食品中A型肉毒梭菌PCR-DHPLC检测方法的建立

目的:建立食品中A型肉毒梭菌的快速检测方法。方法:应用PCR结合变性高效液相色谱(DHPLC)技术,以A型肉毒梭菌的A型肉毒神经毒素基因作为靶基因设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以非A型肉毒梭菌,产气荚膜梭菌等23株参考菌株做特异性实验;A型肉毒梭菌DNA稀释成不同梯度,做灵敏度实验。结果:与传统的检测方法相比,该方法具有很好的特异性,方法灵敏度较高,最低检出限可达到为111ng/tube。结论:该方法可以快速、准确检测A型肉毒梭菌,是食品中致病菌快速检测的新技术。     

食品中A型肉毒梭菌PCR-DHPLC检测方法的建立

目的:建立食品中A型肉毒梭菌的快速检测方法。方法:应用PCR结合变性高效液相色谱(DHPLC)技术,以A型肉毒梭菌的A型肉毒神经毒素基因作为靶基因设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以非A型肉毒梭菌,产气荚膜梭菌等23株参考菌株做特异性实验;A型肉毒梭菌DNA稀释成不同梯度,做灵敏度实验。结果:与传统的检测方法相比,该方法具有很好的特异性,方法灵敏度较高,最低检出限可达到为111ng/tube。结论:该方法可以快速、准确检测A型肉毒梭菌,是食品中致病菌快速检测的新技术。      

石家庄市肉毒梭菌在环境中的分布研究

目的:了解石家庄市肉毒梭菌在环境中的分布情况,为预防、诊断、治疗肉毒梭菌食物中毒提供科学依据.方法:按照地貌,均匀抽取8个县(市、区),采集土壤、食品和水3类样品,检测肉毒梭菌污染情况.结果:采集土壤、水、食品3类样品共350份,肉毒梭菌检出15份,检出率为4.3%,其中,12份为8型肉毒毒素,3份为A型肉毒毒素.发生过肉毒中毒的县(市、区)肉毒梭菌检出率高于未发生过的县(市、区)(P<0.05).山区与平原肉毒梭菌检出率差异无统计学意义.结论:石家庄市外环境中肉毒梭菌污染较以前严重,并且存在A型肉毒毒素.     

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。     

某企业婴儿肉毒中毒乳粉相关样品中肉毒梭菌的分离与分型

目的 对从某企业获取的30批次婴儿配方乳粉样品进行肉毒毒素和肉毒梭菌检测，对分离到的1株B型肉毒梭菌进行全基因组测序分析。方法 参照GB 4789.12—2016《食品安全国家标准 食品微生物学检验 肉毒梭菌及肉毒毒素检验》对样品进行肉毒梭菌分离及肉毒毒素分型实验；对分离到的菌株进行全基因组测序，并分析菌株的遗传特征。结果 30批次样品中均未检出肉毒毒素；将增菌液进行小鼠腹腔注射后，4批次乳粉样品出现了典型小鼠肉毒中毒症状，但仅从1批次乳粉样品中分离到肉毒梭菌。基因组测序分析显示，该菌为Ⅰ群B型肉毒梭菌，毒素基因簇为Ha型，毒素基因为B2亚型。结论 针对背景微生物复杂的婴儿配方乳粉中肉毒梭菌检测，不应以菌种分离作为金标准，而应以增菌液小鼠毒性实验结合肉毒抗血清保护实验作为确认方法。全基因组测序可对分离菌种进行精准鉴定和相关遗传特征分析，为中毒事件处理提供可靠的技术支撑。     

一起肉毒梭菌污染家庭腌制酸肉引起的食物中毒调查处置

目的查找病因,指导临床救治,以便为肉毒梭菌食物中毒事件制定防治措施提供依据。方法应用WS/T 83—1996《肉毒梭菌食物中毒诊断标准及处理原则》,通过现场流行病学调查、实验室检测和启动国家卫计委特效药系统紧急采购肉毒梭菌诊治血清应用于病人,分析致病原因与病程变化关系,确定诊治措施。结果确诊发病病人2例,仅1人住院。在住院患者血清中检出A、B型肉毒梭菌毒素,应用A、B型肉毒梭菌抗毒血清并对症治疗、机械通气,治疗25 d后,症状缓解,逐渐好转并康复出院。结论本次事件是由于进食被A、B型肉毒梭菌毒素污染的自家腌制酸肉导致的食物中毒事件。建议做好饮食安全宣传,加强食物中毒防控。     

