Validation of a clinical assay for botulinum neurotoxins through mass spectrometric detection.

Botulism is a paralytic disease due to the inhibition of acetylcholine exocytosis at the neuromuscular junction, which can be lethal if left untreated. Botulinum neurotoxins (BoNTs) are produced by some spore-forming Clostridium bacteria. The current confirmatory assay to test for BoNTs in clinical specimens is the gold-standard mouse bioassay. However, an Endopep-MS assay method has been developed to detect BoNTs in clinical samples using benchtop mass spectrometric detection. This work demonstrates the validation of the Endopep-MS method for clinical specimens with the intent of method distribution in public health laboratories. The Endopep-MS assay was validated by assessing the sensitivity, robustness, selectivity, specificity, and reproducibility. The limit of detection was found to be equivalent to or more sensitive than the mouse bioassay. Specificity studies determined no cross-reactivity between the different serotypes and no false positives from an exclusivity panel of culture supernatants of enteric disease organisms and non-toxigenic strains of Clostridium. Inter-serotype specificity testing with 19 BoNT subtypes was 100% concordant with the expected results, accurately determining the presence of the correct serotype and the absence of incorrect serotypes. Additionally, a panel of potential interfering substances was used to test selectivity. Finally, clinical studies included clinical specimen stability and reproducibility, which was found to be 99.9% from a multicenter evaluation study. The multicenter validation study also included a clinical validation study, which yielded a 99.4% correct determination rate. Use of the Endopep-MS method will improve the capacity and response time for laboratory confirmation of botulism in public health laboratories.

Inadequate Refrigeration of Some Commercial Foods Is a Continued Cause of Foodborne Botulism in the United States, 1994-2021.

Foodborne botulism is a rapidly progressive potentially fatal paralyzing illness caused by the consumption of botulinum neurotoxin, which is most commonly produced by Clostridium botulinum. Refrigeration is the primary barrier to botulinum neurotoxin production in many processed foods. C. botulinum toxin production has occurred and caused botulism in the United States when foods that were not processed to destroy spores of C. botulinum were stored in an anaerobic environment and not properly refrigerated. We identified 37 cases, including 4 deaths, that occurred during 1994-2021 in the United States from 13 events associated with inadequate refrigeration of commercially produced products. In 11 events, the patient stored the product unrefrigerated at home; in 2 events, a product was kept unrefrigerated at the store before the consumer purchased it. In three events, refrigeration instructions were inadequate or not easily accessible (one label printed on outer but not inner packaging, one label not clearly visible, and one label was not in English). The number of people affected per event ranged from 1 to 16. Using enhanced cost estimates for foodborne botulism cases from a published economic model, these events were estimated to cost >$79M. Potential solutions to this recurring problem include the addition of a secondary barrier, such as an acidifier, to prevent botulinum toxin production, and better labeling to convey risks of refrigerated foods that have not been processed to destroy spores of C. botulinum and to decrease the occurrence of improper storage and handling.

Outbreak of Foodborne Botulism Associated with a Commercially Produced Multipack Potato Product, Colorado: September 2019.

During September 2019, public health authorities in El Paso County, Colorado, were notified of four patients who had presented to nearby hospitals with clinical features consistent with botulism, a paralytic illness caused by botulinum neurotoxin. One patient died soon after presentation; the other three patients required intensive care but recovered after receiving botulism antitoxin. Botulinum toxin type A was detected in serum from all patients. On further investigation, all four patients had shared a meal that included commercially prepared roasted potatoes from an individual package without refrigeration instructions that had been left unrefrigerated for 15 d. Storage of the product at ambient temperature likely allowed botulism spores to produce botulinum toxin, resulting in severe illness and death. The manufacturer improved labeling in response to this outbreak. Public health officials should consider unrefrigerated potato products as a potential source of botulism; clinicians should consider botulism as a possible cause of paralytic illness.

