Infant Botulism: In Search of Clostridium botulinum Spores.

Infant botulism is now the most common form of human botulism in Canada and the United States. Infant botulism is a severe neuroparalytic disease caused by ingestion of the spore-forming neurotoxic clostridia, including Clostridium botulinum that colonize the large intestine and subsequently produce botulinum neurotoxin in situ. It has been over a century since the first surveys documenting the ubiquitous prevalence of C. botulinum in soils around the world. Since then, honey has been identified as the only well-known risk factor for infant botulism despite a multitude of international environmental surveys isolating C. botulinum spores from ground soil, aquatic sediments, and commonly available infant foods. Associations of infant botulism cases with confirmed sources of C. botulinum exposure have primarily implicated outdoor soil and indoor dust, as well as commonly ingested foods including honey, dry cereals, and even powdered infant formula. Yet the origin of infection remains unknown for most infant botulism cases. This review summarizes the various surveys from around the world for C. botulinum in environmental soils and sediments, honey, and other infant foods, as well as laboratory-confirmed associations with documented infant botulism cases. Additional factors are also discussed, including the composition of infant gut microbiota and the practice of breastfeeding. We make several recommendations to better identify sources of exposure to C. botulinum spores that could lead to effective preventive measures and help reduce the incidence of this rare but life-threatening disease.

Antagonistic conflict between transposon-encoded introns and guide RNAs.

TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function as programmable RNA-guided homing endonucleases in bacteria, driving transposon maintenance through DNA double-strand break-stimulated homologous recombination. In this work, we uncovered molecular mechanisms of the transposition life cycle of IS607-family elements that, notably, also encode group I introns. We identified specific features for a candidate "IStron" from Clostridium botulinum that allow the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts evolved an ability to balance competing and mutually exclusive activities that promote selfish transposon spread while limiting adverse fitness costs on the host. Collectively, this work highlights molecular innovation in the multifunctional utility of transposon-encoded noncoding RNAs.

Iatrogenic botulism cases after gastric and axillary application of botulinum toxin and review of literature.

INTRODUCTION: Iatrogenic botulism is a rare, serious disease that progresses with descending paralysis and develops after cosmetic or therapeutic botulinum toxin-A (BoNT-A) application. CASE PRESENTATIONS: In this case series; six cases of iatrogenic botulism followed up in our center are presented. Four of these developed after gastric BoNT-A and two after axillary BoNT-A application. RESULTS: The most important cause for the disease was the use of unlicensed products and high-dose toxin applications. The first symptoms were blurred vision, double vision, difficulty in swallowing, and hoarseness. Symptoms appeared within 4-10 days after the application of BoNT-A. Symptoms progressed in the course of descending paralysis in the following days with fatigue, weakness in extremities and respiratory distress. Diagnosis was based on patient history and clinical findings. The main principles of foodborne botulism therapy were applied in the treatment of iatrogenic botulism. If clinical worsening continued, regardless of the time elapsed after BoNT-A application, the use of botulinum antitoxin made a significant contribution to clinical improvement and was recommended. CONCLUSIONS: Routine and new indications for BoNT-A usage are increasing and, as a result, cases of iatrogenic botulism will be encountered more frequently. Physicians should be alert for iatrogenic botulism in the follow-up after BoNT-A applications and in the differential diagnosis of neurological diseases that are presented with similar findings.

Inhibition of Listeria monocytogenes and Clostridium botulinum in Uncured Shredded Pork and Turkey Packaged Under Reduced Oxygen Conditions.

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.

Botulism in fish: a review.

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.

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

Botulinum neurotoxins (BoNTs) are a class of toxins produced by Clostridium botulinum (C. botulinum) and other species of Clostridia. BoNT/X is a putative novel botulinum neurotoxin identified through genome sequencing and capable of SNARE cleavage, but its neurotoxic potential in humans and vertebrates remained unclear. The C. botulinum strain producing BoNT/X, Strain 111, encodes both a plasmid-borne bont/b2 as well as the chromosomal putative bont/x. This study utilized C. botulinum Strain 111 from Japan as well as recombinantly produced full-length BoNT/X to more fully analyze this putative pathogenic toxin. We confirmed production of full-length, catalytically active native BoNT/X by C. botulinum Strain 111, produced as a disulfide-bonded dichain polypeptide similar to other BoNTs. Both the purified native and the recombinant BoNT/X had high enzymatic activity in vitro but displayed very low potency in human-induced pluripotent stem cell-derived neuronal cells and in mice. Intraperitoneal injection of up to 50 µg of native BoNT/X in mice did not result in botulism; however, mild local paralysis was observed after injection of 2 µg into the gastrocnemius muscle. We further demonstrate that the lack of toxicity by BoNT/X is due to inefficient neuronal cell association and entry, which can be rescued by replacing the receptor binding domain of BoNT/X with that of BoNT/A. These data demonstrate that BoNT/X is not a potent vertebrate neurotoxin like the classical seven serotypes of BoNTs. IMPORTANCE: 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.

