely, but similar to the situation in other animal species, fish can recover from botulism. Fish botulism can cause secondary outbreaks of the disease in birds, as botulism-affected fish stand out from normal fish, and are selectively preyed upon by fish-eating birds, which thus become intoxicated by the BoNT present in sick fish. The source of BoNT in fish has not been definitively confirmed. Fish may ingest C. botulinum spores that then germinate in their digestive tract, but the possibility that fish ingest preformed BoNT from the environment (e.g., dead fish, shellfish, insects) cannot be ruled out. The presumptive diagnosis of botulism in fish is established based on clinical signs, and as in other species, confirmation should be based on detection of BoNT in intestinal content, liver, and/or serum of affected fish. [ABSTRACT FROM AUTHOR]

Dipicolinic acid (DPA) is a major constituent of spores and reportedly provides protection against inactivation by various thermal processes; however, the relationship between DPA and resistance towards pressure-assisted thermal processing is not well understood. Thermal and pressure-assisted thermal inactivation studies of Clostridium botulinum nonproteolytic strains QC-B and 610-F, proteolytic strain Giorgio-A, and thermal surrogate Clostridium sporogenes PA3679 spores suspended in ACES buffer (0.05 M, pH 7.0) were performed to determine if a relationship exists between DPA release and log reduction of spores. Thermal inactivation at 80, 83, and 87 °C for nonproteolytic strains and 101, 105, and 108 °C for the proteolytic strain and thermal surrogate were conducted. Pressure-assisted thermal inactivation for nonproteolytic strains at 83 °C/600 MPa and for the proteolytic strain and thermal surrogate at 105 °C/600 MPa were performed. Surviving spores were enumerated by 5-tube MPN method for log reductions and analyzed for released DPA by liquid chromatography-tandem mass spectrometry. The correlation between MPN log reductions, released DPA, and D-values were calculated. A positive correlation between released DPA and log reduction of spores was observed for QC-B and 610-F at 80 and 83 °C (r = 0.6073 − 0.7755; P 0.05). A strong, positive correlation (r = 0.8359 − 0.9284; P 0.05) was found for PA3679 after pressure-assisted thermal treatment. These results suggest a correlation exists between DPA release and heat resistance; however, the level of correlation varied between strains and temperatures. The findings from this research may aid in the development of spore inactivation strategies targeting the thermal resistance profiles of various strains of C. botulinum spores.

Many bacteria act pathogenic by the release of AB-type protein toxins that efficiently enter human or animal cells and act as enzymes in their cytosol. This leads to disturbed cell functions and the clinical symptoms characteristic for the individual toxin. Therefore, molecules that directly target and neutralize these toxins provide promising novel therapeutic options. Here, we found that the FDA-approved drug disulfiram (DSF), used for decades to treat alcohol abuse, protects cells from intoxication with diphtheria toxin (DT) from Corynebacterium diphtheria, the causative agent of diphtheria, lethal toxin (LT) from Bacillus anthracis, which contributes to anthrax, and C2 enterotoxin from Clostridium botulinum when applied in concentrations lower than those found in plasma of patients receiving standard DSF treatment for alcoholism (up to 20 µM). Moreover, this inhibitory effect is increased by copper, a known enhancer of DSF activity. LT and C2 are binary toxins, consisting of two non-linked proteins, an enzyme (A) and a separate binding/transport (B) subunit. To act cytotoxic, their proteolytically activated B subunits PA63 and C2IIa, respectively, form barrel-shaped heptamers that bind to their cellular receptors and form complexes with their respective A subunits LF and C2I. The toxin complexes are internalized via receptor-mediated endocytosis and in acidified endosomes, PA63 and C2IIa form pores in endosomal membranes, which facilitate translocation of LF and C2I into the cytosol, where they act cytotoxic. In DT, A and B subunits are located within one protein, but DT also forms pores in endosomes that facilitate translocation of the A subunit. If cell binding, membrane translocation, or substrate modification is inhibited, cells are protected from intoxication. Our results implicate that DSF neither affects cellular binding nor the catalytic activity of the investigated toxins to a relevant extend, but interferes with the toxin pore-mediated translocation of the A subunits of DT, LT and C2 toxin, as demonstrated by membrane-translocation assays and toxin pore conductivity experiments in the presence or absence of DSF. Since toxin translocation across intracellular membranes represents a central step during cellular uptake of many bacterial toxins, DSF might neutralize a broad spectrum of medically relevant toxins.

