Search Results (10)

Botulism is a severe muscle paralysis disease mediated by the botulinum toxin. Here, we reported a foodborne botulism case caused by Clostridium botulinum subtype A5(b3) from self-packaged vacuum spicy rabbit heads. Treatment for this case was delayed due to misdiagnosis and insufficient diagnostic capacity in three hospitals, which resulted in progressive clinical deterioration, and eventually, the patient was transferred to Shandong Public Health Clinical Center for specialized therapy. The case was suspected as foodborne botulism by the Qilu Medical-Prevention Innovation Integration pathway and multi-disciplinary consultation. An epidemiological investigation and laboratory confirmation revealed that the botulinum neurotoxin originated from vacuum-packaged spicy rabbit heads distributed via interprovincial cold chain logistics. After treatment with botulism antiserum, the patient’s condition significantly improved, and they were discharged after recovery. We revealed that this foodborne botulism outbreak was caused by the Clostridium botulinum A5(b3) subtype from food by whole-genome sequencing and SNP typing. All the strains belonged to Group I carrying the botulinum neurotoxin gene classified as the ha cluster. Toxin A was confirmed by MBA and other methods, while toxin B was non-functional due to the truncated bont/B gene. Other virulence genes and antibiotic resistance genes were also detected. Our findings indicate that self-packaged vacuum meat products represent an emerging risk factor for botulism transmission when stored improperly. Importantly, the recurrent misdiagnosis in this case underscored the urgent need to enhance the training of healthcare professionals in medical institutions to improve the diagnostic accuracy and clinical management of botulism. Full article

Allicin is a chemically complex bioactive compound synthesized in many varieties of garlic. The wide range of biological properties of allicin provides the basis for its potential use as an alternative to antibiotic growth promoters that are currently prohibited in farm animal breeding. Among the many benefits resulting from the use of allicin in animal breeding, especially poultry, its modulating effect on intestinal microbiota, which includes the anaerobic spore-forming bacteria of the genus Clostridium spp., seems to be important. The material for this study consists of intestinal content collected from the caecums of Japanese quails (Coturnix japonica). Culture methods were used to isolate the strains, and the obtained isolates were identified based on their phenotypic characteristics. In addition, PCR methods were used for the detection of the ntnh gene-encoding non-haemagglutinin component of botulinum neurotoxins (BoNTs), the detection of individual genes responsible for the production of major toxins by Clostridium perfringens, and the amplification of conservative 16S rDNA genes. The 16S rDNA amplicons were subsequently submitted to Sanger sequencing. The obtained sequences were analyzed using the Basic Local Alignment Search Tool (BLAST). The ntnh gene was not found in the genetic material of the isolated strains. Among the isolates suspected of belonging to the Clostridium perfringens species, the plc gene determining the production of the alpha toxin was detected, which justifies the classification of the strains into toxotype A. The Sanger sequencing results confirm the presence of mainly saprophytic species in the studied material. The statistical analysis indicated a statistically significant reduction in the level of Clostridium spp., obtained by the use of an appropriate dose of allicin. The presented research results indicate the significant impact of an appropriate dose of allicin on reducing the occurrence of anaerobic intestinal microbiota, while providing important information on the potential application of this compound in animal production in the future. Full article

Clostridium botulinum neurotoxins (BoNTs) are the most potent toxins known, causing the deadly disease botulism. They function through Zn²⁺-dependent endopeptidase cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, preventing vesicular fusion and subsequent neurotransmitter release from motor neurons. Several serotypes of BoNTs produced by Clostridium botulinum (BoNT/A-/G and/X) have been well-characterised over the years. However, a BoNT-like gene (homologue of BoNT) was recently identified in the non-clostridial species, Enterococcus faecium, which is the leading cause of hospital-acquired multi-drug resistant infections. Here, we report the crystal structure of the catalytic domain of a BoNT homologue from Enterococcus faecium (LC/En) at 2.0 Å resolution. Detailed structural analysis in comparison with the full-length BoNT/En AlphaFold2-predicted structure, LC/A (from BoNT/A), and LC/F (from BoNT/F) revealed putative subsites and exosites (including loops 1–5) involved in recognition of LC/En substrates. LC/En also appears to possess a conserved autoproteolytic cleavage site whose function is yet to be established. Full article

The diversity of BoNT-producing Clostridia is still a worrying problem for specialists who explore the evolutionary and taxonomic diversity of C. botulinum. It is also a problem for epidemiologists and laboratory staff conducting investigations into foodborne botulism in humans and animals, because their genetic and phenotypic heterogeneity cause complications in choosing the proper analytical tools and in reliably interpreting results. Botulinum neurotoxins (BoNTs) are produced by several bacterial groups that meet all the criteria of distinct species. Despite this, the historical designation of C. botulinum as the one species that produces botulinum toxins is still exploited. New genetic tools such as whole-genome sequencing (WGS) indicate horizontal gene transfer and the occurrence of botulinum gene clusters that are not limited only to Clostridium spp., but also to Gram-negative aerobic species. The literature data regarding the mentioned heterogeneity of BoNT-producing Clostridia indicate the requirement to reclassify C. botulinum species and other microorganisms able to produce BoNTs or possessing botulinum-like gene clusters. The aim of this study was to present the problem of the diversity of BoNT-producing Clostridia over time and new trends toward obtaining a reliable classification of these microorganisms, based on a complex review of the literature. Full article