Molecular Characterization of Clostridium botulinum Harboring the bont/B7 Gene.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the causative agent of botulism, a rare but serious disease that can result in death if not treated. Infant botulism occurs when C. botulinum colonizes the intestinal tract of infants and produces BoNT. It has been proposed that infants under the age of 1 year are uniquely susceptible to colonization by C. botulinum as their intestinal microbiota is not fully developed and provides little competition, allowing C. botulinum to thrive and produce BoNT in the gut. There are seven well-characterized serotypes (A-G) of BoNT identified by the ability of specific antitoxins to neutralize BoNTs. Molecular technology has allowed researchers to narrow these further into subtypes based on nucleic acid sequences of the botulinum toxin (bont) gene. One of the most recently recognized subtypes for bont/B is subtype bont/B7. We identified through whole genome sequencing five C. botulinum isolates harboring bont/B7 from CDC's strain collection, including patient isolates and an epidemiologically linked isolate from an opened infant formula container. In this study, we report the results of whole genome sequencing analysis of these C. botulinum subtype bont/B7 isolates. Average nucleotide identity and high quality single nucleotide polymorphism (hqSNP) analysis resulted in two major clades. The epidemiologically linked isolates differed from each other by 2-6 hqSNPs, and this clade separated from the other isolates by 95-119 hqSNPs, corroborating available epidemiological evidence.

Pulsotype Diversity of Clostridium botulinum Strains Containing Serotypes A and/or B Genes.

Clostridium botulinum strains are prevalent in the environment and produce a potent neurotoxin that causes botulism, a rare but serious paralytic disease. In 2010, a national PulseNet database was established to curate C. botulinum pulsotypes and facilitate epidemiological investigations, particularly for serotypes A and B strains frequently associated with botulism cases in the United States. Between 2010 and 2014 we performed pulsed-field gel electrophoresis (PFGE) using a PulseNet protocol, uploaded the resulting PFGE patterns into a national database, and analyzed data according to PulseNet criteria (UPGMA clustering, Dice coefficient, 1.5% position tolerance, and 1.5% optimization). A retrospective data analysis was undertaken on 349 entries comprised of type A and B strains isolated from foodborne and infant cases to determine epidemiological relevance, resolution of the method, and the diversity of the database. Most studies to date on the pulsotype diversity of C. botulinum have encompassed very small sets of isolates; this study, with over 300 isolates, is more comprehensive than any published to date. Epidemiologically linked isolates had indistinguishable patterns, except in four instances and there were no obvious geographic trends noted. Simpson's Index of Diversity (D) has historically been used to demonstrate species diversity and abundance within a group, and is considered a standard descriptor for PFGE databases. Simpson's Index was calculated for each restriction endonuclease (SmaI, XhoI), the pattern combination SmaI-XhoI, as well as for each toxin serotype. The D values indicate that both enzymes provided better resolution for serotype B isolates than serotype A. XhoI as the secondary enzyme provided little additional discrimination for C. botulinum. SmaI patterns can be used to exclude unrelated isolates during a foodborne outbreak, but pulsotypes should always be considered concurrently with available epidemiological data.

A Novel Botulinum Neurotoxin, Previously Reported as Serotype H, Has a Hybrid-Like Structure With Regions of Similarity to the Structures of Serotypes A and F and Is Neutralized With Serotype A Antitoxin.

Botulism is a potentially fatal paralytic disease caused by the action of botulinum neurotoxin (BoNT) on nerve cells. There are 7 known serotypes (A-G) of BoNT and up to 40 genetic variants. Clostridium botulinum strain IBCA10-7060 was recently reported to produce BoNT serotype B (BoNT/B) and a novel BoNT, designated as BoNT/H. The BoNT gene (bont) sequence of BoNT/H was compared to known bont sequences. Genetic analysis suggested that BoNT/H has a hybrid-like structure containing regions of similarity to the structures of BoNT/A1 and BoNT/F5. This novel BoNT was serologically characterized by the mouse neutralization assay and a neuronal cell-based assay. The toxic effects of this hybrid-like BoNT were completely eliminated by existing serotype A antitoxins, including those contained in multivalent therapeutic antitoxin products that are the mainstay of human botulism treatment.