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A-G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1-OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.

Abstract Background Intestinal botulism is primarily reported in small babies as a condition known as infant botulism. The condition results from the ingestion of environmental or foodborne spores of botulinum neurotoxin (BoNT) producing Clostridia, usually Clostridium botulinum, and subsequent spore germination into active botulinum neurotoxinogenic cultures in the gut. It is generally considered that small babies are susceptible to C. botulinum colonization because of their immature gut microbiota. Yet, it is poorly understood which host factors contribute to the clinical outcome of intestinal botulism. We previously reported a case of infant botulism where the infant recovered clinically in six weeks but continued to secrete C. botulinum cells and/or BoNT in the feces for seven months. Case presentation To further understand the microbial ecology behind this exceptionally long-lasting botulinum neurotoxinogenic colonization, we characterized the infant fecal microbiota using 16S rRNA gene amplicon sequencing over the course of disease and recovery. C. botulinum could be detected in the infant fecal samples at low levels through the acute phase of the disease and three months after recovery. Overall, we observed a temporal delay in the maturation of the infant fecal microbiota associated with a persistently high-level bifidobacterial population and a low level of Lachnospiraceae, Bacteroidaceae and Ruminococcaceae compared to healthy infants over time. Conclusion This study brings novel insights into the infant fecal composition associated with intestinal botulism and provides a basis for a more systematic analysis of the gut microbiota of infants diagnosed with botulism. A better understanding of the gut microbial ecology associated with infant botulism may support the development of prophylactic strategies against this life-threatening disease in small babies.

ObjectiveTo provide a scientific basis for the prevention and control of botulism， we analyzed the epidemic characteristics and influencing factors of foodborne botulism incidents in Qinghai Province over the years.MethodsBotulism data from 1959 to 2022 were collected， and epidemiological characteristics were analyzed using Microsoft Excel 2021 software. The Mann-Kendall trend test was conducted to analyze the number of botulism events and case fatality rates using R 4.2.3 software. The ArcGis 10.2 software was used to create a spatial distribution map of botulism incidents in Qinghai Province.ResultsA total of 86 events of foodborne botulism occurred from 1959 to 2022， with 348 cases and 195 deaths. The case fatality rate was 56.03%. The incidence was mainly concentrated in the second and third quarters， with herders being the major affected population. The chief type of botulinum toxin was the E type and “air-dried meat” was the major toxic food source. Raw and ready-to-eat foods were the major products consumed.ConclusionAs a region with a high incidence of foodborne botulism， the cases of botulism and fatality rates have decreased significantly from 1959 to 2022. However， the case fatality rate remains high. Considering the particularity and high case fatality rate and geographical location of botulism， food safety education must be strengthened for high-risk groups， attract the attention of grassroots personnel in high-incidence areas， and improve the ability of grassroots medical institutions to treat this disease.

Clostridium botulinum strain Hall produces potent botulinum neurotoxin type A1, which causes food-borne, infant, and wound botulism in humans. Antibiotics and botulinum antitoxins can control growth and prevent botulinum toxicity. However, limited information on a protein with an unknown function hinders the discovery of new drug targets for this disease. In this study, a combined bioinformatics approach with literature support was applied to predict, assign, and validate operome functions. Our functional annotation scheme was based on sequence motifs, conserved domains, structures, protein folds, and evolutionary relationships. Approximately 14.62 % of the operome exhibited sequence similarity to known proteins, with 6.65 % predicted functions for 293 proteins, including 121 proteins exclusive to C. botulinum. Structural analysis revealed a significant presence of the Rossmann fold (26 %) and miscellaneous folds (43 %) among the operome. Transporters (>85) and transcriptional regulators (>45) were prevalent, underscoring their importance in C. botulinum adaptive strategies. The newly identified operome contributed to the diverse cellular and metabolic processes of this organism. The function of its operome was involved in amino acid metabolism and botulinum neurotoxin biosynthesis. In this study, we identified and characterized 13 new virulence proteins from the operome to determine their structure–function relationships. These new metabolic and virulence proteins allow the organism to colonize and interact with the human gastrointestinal tract. This study provides a quest for new drugs and targets for treating the underlying diseases of C. botulinum in humans.