Oil addition increases the heat resistance of Clostridium sporogenes spores in braised sauce beef: Perspectives from spore surface characteristics and microstructure.

During thermal processing of braised sauce beef, the lipid content of circularly used sauce increased accordingly because of lipid migration from beef to sauce, which may impact the bacterial heat resistance in the products. This study aims to characterize the heat resistance of Clostridium sporogenes spores in braised sauce beef, and investigate the effects of oil on the spore surface characteristics and microstructure. The results indicated that the heat resistance of C. sporogenes spores in beef was significantly higher than that in sauce. Oil addition remarkably enhanced the spore heat resistance in sauce, with D95°C value three times more than that without oil added, and even higher than that in beef. The results of spore surface characteristics indicated that oil addition led to an increase of hydrophobicity and a decrease of zeta potential, which ultimately increased spore heat resistance. Microstructure analysis indicated that exosporium maintenance and cortex expansion induced by oil addition might contribute to the increase of spore heat resistance. This study has sufficiently verified the importance of oil content on the heat resistance of C. sporogenes spores, which should be taken into consideration when developing thermal processes for controlling the spores in food matrices.

Iatrogenic botulism. / Iatrogen botulisme.

This case study describes severe iatrogenic botulism following treatment with a botulinum toxin injection at a private clinic abroad.

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.

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.

Emulsified and Nanoemulsified Essential Oils in the Control of Clostridium botulinum and Clostridium sporogenes in Mortadella.

Clostridium botulinum is a foodborne pathogen responsible for severe neuroparalytic disease associated with the ingestion of pre-formed toxin in food, with processed meats and canned foods being the most affected. Control of this pathogen in meat products is carried out using the preservative sodium nitrite (NaNO2), which in food, under certain conditions, such as thermal processing and storage, can form carcinogenic compounds. Therefore, the objective was to use nanoemulsified essential oils (EOs) as natural antimicrobial agents, with the aim of reducing the dose of NaNO2 applied in mortadella. The antimicrobial activity of nanoemulsions prepared with mixtures of EOs of garlic, clove, pink pepper, and black pepper was evaluated on endospores and vegetative cells of C. botulinum and Clostridium sporogenes (surrogate model) inoculated in mortadella prepared with 50 parts per million NaNO2. The effects on the technological (pH, water activity, and color) and sensory characteristics of the product were also evaluated. The combinations of EOs and their nanoemulsions showed sporicidal effects on the endospores of both tested microorganisms, with no counts observed from the 10th day of analysis. Furthermore, bacteriostatic effects on the studied microorganisms were observed. Regarding the technological and sensorial characteristics of the product, the addition of the combined EOs had a negative impact on the color of the mortadella and on the flavor/aroma. Despite the strong commercial appeal of adding natural preservatives to foods, the effects on flavor and color must be considered. Given the importance of controlling C. botulinum in this type of product, as well as the reduction in the amount of NaNO2 used, this combination of EOs represents a promising antimicrobial alternative to this preservative, encouraging further research in this direction.

Efficacy and Safety Analysis of Botox Application and Iatrogenic Botulism: Panacea or Peril?

Clostridium botulinum toxin-A (BoNT-A) creates temporary paralysis in the muscles by acting on the muscle-nerve junction. It is injected into the mimic muscles when a decrease in the movements of the mimic muscles is desired. Despite many favorable applications, the use of BoNT-A is not without drawbacks. Although there is no expected serious side effect on health in BoNT-A treatments, various problems can be encountered in patients treated for aesthetic purposes. Botulism is a rare but potentially life-threatening syndrome, which is caused by the toxin produced by the bacterium Clostridium botulinum, which acts on the nervous system, vegetative forms of C. botulinum can only survive in anaerobic conditions, while spore forms are common in nature and can withstand harsh conditions. Botulism can stem from bacterial spores which release toxin in the body; in the form of enteric botulism, and wound botulism. The cases that develop 'iatrogenic botulism' after such procedures are usually those receiving high-dose toxin for therapeutic purposes. The treatment of botulism mainly consists of anti-toxin therapy and, if necessary, intensive care to prevent organ failures, including respiratory support. This article aims to cover all these issues related to botulism and other adverse outcomes related to BoNT-A injection in light of the most recent literature.

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.

The cryo-EM structure of the BoNT/Wo-NTNH complex reveals two immunoglobulin-like domains.