Functional characterization of botulinum neurotoxin serotype H as a hybrid of known serotypes F and A (BoNT F/A).

A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins: botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known subtype F5, while the C-terminus of the heavy chain (HC) most resembled the binding domain of serotype A. We evaluated the functionality of both toxins produced in culture by first incorporating an immunoaffinity step using monoclonal antibodies to purify BoNT from culture supernatants and tested each immune-captured neurotoxin with full-length substrates vesicle-associated membrane protein 2 (VAMP-2), synaptosomal-associated protein 25 (SNAP-25), syntaxin, and shortened peptides representing the substrates. The BoNT/B produced by this strain behaved as a typical BoNT/B, having immunoaffinity for anti-B monoclonal antibodies and cleaving both full length VAMP-2 and a peptide based on the sequence of VAMP-2 in the expected location. As expected, there was no activity toward SNAP-25 or syntaxin. The novel BoNT demonstrated immunoaffinity for anti-A monoclonal antibodies but did not cleave SNAP-25 as expected for BoNT/A. Instead, the novel BoNT cleaved VAMP-2 and VAMP-2-based peptides in the same location as BoNT/F5. This is the first discovery of a single botulinum neurotoxin with BoNT/A antigenicity and BoNT/F light chain function. This work suggests that the newly reported serotype H may actually be a hybrid of previously known BoNT serotype A and serotype F, specifically subtype F5.

Laboratory Investigation of the First Case of Botulism Caused by Clostridium butyricum Type E Toxin in the United States.

We report here the laboratory investigation of the first known case of botulism in the United States caused by Clostridium butyricum type E. This investigation demonstrates the importance of extensive microbiological examination of specimens, which resulted in the isolation of this organism.

First report of an infant botulism case due to Clostridium botulinum type Af.

Most infant botulism cases worldwide are due to botulinum toxin types A and B. Rarely, Clostridium botulinum strains that produce two serotypes (Ab, Ba, and Bf) have also been isolated from infant botulism cases. This is the first reported case of infant botulism due to C. botulinum type Af worldwide.

National outbreak of type a foodborne botulism associated with a widely distributed commercially canned hot dog chili sauce.

BACKGROUND: On 7 and 11 July 2007, health officials in Texas and Indiana, respectively, reported 4 possible cases of type A foodborne botulism to the US Centers for Disease Control and Prevention. Foodborne botulism is a rare and sometimes fatal illness caused by consuming foods containing botulinum neurotoxin. METHODS: Investigators reviewed patients' medical charts and food histories. Clinical specimens and food samples were tested for botulinum toxin and neurotoxin-producing Clostridium species. Investigators conducted inspections of the cannery that produced the implicated product. RESULTS: Eight confirmed outbreak associated cases were identified from Indiana (n = 2), Texas (n = 3), and Ohio (n = 3). Botulinum toxin type A was identified in leftover chili sauce consumed by the Indiana patients and 1 of the Ohio patients. Cannery inspectors found violations of federal canned-food regulations that could have led to survival of Clostridium botulinum spores during sterilization. The company recalled 39 million cans of chili. Following the outbreak, the US Food and Drug Administration inspected other canneries with similar canning systems and issued warnings to the industry about the danger of C. botulinum and the importance of compliance with canned food manufacturing regulations. CONCLUSIONS: Commercially produced hot dog chili sauce caused these cases of type A botulism. This is the first US foodborne botulism outbreak involving a commercial cannery in >30 years. Sharing of epidemiologic and laboratory findings allowed for the rapid identification of implicated food items and swift removal of potentially deadly products from the market by US food regulatory authorities.

Unintended consequences: Renaming botulinum neurotoxin-producing species of clostridium and related species.