ABSTRACTBotulism is a fatal neurologic disease caused by the botulinum toxin (BoNT) produced by Clostridium botulinum. It is a rare but highly toxic disease with symptoms, such as cramps, nausea, vomiting, diarrhea, dysphagia, respiratory failure, muscle weakness, and even death. Currently, two types of antitoxin are used: equine-derived heptavalent antitoxin and human-derived immunoglobulin (BabyBIG®). However, heptavalent treatment may result in hypersensitivity, whereas BabyBIG®, has a low yield. The present study focused on the development of three anti-BoNT monoclonal antibodies (mAbs), 1B18, C25, and M2, in Nicotiana benthamiana. The plant-expressed mAbs were purified and examined for size, purity and integrity by SDS-PAGE, western blotting and size-exclusion chromatography. Analysis showed that plant-produced anti-BoNT mAbs can fully assemble in plants, can be purified in a single purification step, and mostly remain as monomeric proteins. The efficiency of anti-BoNT mAbs binding to BoNT/A and B was then tested. Plant-produced 1B18 retained its ability to recognize both mBoNT/A1 and ciBoNT/B1. At the same time, the binding specificities of two other mAbs were determined: C25 for mBoNT/A1 and M2 for ciBoNT/B1. In conclusion, our results confirm the use of plants as an alternative platform for the production of anti-BoNT mAbs. This plant-based technology will serve as a versatile system for the development botulism immunotherapeutics.

Background: Intestinal botulism is primarily reported in small babies as a condition known as infant botulism. The condition results from the ingestion of environmental or foodborne spores of botulinum neurotoxin (BoNT) producing Clostridia, usually Clostridium botulinum, and subsequent spore germination into active botulinum neurotoxinogenic cultures in the gut. It is generally considered that small babies are susceptible to C. botulinum colonization because of their immature gut microbiota. Yet, it is poorly understood which host factors contribute to the clinical outcome of intestinal botulism. We previously reported a case of infant botulism where the infant recovered clinically in six weeks but continued to secrete C. botulinum cells and/or BoNT in the feces for seven months. Case presentation: To further understand the microbial ecology behind this exceptionally long-lasting botulinum neurotoxinogenic colonization, we characterized the infant fecal microbiota using 16S rRNA gene amplicon sequencing over the course of disease and recovery. C. botulinum could be detected in the infant fecal samples at low levels through the acute phase of the disease and three months after recovery. Overall, we observed a temporal delay in the maturation of the infant fecal microbiota associated with a persistently high-level bifidobacterial population and a low level of Lachnospiraceae, Bacteroidaceae and Ruminococcaceae compared to healthy infants over time. Conclusion: This study brings novel insights into the infant fecal composition associated with intestinal botulism and provides a basis for a more systematic analysis of the gut microbiota of infants diagnosed with botulism. A better understanding of the gut microbial ecology associated with infant botulism may support the development of prophylactic strategies against this life-threatening disease in small babies. [ABSTRACT FROM AUTHOR]

Despite the technologies applied to food production, microbial contamination and chemical deterioration are still matters of great concern. In order to limit these phenomena, new natural approaches should be applied. In this context, the present study aimed to assess the antioxidant and anti-Clostridial effects of two different polyphenolic extracts derived from olive mill vegetation water, one liquid (LE) and one encapsulated (EE). The extracts have been preliminary characterized using Liquid Chromatography Quadrupole Time-Of Flight spectrometry. The Oxygen Radical Absorbance Capacity method was used to determine the antioxidant capacity, registering a higher value for EE compared to that for LE (3256 ± 85 and 2446 ± 13 µgTE/g, respectively). The antibacterial activity against C. perfringens, C. botulinum and C. difficile was studied by the agar well diffusion method, MIC and MBC determination and a time-kill test. The results confirm that EE and LE are able to limit microbial growth, albeit with minor effects when the phenolic compounds are encapsulated. Further studies are needed to evaluate the possible application of these extracts in food systems. [ABSTRACT FROM AUTHOR]

