PCR assay for differentiating between Group I (proteolytic) and Group II (nonproteolytic) strains of Clostridium botulinum

Groups I (proteolytic) and II (nonproteolytic) C. botulinum are genetically and physiologically distinct groups of organisms, with both groups being involved with human botulism. Due to differences in spore heat resistance and growth characteristics, the two groups possess different types of human health risks through foods, drink, and the environment. The epidemiology of human botulism due to Groups I and II C. botulinum is poorly understood, largely due to insufficient characterization of disease isolates, and warrants thorough outbreak investigation with a particular attention to discrimination between the different physiological groups of C. botulinum. In this study, a PCR assay was developed to discriminate between Group I and Group II C. botulinum. The assay is based on the fldB associated with phenylalanine metabolism in proteolytic clostridia, and employs an internal amplification control targeted to conservative regions of 16S rrn in Groups I and II C. botulinum. The assay correctly identified all 36 Group I and 24 Group II C. botulinum strains, possessing a 100% exclusivity and inclusivity. The assay provides a substantial improvement in discriminating between the Groups I and II C. botulinum, which traditionally is based on a time-consuming and error-prone culture method. Differentiation between the physiological groups of C. botulinum is an essential step in investigation of human botulism outbreaks, and should be considered as a diagnostic corner-stone in order to improve our epidemiological understanding of human botulism.     

四川省肉毒梭菌PCR基因分型方法比较研究

目的比较分析4种肉毒毒素(botulinum neurotoxins,Bo NT)基因分型方法,为四川省监测和食物中毒中肉毒梭菌(Clostridium botulinum)分型鉴定提供可靠的方法。方法使用本实验室保存的6株肉毒梭菌(包括A、B、E型)验证4种肉毒梭菌PCR基因分型鉴定方法——美国食品药品监督管理局(FDA)多重PCR分型方法、国际标准化组织(ISO)的两种多重PCR分型方法和一种实时荧光PCR分型方法,比较方法间的差异,并初步分析差异原因。结果 3种多重PCR方法均可在一个反应中同时检测A、B、E 3种型别肉毒梭菌。ISO多重PCR方法 1中A型检测虽能获得预期条带,但结果条带不清晰。其余两种多重PCR方法在分型检测肉毒梭菌时,可获得清晰的预期条带。实时荧光PCR分型方法能在多重反应体系中同时检测到不同型的肉毒梭菌,但由于荧光标记相同,要获得分型结果需要分别检测各毒素型别。结论美国FDA多重PCR方法和ISO多重PCR方法 2操作较简单易行,可在四川省肉毒梭菌监测中推荐使用。     

Production and Stability Studies of a Neurotoxin Produced by Clostridium sp. RKD

ABSTRACT: A neurotoxigenic Clostridium sp. (RKD) isolated from intestine of decaying fish produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) when tested by mouse protection bioassay. An amplicon of expected size (approximately 700 bp) was generated with primers specific for BoNT/B. Toxin was maximally released in the culture supernatant in the late stationary phase and was dependent on media composition. Growth was optimal in trypticase peptone yeast-extract glucose (TPYG) medium in a pH range of 7.5 to 8.0 and at a temperature between 35°C to 40°C while toxin production was optimum at 37°C (3 to 4 × 10³ minimum lethal dose per milliliter) without any protease treatment. There was no correlation between growth and toxin production when cells were grown in media containing different concentrations of NaCl (0% to 5%). Toxin in the culture supernatant was more stable (50% reduction at 50°C in 90 min) than the partially purified fraction. Toxicity was destroyed gradually after increasing the number of freeze-thaw cycles and was almost completely inactivated after 5 cycles. It was completely inactivated by overnight treatment of 1 N NaOH while it retained 1.5% activity with a similar treatment with 1 N HCl.     

