Growth and toxigenesis of C. botulinum type E in fishes packaged under modified atmospheres

Modified atmosphere packaging of fresh fish is used to market high quality products in some European countries. The potential risk of C. botulinum growth in these extended shelf-life foods is still a concern; especially since toxigenesis may precede organoleptic spoilage. This paper will present toxigenic data from rockfish, salmon and sole muscle tissues which were inoculated with a pool of non-proteolytic C. botulinum type E at seven levels (10⁻² −10⁴ spores/sample), and stored under vacuum and 100% CO₂, at incubation temperatures between 30 and 4°C, for up to 60 days. Factorial experimental design allowed predictive formulae to be developed able to describe the lag time prior to C. botulinum toxigenesis and the probability of one spore to initiate toxigenesis based upon the storage conditions. Accurate characterization of the microbial ecology of C. botulinum in modified atmosphere-packaged fish, will support safe exploitation of these packaging systems in the market place, and identify critical control points for potential product or process abuses.     

乙酰胆碱受体δ、r亚基上Loop F高可靠性结构模型的构建

报导从单斑眼镜蛇(monocellete cobra，Naja kaouthia)毒中分离纯化出一个新的短链神经毒cobrotoxin c(CBT C)，并确定了一个新的功能氨基酸Arg56．根据笔者测定的NMR液相三级结构和近年来神经毒素与受体结合机制研究的进展，推断Arg56及公认的功能残基Lys27、Lys47一起与α1型乙酰胆碱受体δ/r亚基上的Loop F相互作用．基于此，笔者以MODEILER 6V2软件构建了这种蛇毒的包括CBT C在内的五个短链神经毒与Loop F结合的模型．五个模型中的Loop F结构具有高度的一致性，RMSD值在0．22nm左右，从而断定此模型有较高的可靠性．构建出的Loop F的结构模型将对构建高可靠性α1型AChR结构模型、研究离子通道开闭机制等都具非常重要的意义．     

Three‐Dimensional Database Mining Identifies a Unique Chemotype that Unites Structurally Diverse Botulinum Neurotoxin Serotype A Inhibitors in a Three‐Zone Pharmacophore

A search query consisting of two aromatic centers and two cationic centers was defined based on previously identified small molecule inhibitors of the botulinum neurotoxin serotype A light chain (BoNT/A LC) and used to mine the National Cancer Institute Open Repository. Ten small molecule hits were identified, and upon testing, three demonstrated inhibitory activity. Of these, one was structurally unique, possessing a rigid diazachrysene scaffold. The steric limitations of the diazachrysene imposed a separation between the overlaps of previously identified inhibitors, revealing an extended binding mode. As a result, the pharmacophore for BoNT/A LC inhibition has been modified to encompass three zones. To demonstrate the utility of this model, a novel three‐zone inhibitor was mined and its activity was confirmed.     

Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.     

Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1–A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol. Steady-state and live imaging of LC/A3-A1 chimeras identified a two-step process where the LC/A N terminus bound intracellular vesicles, which facilitated an internal α-helical-rich domain to mediate LC/A plasma membrane association. The propensity of LC/A variants for membrane association correlated with enhanced BoNT/A potency. Understanding the basis for light chain intracellular localization provides insight to mechanisms underlying BoNT/A potency, which can be extended to applications as a human therapy.     

Reduction efficiency of the neurotoxin β‐ODAP in low‐toxin varieties of Lathyrus sativus seeds by solid state fermentation with Aspergillus oryzae and Rhizopus microsporus var chinensis

Solid state fermentation of several low‐toxin varieties of grass pea (Lathyrus sativus L) seeds with Aspergillus oryzae and Rhizopus microsporus var chinensis removed the neurotoxin β‐ODAP (3‐N‐oxalyl‐L ‐2,3‐diaminopropanoic acid) to a considerable degree from the seed meal. The detoxification efficiency was statistically significant and ranged from 52.4% (p < 0.01) to 82.2% (p < 0.001), which was lower than for a high‐toxin variety processed by the same fermentation procedure (94.8%, p < 0.001). While the content of β‐ODAP decreased, those of other free protein amino acids, especially glutamic acid, histidine, threonine, tyrosine, valine and lysine, increased dramatically in the fermented seeds. Efforts to remove the neurotoxin from Lathyrus sativus either by breeding or by food processing to obtain toxin‐free grass pea seeds have been made worldwide for several decades. The efficiencies of various reported processing methods are summarised and compared. © 2000 Society of Chemical Industry     

1959—2022年青海省食源性肉毒中毒事件流行病学分析

目的 分析青海省食源性肉毒中毒事件流行特征及影响因素,为防控提供科学依据。方法 收集1959—2022年青海省肉毒中毒数据,应用Excel 2021进行流行病学特征分析,利用R 4.2.3对事件数和病死率进行Mann-Kendall趋势检验,运用ArcGis 10.2软件制作空间分布图。结果 1959—2022年发生食源性肉毒中毒事件86起,发病348人,死亡195人,病死率为56.03%。中毒主要发生在第二、三季度,牧民为主要的发病人群,肉毒毒素型别主要为E型,“风干肉”为主要的中毒食品,生食和开锅即食为主要的食用方式。结论 青海省食源性肉毒中毒病死率整体呈下降趋势,发病地区也逐渐缩小,但作为我国高发地区之一,病死率仍处于较高水平,应进一步加强对高危人群食品安全教育,提高基层医疗机构对肉毒中毒的救治能力,减少中毒事件的发生和死亡。     

Caffeic Acid Activity Against Clostridium botulinum Spores

Caffeic acid (CA) is widely distributed among higher fruits and vegetables. While CA has antimicrobial activity, little information exists on its utility as a food additive. As such, CA was tested for activity against Clostridium botulinum spores. At 0.78 and 3.25 mM, CA inhibited germination for 6 and 24 hr, respectively, with >100 mM required to render spores nonviable. CA concentrations a 50mM reduced 80°C spore thermal resistance. Sporostatic activity was retained when tested in commercial meat broths, and 5.0 mM CA delayed toxigenesis. Caffeic acid has potential as a food additive to inhibit growth of C. botulinum, and reduce thermal processing requirements of heat sensitive foods.     

Probabilistic representation of the exposure of consumers to Clostridium botulinum neurotoxin in a minimally processed potato product

We have examined the potential of a well-specified, minimally processed potato product as a vehicle for the exposure of consumers to Clostridium botulinum neurotoxin. The product is a relatively simple combination of raw potato flakes, flour, starch and other minor ingredients and has an extended lifetime under refrigeration conditions. A combination of information and data, from a variety of sources that includes the manufacturer, has shown that the product is particularly safe with respect to non-proteolytic C. botulinum hazards. The model concentrates on a simple end point, the toxicity of an individual retail unit of the product at the point of consumer preparation, which is related to an individual risk. The probabilistic analysis was built using Bayesian Belief Network (BBN) techniques.