Botulism, caused by a toxin produced by Clostridium botulinum, is a disease of great importance in public and animal health, being the most common cause of death in wild birds worldwide, mainly affecting aquatic and shore birds. In October 2022, a significant increase in clinical cases of avian botulism was identified at the Pelican Harbor Seabird Station, in Miami, Florida, the United States of America. The objective of the present study was to analyze report cases of avian botulism and its association with water quality from Biscayne Bay of Miami, Florida. Between June and October 2022, 130 animals with a presumptive clinical diagnosis of botulism were received at the Wildlife Center of the PHSS. The clinical histories were obtained, which were systematized in a database to perform descriptive analyses and estimate prevalence, lethality, and mortality indicators. The association of the presence of botulism and water quality was carried out using Spearman´s correlation coefficient for non-parametric data and risk analysis with the calculation of chi-squared test (X²) and odds ratio (OR). The results showed that the prevalence of avian botulism was 20.7% in the period, with the mortality varying between 9% and 19% and lethality between 54% to 90%. The most affected species were Eudocimus albus (American white ibis) and Leucophaeus atricilla (laughing gull). The main clinical signs observed were limb paralysis (92%). An association was found between the disease and combined water quality, water clarity, and nitrogen. The risk analysis was statistically significant for combined water quality (OR=1.96) and nitrogen (OR=0.21). A possible association between the disease and water was identified, however, more studies are needed to determine the influence of different water quality parameters and the bird botulism. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs), derived from Clostridium botulinum, have been employed to treat a range of central and peripheral neurological disease. Some studies indicate that BoNT may be beneficial for pain conditions as well. It has been hypothesized that BoNTs may exert their analgesic effects by preventing the release of pain‐related neurotransmitters and neuroinflammatory agents from sensory nerve endings, suppressing glial activation, and inhibiting the transmission of pain‐related receptors to the neuronal cell membrane. In addition, there is evidence to suggest that the central analgesic effects of BoNTs are mediated through their retrograde axonal transport. The purpose of this review is 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. Most of the studies reviewed in this article were conducted using BoNT/A. The PubMed database was searched from 1995 to December 2022 to identify relevant literature. [ABSTRACT FROM AUTHOR]

Clostridium botulinum has become one of the most well-known microorganisms in medical history, thanks to both fatal botulism epidemics and the recent medical use of botulinum neurotoxin. It is a Gram-positive, anaerobic, spore-forming bacterium that causes classic foodborne botulism, infant botulism, wound botulism, and intestinal colonization botulism, a serious neuroparalytic disease. The most common type of botulism is foodborne botulism, which is caused by eating botulinum toxin-contaminated foods. Just a few micrograms of toxin are enough to cause symptoms and, if untreated, death. Rapid diagnosis of the condition is critical to avoiding fatal outcomes. This article describes a clinical case of a patient who presented to us in June with typical symptoms of botulism. Early clinical diagnosis is based on a thorough medical history, including a meticulous reconstruction of the patient's food history in the days preceding symptom onset, as well as a careful physical examination, which can be highly suggestive of botulinum intoxication. Botulism treatment consists of symptom control, mechanical respiratory support, and Botulinum Antitoxin (BAT) administered intravenously. Epidemiological investigation is critical for quickly identifying the food vehicle causing the intoxication. [ABSTRACT FROM AUTHOR]

In the fall of 2021, a significant mortality event in free-ranging Southern Lapwing (Vanellus chilensis) occurred on a soccer field in southern Brazil. Approximately 130 adult southern lapwings died after showing weakness and flaccid paralysis, characterized by the inability to move or fly and drooped wings. Due to the large number of animals affected, there was concern that they had been criminally poisoned. The affected birds were found to have ingested maggots in fresh poultry litter incorporated into the grass surface. Postmortem examinations of four southern lapwings revealed no significant gross and histological findings. Polymerase Chain Reaction (PCR) for influenza A virus, flavivirus, and paramyxovirus was negative. Based on the epidemiological and clinical findings and the negative viral results, a presumptive diagnosis of botulism was made. This diagnosis was confirmed through mouse bioassay and seroneutralization, which detected botulinum toxin type C. Maggots loaded with botulinum neurotoxins were the probable vehicle for intoxication in the outbreak. Considering the impact of avian botulism on wild bird populations, our results may help prevent similar outbreaks in the future. [ABSTRACT FROM AUTHOR]