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

At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense. Full article

A feeding experiment was carried out with late-lactating cows over 12 weeks to evaluate the feeding value of a basic diet with maize and grass silage (MS, GS) when combined with varying portions of concentrate in the ration (20% and 60% on a dry matter basis) and to test the effects on health and performance, the transfer of important Fusarium toxins to blood and milk, the total and Shiga toxin (stx)-forming E. coli counts, and the presence of Clostridium botulinum neurotoxin (BoNT) genes in rectal fecal samples. MS was contaminated by a broader spectrum of fungal and other metabolites compared to GS. MS contained higher concentrations of the important Fusarium toxins deoxynivalenol (DON) and zearalenone (ZEN). Blood and milk levels of DON and ZEN residues generally reflected the differences in exposure at a low level. Feeding of MS with 60% concentrate feed induced subacute ruminal acidosis (SARA) associated with a marked drop in dry matter intake, fat corrected milk yield and a fat to protein ratio in milk of lower than 1. The SARA-associated higher ruminal LPS concentration did not affect the circulating concentrations of haptoglobin as an indicator of systemic inflammation. Lower rumen pH values in both MS-fed groups were associated with lower pH values, higher absolute E. coli counts and increased proportions of stx-positive E. coli in rectal feces. BoNT genes A, B, C, D, E and F remained undetectable in any of the fecal samples suggesting that feedstuffs were virtually free of the corresponding C. botulinum strains. In conclusion, maize feedstuff (silage, grains, starch-containing byproducts)-dominated rations for dairy cows should be avoided to reduce adverse effects on health and food safety. Full article

The presence of botulinum neurotoxin-producing Clostridia (BPC) in food sources is a public health concern. In favorable environmental conditions, BPC can produce botulinum neurotoxins (BoNTs) outside or inside the vertebrate host, leading to intoxications or toxico-infectious forms of botulism, respectively. BPC in food are almost invariably detected either by PCR protocols targeted at the known neurotoxin-encoding genes, or by the mouse test to assay for the presence of BoNTs in the supernatants of enrichment broths inoculated with the tested food sample. The sample is considered positive for BPC when the supernatant contains toxic substances that are lethal to mice, heat-labile and neutralized in vivo by appropriate polyclonal antibodies raised against purified BoNTs of different serotypes. Here, we report the detection in a food sample of a Clostridium tetani strain that produces tetanus neurotoxins (TeNTs) with the above-mentioned characteristics: lethal for mice, heat-labile and neutralized by botulinum antitoxin type B. Notably, neutralization occurred with two different commercially available type B antitoxins, but not with type A, C, D, E and F antitoxins. Although TeNT and BoNT fold very similarly, evidence that antitoxin B antiserum can neutralize the neurotoxic effect of TeNT in vivo has not been documented before. The presence of C. tetani strains in food can produce misleading results in BPC detection using the mouse test. Full article

Botulism is a disease involving intoxication with botulinum neurotoxins (BoNTs), toxic proteins produced by Clostridium botulinum and other clostridia. The 150 kDa neurotoxin is produced in conjunction with other proteins to form the botulinum progenitor toxin complex (PTC), alternating in size from 300 kDa to 500 kDa. These progenitor complexes can be classified into hemagglutinin positive or hemagglutinin negative, depending on the ability of some of the neurotoxin-associated proteins (NAPs) to cause hemagglutination. The hemagglutinin positive progenitor toxin complex consists of BoNT, nontoxic non-hemagglutinin (NTNH), and three hemagglutinin proteins; HA-70, HA-33, and HA-17. Hemagglutinin negative progenitor toxin complexes contain BoNT and NTNH as the minimally functional PTC (M-PTC), but not the three hemagglutinin proteins. Interestingly, the genome of hemagglutinin negative progenitor toxin complexes comprises open reading frames (orfs) which encode for three proteins, but the existence of these proteins has not yet been extensively demonstrated. In this work, we demonstrate that these three proteins exist and form part of the PTC for hemagglutinin negative complexes. Several hemagglutinin negative strains producing BoNT/A, /E, and /F were found to contain the three open reading frame proteins. Additionally, several BoNT/A-containing bivalent strains were examined, and NAPs from both genes, including the open reading frame proteins, were associated with BoNT/A. The open reading frame encoded proteins are more easily removed from the botulinum complex than the hemagglutinin proteins, but are present in several BoNT/A and /F toxin preparations. These are not easily removed from the BoNT/E complex, however, and are present even in commercially-available purified BoNT/E complex. Full article