The botulinum neurotoxin-like toxin from Weissella oryzae (BoNT/Wo) is one of the BoNT-like toxins recently identified outside of the Clostridium genus. We show that, like the canonical BoNTs, BoNT/Wo forms a complex with its non-toxic non-hemagglutinin (NTNH) partner, which in traditional BoNT serotypes protects the toxin from proteases and the acidic environment of the hosts' guts. We here report the cryo-EM structure of the 300 kDa BoNT/Wo-NTNH/Wo complex together with pH stability studies of the complex. The structure reveals molecular details of the toxin's interactions with its protective partner. The overall structural arrangement is similar to other reported BoNT-NTNH complexes, but NTNH/Wo uniquely contains two extra bacterial immunoglobulin-like (Big) domains on the C-terminus. Although the function of these Big domains is unknown, they are structurally most similar to bacterial proteins involved in adhesion to host cells. In addition, the BoNT/Wo protease domain contains an internal disulfide bond not seen in other BoNTs. Mass photometry analysis revealed that the BoNT/Wo-NTNH/Wo complex is stable under acidic conditions and may dissociate at neutral to basic pH. These findings established that BoNT/Wo-NTNH/Wo shares the general fold of canonical BoNT-NTNH complexes. The presence of unique structural features suggests that it may have an alternative mode of activation, translocation and recognition of host cells, raising interesting questions about the activity and the mechanism of action of BoNT/Wo as well as about its target environment, receptors and substrates.

Clostridium botulinum C3bot mediated effects on cytokine-induced psoriasis-like phenotype in full-thickness skin model.

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.

Heterologous expression of NoxA confers aerotolerance in Clostridium sporogenes.

Clostridium is a genus of gram-positive obligate anaerobic bacteria. Some species of Clostridium, including Clostridium sporogenes, may be of use in bacteria-mediated cancer therapy. Spores of Clostridium are inert in healthy normoxic tissue but germinate when in the hypoxic regions of solid tumors, causing tumor regression. However, such treatments fail to completely eradicate tumors partly because of higher oxygen levels at the tumor's outer rim. In this study, we demonstrate that a degree of aerotolerance can be introduced to C. sporogenes by transfer of the noxA gene from Clostridium aminovalericum. NoxA is a water-forming NADH oxidase enzyme, and so has no detrimental effect on cell viability. In addition to its potential in cancer treatment, the noxA-expressing strain described here could be used to alleviate challenges related to oxygen sensitivity of C. sporogenes in biomanufacturing.

NTNH protein: more than a bodyguard for botulinum neurotoxins.

As one of the most fatal substances, botulinum neurotoxins (BoNTs) have never acted solo to accomplish their formidable missions. Most notably, nontoxic nonhemagglutinin (NTNH), a protein co-secreted with BoNT by bacteria, plays critical roles to stabilize and protect BoNT by tightly associating with it to form the minimal progenitor toxin complex (M-PTC). A new cryo-EM structure of the M-PTC of a BoNT-like toxin from Weissella oryzae (BoNT/Wo) reveals similar assembly modes between M-PTC/Wo and that of other BoNTs, yet also reveals some unique structural features of NTNH/Wo. These findings shed new light on the potential versatile roles of NTNH during BoNT intoxication.

A DARPin promotes faster onset of botulinum neurotoxin A1 action.

In this study, we characterize Designed Ankyrin Repeat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin A1 (BoNT/A1) structure and function. We identify DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro. X-ray crystallography reveals that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and ß-exosite. This DARPin does not block substrate cleavage of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. BoNT/A1 Glu-171 plays a critical role in the interaction with DARPin-F5 and its mutation to Asp, the residue found in BoNT/A3, results in a loss of inhibition of substrate cleavage. In contrast to the in vitro results, DARPin-F5 promotes faster substrate cleavage of BoNT/A1 in primary neurons and muscle tissue by increasing toxin translocation. Our findings could have important implications for the application of BoNT/A1 in therapeutic areas requiring faster onset of toxin action combined with long persistence.

A Novel Prophage-like Insertion Element within yabG Triggers Early Entry into Sporulation in Clostridium botulinum.

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.

An intravenous pancreatic cancer therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi-Non Toxic.

Clostridium novyi has demonstrated selective efficacy against solid tumors largely due to the microenvironment contained within dense tumor cores. The core of a solid tumor is typically hypoxic, acidic, and necrotic-impeding the penetration of current therapeutics. C. novyi is attracted to the tumor microenvironment and once there, can both lyse and proliferate while simultaneously re-activating the suppressed immune system. C. novyi systemic toxicity is easily mitigated by knocking out the phage DNA plasmid encoded alpha toxin resulting in C. novyi-NT; but, after intravenous injection spores are quickly cleared by phagocytosis before accomplishing significant tumor localization. C. novyi-NT could be designed to accomplish intravenous delivery with the potential to target all solid tumors and their metastases in a single dose. This study characterizes CRISPR/Cas9 modified C. novyi-NT to insert the gene for RGD, a tumor targeting peptide, expressed within the promoter region of a spore coat protein. Expression of the RGD peptide on the outer spore coat of C. novyi-NT indicates an increased capacity for tumor localization of C. novyi upon intravenous introduction based on the natural binding of RGD with the αvß3 integrin commonly overexpressed on the epithelial tissue surrounding a tumor, and lead to immune stimulation.