Background: Botulinum toxin, produced by the Gram-positive anaerobe Clostridium botulinum, is composed of seven antigenic subtypes (A, B, C, D, E, F, and G). Currently, only Botulinum toxin type A, commonly referred to as "Botox," is approved for clinical use, given its relatively safe clinical profile. Botulinum toxin type A has a wide range of therapeutic indications, including treatment for dystonia, migraine headache, neurogenic bladder, and large muscle spastic disorders. However, the toxin is most widely known for its cosmetic effects in treating wrinkles and facial lines. Case presentation: This article describes a 62-year-old Caucasian female who presented for investigation and workup of an isolated lytic lesion of her frontal bone a few weeks after administration of botulinum toxin injection into the corresponding site in the frontalis muscle. This presented as a large, palpable, painless forehead lump causing significant psychological distress. After no neoplastic cause for the lesion was found and histopathology was performed, our researchers concluded that the most likely explanation was that the bony lytic lesion resulted from inadvertent injection of the "Botox" neurotoxin through the intended target muscle and into the cortex of the underlying bone. Conclusions: Our search of the literature failed to identify any previous cases of this occurring. However, as the popularity of this cosmetic procedure only increases, we believe that this represents an important possible differential for isolated lytic lesion after administration of "Botox" injection. [ABSTRACT FROM AUTHOR]

The toxicity of botulinum multi-domain neurotoxins (BoNTs) arises from a sequence of molecular events, in which the translocation of the catalytic domain through the membrane of a neurotransmitter vesicle plays a key role. A recent structural study of the translocation domain of BoNTs suggests that the interaction with the membrane is driven by the transition of an α helical switch towards a β hairpin. Atomistic simulations in conjunction with the mesoscopic Twister model are used to investigate the consequences of this proposition for the toxin–membrane interaction. The conformational mobilities of the domain, as well as the effect of the membrane, implicitly examined by comparing water and water–ethanol solvents, lead to the conclusion that the transition of the switch modifies the internal dynamics and the effect of membrane hydrophobicity on the whole protein. The central two α helices, helix 1 and helix 2, forming two coiled-coil motifs, are analyzed using the Twister model, in which the initial deformation of the membrane by the protein is caused by the presence of local torques arising from asymmetric positions of hydrophobic residues. Different torque distributions are observed depending on the switch conformations and permit an origin for the mechanism opening the membrane to be proposed. [ABSTRACT FROM AUTHOR]

Retort processing is a food preservation technique to address the challenge posed by Clostridium botulinum for commercial sterility of a food product to get microbiologically safe and stable products by heating. This review aims to explore the journey of retort processing, starting from its early use in single‐batch canned foods and progressing to its contemporary applications with different types of containers and heating mediums. Additionally, it will delve into the adaptability of retort equipment, including its ability to operate in stationary and various agitation states, as well as its flexibility in processing speed for both single‐batch and continuous operations. [ABSTRACT FROM AUTHOR]

Clostridium botulinum C3 exoenzyme (C3bot) exclusively inhibits RhoA, B and C by ADP-ribosylation and is therefore used as a cell-permeable tool for investigating the cellular role of these Rho-GTPases. Rho-GTPases represent a molecular switch integrating different receptor signalling to downstream cascades including transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton and cell proliferation. C3bot-induced inhibition of RhoA leads to reorganization of the actin cytoskeleton, morphological changes, and inhibition of cell proliferation as well as modulation of inflammatory response. In this study, we characterized the C3bot-mediated effects on a full-thickness skin model exhibiting a psoriasis-like phenotype through the addition of cytokines. Indeed, after the addition of cytokines, a decrease in epidermal thickness, parakeratosis, and induction of IL-6 was detected. In the next step, it was studied whether C3bot caused a reduction in the cytokine-induced psoriasis-like phenotypes. Basal addition of C3bot after cytokine induction of the full-thickness skin models caused less epidermal thinning and reduced IL-6 abundance. Simultaneous basal incubation with cytokines and C3bot, IL-6 abundance was inhibited, but epidermal thickness was only moderately affected. When C3bot was added apically to the skin model, IL-6 abundance was reduced, but no further effects on the psoriasis-like phenotype of the epidermis were observed. In summary, C3bot inhibits the cytokine-induced expression of IL-6 and thus may have an impact on the pro-inflammatory immune response in the psoriasis-like phenotype. [ABSTRACT FROM AUTHOR]