Botulinum neurotoxin-producing species of Clostridium are highly diverse. Clostridium botulinum could represent at least four different species of Clostridium. In addition, strains that do not produce botulinum neurotoxin are closely related to toxigenic strains, probably representing the same species. Although reclassification of these organisms has been proposed in the past, their species names have remained unchanged, mainly because of the premise that changing names of medically relevant organisms might cause confusion in the healthcare and scientific community. In this review, we discuss the possible unintended consequences of reclassifying botulinum neurotoxin-producing species of Clostridium, which are of public health, medical, and biodefense interest.

Draft Genome Sequences of 20 Clostridium botulinum Type A Isolates from Foodborne Botulism Outbreaks.

Here, we present 20 draft genome sequences of Clostridium botulinum type A isolates originating from foodborne outbreaks in the United States and Ethiopia. Publicly available genomes enhance our understanding of C. botulinum genomics and are an asset in bioterrorism preparedness.

Draft Genome Sequence of Clostridium botulinum Subtype bont/A5(B2').

Here, we report the draft genome sequence of Clostridium botulinum strain CDC76130, which harbors a rare botulinum toxin gene (bont) complex arrangement of bont/A5 and truncated bont/B2 within the same ha toxin gene cluster.

Clostridium botulinum Type B Isolated From a Wound Botulism Case Due to Injection Drug Use Resembles Other Local Strains Originating From Hawaii.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which can lead to death if untreated. In the United States, over 90% of wound botulism cases are associated with injection drug use of black tar heroin. We sought to determine the phylogenetic relatedness of C. botulinum isolated from an injection drug use wound botulism case and isolates from endogenous infant botulism cases in Hawaii. Nineteen C. botulinum type B isolates from Hawaii and one type B isolate from California were analyzed by whole-genome sequencing. The botulinum toxin gene (bont) subtype was determined using CLC Genomics Workbench, and the seven-gene multi-locus sequence type (MLST) was identified by querying PubMLST. Mashtree and pairwise average nucleotide identity were used to find nearest neighbors, and Lyve-SET approximated a phylogeny. Eighteen of the isolates harbored the bont/B5 gene: of those, 17 were classified as sequence type ST36 and one was classified as ST104. A single isolate from Hawaii harbored bont/B1 and was determined to belong to ST110, and the isolate from California harbored bont/B1 and belonged to ST30. A tree constructed with Lyve-SET showed a high degree of homology among all the Hawaiian C. botulinum isolates that harbor the bont/B5 gene. Our results indicate that the bont/B-expressing isolates recovered from Hawaii are closely related to each other, suggesting local contamination of the drug paraphernalia or the wound itself with spores rather than contamination of the drug at manufacture or during transport. These findings may assist in identifying interventions to decrease wound botulism among persons who inject drugs.

Mild Botulism From Illicitly Brewed Alcohol in a Large Prison Outbreak in Mississippi.

Botulism is typically described as a rapidly progressing, severe neuroparalytic disease. Foodborne botulism is transmitted through consuming food or drink that has been contaminated with botulinum toxin. During a botulism outbreak linked to illicitly brewed alcohol (also known as "hooch" or "pruno") in a prison, 11 (35%) of 31 inmates that consumed contaminated hooch had mild illnesses. This includes 2 inmates with laboratory confirmed botulism. The most frequently reported signs and symptoms among the 11 patients with mild illness included dry mouth (91%), hoarse voice (91%), difficulty swallowing (82%), fatigue (82%), and abdominal pain (82%). Foodborne botulism is likely underdiagnosed and underreported in patients with mild illness. Botulism should be considered on the differential diagnosis for patients with cranial nerve palsies.

First report worldwide of an infant botulism case due to Clostridium botulinum type E.

Clostridium botulinum type E has been associated with botulism in adults but never in infants. Infant botulism type E cases have been associated with neurotoxigenic strains of C. butyricum. We report the first infant botulism case due to C. botulinum type E worldwide.