Special Delivery: Potential Mechanisms of Botulinum Neurotoxin Uptake and Trafficking within Motor Nerve Terminals

Botulinum neurotoxins (BoNTs) are highly potent, neuroparalytic protein toxins that block the release of acetylcholine from motor neurons and autonomic synapses. The unparalleled toxicity of BoNTs results from the highly specific and localized cleavage of presynaptic proteins required for nerve transmission. Currently, the only pharmacotherapy for botulism is prophylaxis with antitoxin, which becomes progressively less effective as symptoms develop. Treatment for symptomatic botulism is limited to supportive care and artificial ventilation until respiratory function spontaneously recovers, which can take weeks or longer. Mechanistic insights into intracellular toxin behavior have progressed significantly since it was shown that toxins exploit synaptic endocytosis for entry into the nerve terminal, but fundamental questions about host-toxin interactions remain unanswered. Chief among these are mechanisms by which BoNT is internalized into neurons and trafficked to sites of molecular toxicity. Elucidating how receptor-bound toxin is internalized and conditions under which the toxin light chain engages with target SNARE proteins is critical for understanding the dynamics of intoxication and identifying novel therapeutics. Here, we discuss the implications of newly discovered modes of synaptic vesicle recycling on BoNT uptake and intraneuronal trafficking.     

Construction of a computer-controlled thermoresistometer for the determination of the heat resistance of bacterial spores over the temperature range 100 to 150°C

A computer-controlled thermoresistometer for the determination of the heat resistance of bacterial spores has been designed and constructed. The thermoresistometer is capable of operating between 100 and 150°C0.1°C with exposusre times as short as 0.1 sec. Five samples (0.01 ml) of spore suspension can be exposed to steam at each time/temperature combination using a computer-controlled piston-driven exposure system. Temperature control of the apparatus is achieved using platinum resistance probes linked to the computer, which then actuates a variable steam control valve.     

肉毒毒素的应用现状

肉毒毒素是肉毒杆菌在生长繁殖中产生的一种神经毒素。注入肌肉后，能迅速与突触前胆碱能神经终板结合，减少周围运动神经末梢和神经肌肉接头处乙酰胆碱的释放，抑制细胞外乙酰胆碱，造成肌肉松弛和麻痹。20世纪70年代末肉毒毒素被开发并逐步应用于临床，以治疗某些神经肌肉疾思。目前，肉毒毒素已经用于眼科、神经科、康复科、消化科及皮肤科(多汗症、美容)等领域50余种病症的治疗，取得了诸多成果。本文就肉毒毒素在临床医疗、医学美容、生物农药等方面的应用情况进行了概述。     

Nisin-resistant (Nisr) Listeria monocytogenes and Nisr Clostridium botulinum Are Not Resistant to Common Food Preservatives

Nisin‐resistant (Nisr) strains of Clostridium botulinum and Listeria monocytogenes may arise as nisin becomes more widely used as an additional safety barrier in minimally‐processed foods. The sensitivity of Nisr L. monocytogenes ATCC 700301 and ATCC 700302 and toxigenic Nisr C. botulinum 169B to low pH, salt, sodium nitrite, and potassium sorbate was assayed using discontinuous gradients in broth and compared to the parental wild‐type strains. The nisin‐resistant strains did not have intrinsic resistance to low pH, sodium chloride, potassium sorbate, or sodium nitrite. In no case were the Nisr L. monocytogenes and C. botulinum strains examined more resistant to inhibitors than the parental strains.     

治疗用A型肉毒梭菌神经毒素全基因克隆及序列分析

目的对我国治疗用A型肉毒毒素生产用菌株Hall株神经毒素(BoNT)全基因进行克隆及序列分析,了解其遗传特性,为该制剂的质量控制提供依据。方法取生产用主代种子、工作种子,通过PCR扩增BoNT全基因片段,将其克隆至载体pGEM-T中,构建重组克隆质粒pGEM-T-BoNT,对克隆的BoNT基因进行序列测定与分析。结果测得的Hall株BoNT全基因序列3891bp,共编码1297个氨基酸。4种核苷酸的比例分别为:A:40.58%,G:16.17%,T:33.13%,C:10.13%;GC含量为26.29%,AT含量为73.71%。主代种子、工作种子核苷酸序列3591位的G变成A,为无义突变,未导致其推导的氨基酸的改变。序列测定结果与GenBank中登录的Hall标准株进行比较,主代种子、工作种子核苷酸和氨基酸序列的同源性分别为99.99%和100%。结论 A型肉毒梭菌Hall株在实验室长期的保存和生产传代过程中,BoNT基因遗传特性非常稳定。     

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。