Summary: With the growing demand for convenient and shelf‐stable canned beans, understanding the changes in starch digestibility due to retort processing is crucial for optimising nutritional quality and enhancing consumer health. The present study investigated the impact of retort processing, specifically container type and agitation, on the digestibility of black bean starch. Retort pouches exhibited a more rapid heat transfer rate to the enclosed product than cans, which reduced the overall processing duration. Also, the processing time to achieve a Fo = 6 of Clostridium botulinum was almost 2.5 min (pouches) and 3.2 min (cans) faster for the oscillating than the static mode. The in vitro digestibility data revealed that the retort processing of black beans led to an increase in rapidly digestible starch (RDS) from 10.51% to 32.11%. However, slowly digestible starch (SDS) decreased from 15.67% to 7.89%, and resistant starch (RS) decreased from 47.91% to 11.86%, regardless of the container type and motion involved. Processing enhanced starch digestibility in all the combinations of container and motions; nevertheless, black beans in pouches with oscillatory motion still contain a relatively significant amount of SDS and RS than the other combinations. These findings have important implications for the food industry in optimising processing techniques and packaging strategies to preserve the nutritional value of black beans. The findings of this study can have practical implications for food producers engaged in the retort processing of foods contained in pouches and cans. The investigation will provide insights into how the process of retort sterilisation affects digestibility, including slowly digestible starch and resistant starch of different types of starch. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cooked, uncured meat products packaged under reduced oxygen packaging conditions require the control of anaerobic and facultative anaerobic pathogens if they are held at temperatures greater than 3°C at retail or consumer level. The objective of this study was to determine the inhibition of Listeria monocytogenes and Clostridium botulinum in cooked, uncured shredded turkey and pork formulated with synthetic or clean-label antimicrobials. Treatments of shredded meat products were prepared with or without antimicrobials using turkey thigh or breast that were cooked to 85°C, shredded, and chilled before inoculation with the target pathogen. L. monocytogenes inoculated samples were stored at 7.2°C, whereas C. botulinum samples were stored at 12.8°C; triplicate samples were assayed every 2 weeks. In the first set of experiments, L. monocytogenes populations increased 2 to 3 logs within 2 weeks of storage at 7.2°C in both meat control treatments without antimicrobials and in pork with 4% lactate-diacetate blend (LD). A 1-log increase was observed in turkey with 4% LD and Pork with 2% cultured dextrose-vinegar-rosemary (CDVR) under the same storage conditions; a 1-log increase was observed in turkey with CDVR at 4 weeks. The second set of experiments tested the effect of pH reduction (to less than 5.5 by the addition of 0.5% citric acid) in combination with 2% CDVR when added to the brine precook or postcook during shredding. Populations of L. monocytogenes increased 4-log within 2 and 4 weeks at 7.2°C for the control turkey and pork formulations, respectively. No growth was observed in 12 weeks for any antimicrobial CDVR-CA treatments regardless of how antimicrobial was added. Similarly, botulinum toxin was detected in both control treatments at week 2 at 12.8°C, but no toxicity was observed in either antimicrobial treatment through 12 weeks. These data suggest that a combination of 2% cultured dextrose-vinegar-rosemary extract plus 0.5% citric acid to reduce pH inhibits the growth of L. monocytogenes and toxin production of C. botulinum in uncured shredded turkey and pork products stored under mild temperature abuse conditions for up to 12 weeks in reduced oxygen packaging.

The role of Real-Time PCR assays for surveillance and rapid screening for pathogens is garnering more and more attention because of its versatility and ease of adoption. The goal of this study was to design, test, and evaluate Real-Time TaqMan PCR assays for the detection of botulinum neurotoxin (bont/A-G) genes from currently recognized BoNT subtypes. Assays were computationally designed and then laboratory tested for sensitivity and specificity using DNA preparations containing bont genes from 82 target toxin subtypes, including nine bivalent toxin types; 31 strains representing other clostridial species; and an extensive panel that consisted of DNA from a diverse set of prokaryotic (bacterial) and eukaryotic (fungal, protozoan, plant, and animal) species. In addition to laboratory testing, the assays were computationally evaluated using in silico analysis for their ability to detect bont gene sequences from recently identified toxin subtypes. Seventeen specific assays (two for each of the bont/C, bont/D, bont/E, and bont/G subtypes and three for each of the bont/A, bont/B, and bont/F subtypes) were designed and evaluated for their ability to detect bont genes encoding multiple subtypes from all seven serotypes. These assays could provide an additional tool for the detection of botulinum neurotoxins in clinical, environmental and food samples that can complement other existing methods used in clinical diagnostics, regulatory, public health, and research laboratories.

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Simple Summary: This work addresses the significance of Clostridium botulinum in avian botulism. Its importance is underscored due to the numerous outbreaks affecting wild birds and the potential risks it poses to biodiversity. To comprehend this pathogen and effectively fight against it, this work has conducted a meticulous compilation of the most cutting-edge scientific studies to date. This allows the reader to gain insights into the most relevant microbiological characteristics of this bacterium, the conditions that most promote its proliferation, its special and effective epidemiological cycle, the avian species most susceptible to it, the most characteristic clinical manifestations and more. All of this is geared towards equipping us with the necessary tools to implement effective surveillance and control strategies, thereby preventing the death of individuals belonging to endangered species through accurate diagnosis and treatment. The aim is to minimise, as far as possible, the presence of this pathogen in the habitats of these birds. 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. [ABSTRACT FROM AUTHOR]

Smoked fish roes within cold smoked herring are considered a very popular ready-to-eat food in Egypt. Therefore, the microbial quality of fish roes should be of concern. The purposes of this study were to conduct bacterial analysis and the possibility of detection of foodborne pathogens. With limited application of dry heating at 85°C for 1 and 5 minutes to improve the safety of roes. To study the biogenic amines (BA) including spermine, putrescine, cadaverine, histamine, tyramine, and spermidine in fresh, heated at 85°C for 5 min, and stored at 0°C for 6 months smoked roes. Vibrio spp. including Vibrio vulnificus, Vibrio furnissii, and Vibrio carcbariae were found in 5 samples (9.6%). One Salmonella spp. (1.9%) and one Listeria monocytogenes (1.9%), 4 Staphylococcus aureus (7.7%), and 3 Bacillus cereus (5.8%) pathogens. While Clostridium botulinum (vegetative forms) was not detected. None of the aerobic, anaerobic, or selective bacterial counts existed after heating smoked roes at 85°C for 5 minutes. The average of each BA in all samples was below 3mg/100g, which is less than the 5.0 mg/100g acceptable limit recommended by the US Food and Drug Administration. Histamine and tyramine only appeared after cold storage of roes at 0°C for 6 months. Cold smoked herring roe was low in acidity (pH: 5.92), high in moisture (68.5%), protein (61.37%), and lipid content (28.7%), thus it could support the bacteria growth. Additional measures are needed to reduce the possible health risks for fish roe consumers. [ABSTRACT FROM AUTHOR]

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent biotoxins ever known. Its entry into neurons could block vesicle exocytosis to abolish the release of neurotransmitters from nerve terminals, thus leading to muscle paralysis. Although there are so many peptides, antibodies and chemical compounds claimed to have anti-toxin activity, no drug is available in the clinical application except equine antitoxin serum. In the present work, a short peptide inhibitor RRGW of BoNT/A was firstly identified by computer-aided ligand-receptor binding simulation, then an RRGW derived peptide was rational designed based on the fragment of SNAP-25 (141–206 aa). Proteolytic assay showed that the anti-toxin activity of the RRGW derived peptide was much higher than that of RRGW. Digit abduction score assay demonstrated that the derived peptide delayed BoNT/A-induced muscle paralysis at a lower concentration by 20-fold than RRGW. The results supported that RRGW derived peptide can be a potential BoNT/A inhibitor candidate for further treating botulism. [ABSTRACT FROM AUTHOR]

Sporulation is a finely regulated morphogenetic program important in the ecology and epidemiology of Clostridium botulinum. Exogenous elements disrupting sporulation-associated genes contribute to sporulation regulation and introduce diversity in the generally conserved sporulation programs of endospore formers. We identified a novel prophage-like DNA segment, termed the yin element, inserted within yabG, encoding a sporulation-specific cysteine protease, in an environmental isolate of C. botulinum. Bioinformatic analysis revealed that the genetic structure of the yin element resembles previously reported mobile intervening elements associated with sporulation genes. Within a pure C. botulinum culture, we observed two subpopulations of cells with the yin element either integrated into the yabG locus or excised as a circular DNA molecule. The dynamics between the two observed conformations of the yin element was growth-phase dependent and likely mediated by recombination events. The yin element was not required for sporulation by C. botulinum but triggered an earlier entry into sporulation than in a related isolate lacking this element. So far, the yin element has not been found in any other C. botulinum strains or other endospore-forming species. It remains to be demonstrated what kind of competitive edge it provides for C. botulinum survival and persistence. [ABSTRACT FROM AUTHOR]

We report a case of an 80-year-old woman with botulism from 2020 in Osaka, Japan. The patient complained of dysarthria and dizziness. On the same day, the patient developed respiratory failure, and was intubated and placed on mechanical ventilation. Subsequently, ophthalmoparesis and quadriparesis progressed rapidly. Ten days after onset, the patient failed to respond to any external stimulation. Blood tests showed anemia, and computed tomography revealed undiagnosed cervical cancer. Initially, diagnosis of neuromuscular junction disorder and acute motor neuropathy, including paraneoplastic syndrome, were considered. However, intravenous immunoglobulin therapy and plasma exchange were ineffective. A fecal sample on day 30 showed a large number of C. botulinum spores. On day 34, a mouse bioassay revealed botulinum toxin type A in the patient's serum; therefore, a botulinum antitoxin was administered. Later, the patient's muscle strength was gradually improved. However, severe muscle paralysis persisted, and the patient died of cachexia owing to cervical cancer on day 196. The etiology of this case was unknown because no contaminated food was identified during an inspection of the patient's home. Fecal 16S rRNA gene sequencing revealed dysbiosis of the intestinal microbiota with abundant Enterococcus species. Long-lasting excretion of substantial botulinum spores even on day 30 indicated colonization of C. botulinum in the intestinal tract. This case suggests that C. botulinum colonization with co-existing intestinal dysbiosis may be associated with severe and prolonged symptoms of botulism. [ABSTRACT FROM AUTHOR]

For dairy processors, spoilage and pathogenic spore‐forming bacteria are key sources of concern, not only due to their ability to remain dormant in a desiccated state in powders and to survive heat treatments, but also their ability to form biofilms in the vegetative state that lead to contamination of foods. These include members of the genera Bacillus, Geobacillus, Anoxybacillus, Brevibacillus, Paenibacillus and Clostridium, many of which are associated with food poisoning and spoilage. Here, we review the common bacterial species that form spores in whey powders and their sources and provide insights into their risks and strategies to control them. [ABSTRACT FROM AUTHOR]

Botulinum toxin is a protein deriving from the bacteria Clostridium botulinum and it is widely used for the treatment of a variety of muscle hyperactivity syndromes and for cosmetic indications. Having a long-lasting effect, Botulinum toxin type A (BTA) is one of the most botulin toxin products used. Even if BTA has shown benefits in reducing the vertical lines between the eyebrows, Adverse Drug Reactions (ADRs) have been experienced as well, of which the most common ones are headache and drooping eyelids. In addition, since other local and systemic risks have been identified, a non-interventional post-authorization safety study (PASS) has been started. The aim of the present study was to report cases of skin toxicity associated with this drug, considering Individual Case Safety Reports (ICSRs) existing on the Eudravigilance website. Among 1464 ICSRs sent to the EV database, 718 ICSRs, including 5154 PTs, reported BTA as a suspected drug associated with cutaneous toxicity. The majority of patients experiencing BTA-induced skin toxicity were female (92.1%) belonging mostly to the age group of 18–64 years. The most serious criteria, when reported, were "Other Medically Important Condition" and "Caused/prolonged hospitalization", although the outcome was mainly reported as "Unknown". The most reported PTs, related to skin disorders, were: "Erythema", "Rash", "Pruritus", "Urticaria", "Swelling face", "Brow ptosis", "Eyelid ptosis", "Injection site pain", and "Angioedema". Considering that in most ICSRs, ADRs related to skin disorders were symptoms of hypersensitivity reactions which in some conditions could be life-threatening, further studies are required to better define the safety profile of BTA used for aesthetic procedures. [ABSTRACT FROM AUTHOR]