市售婴幼儿配方奶粉和婴幼儿米粉中梭状芽胞杆菌的分离和综合鉴定分析

目的 对57件市售婴幼儿配方奶粉、50件婴幼儿米粉进行梭状芽胞菌分离鉴定分析及毒素基因检测,获得梭状芽胞杆菌的污染水平数据,并评估各鉴定方法。方法 通过分离菌株的生长特性、革兰染色、普通显微镜下形态特征等表型特征,应用微生物飞行时间质谱（MALDI-TOF-MS）、16S rRNA基因测序技术、全基因组测序技术对分离菌株进行综合鉴定,通过PCR对分离的梭状芽胞杆菌进行肉毒毒素基因检测,对肉毒毒素基因PCR阳性片段进行测序和比对分析。结果 57份市售婴儿配方奶粉中有26份样品中检出38株梭状芽胞杆菌,其中9份样品中同时检出超过两种细菌。50份市售婴幼儿米粉中有5份样品检出5株梭状芽胞杆菌。奶粉中分离的一株楔形梭菌E型肉毒梭菌毒素基因PCR扩增阳性,扩增片段测序比对结果显示这段序列并不是E型肉毒梭菌毒素基因,全基因组测序结果也证实。43株梭状芽胞杆菌均未检出肉毒梭菌的毒素基因。结论 梭状芽胞杆菌鉴定需要多种方法的综合分析。市售婴幼儿配方奶粉、婴幼儿米粉中存在梭状芽胞杆菌的污染,应加强婴幼儿配方食品中重要梭状芽胞杆菌的监测,为风险评估提供数据支持。     

一起由食用家庭自制辣椒酱引起肉毒中毒的实验室诊断

目的 对1例疑似肉毒中毒病例的相关样品进行实验室检测。方法 参照GB 4789.12—2016对病例暴露食品样品（自制辣椒酱、咸菜、芝麻酱、卤猪蹄）和粪便标本进行前处理,应用实时荧光定量聚合酶链式反应（PCR）检测样品/标本中肉毒毒素基因,通过动物试验进行毒素检测和型别确认,经接种疱肉培养基和TPGYT培养基增菌培养,采用血琼脂平板进行分离、纯化,并做菌种鉴定。结果 5份样品/标本经实时荧光定量PCR法检测,仅在自制辣椒酱样品中检测到A型肉毒毒素基因;通过动物试验进行毒素检测,在自制辣椒酱样品中检测到A型肉毒毒素,其他样品/标本中未检测到肉毒毒素;5份样品/标本中,从自制辣椒酱和卤猪蹄样品中均分离到A型肉毒梭菌。结论 该起中毒事件是由食用肉毒梭菌污染的家庭自制辣椒酱引起。     

昆明市首起由疑似肉毒梭菌引起食物中毒的实验室快速检测

目的快速查明昆明市一起疑似由肉毒梭菌引起食物中毒事件的病原菌的具体类别。方法根据GB4789.12-2016《食品安全国家标准食品微生物学检验肉毒梭菌及肉毒毒素检验》,样品在庖肉培养基中35℃厌氧增菌5 d后镜检,用2个不同生产厂家的肉毒梭菌实时荧光定量PCR(Real-time PCR)检测试剂进行检测并进行肉毒毒素分型。结果镜检符合典型的肉毒梭菌的特征,2个不同生产厂家的肉毒梭菌Real-time PCR检测结果均为阳性,且均检测出E型肉毒毒素。Real-time PCR检测只需在增菌后4～6 h的检测时间,即可得到结果,且无需进行菌体的纯化培养,鉴定时间节约至少96 h。结论此次事件为食用被E型肉毒梭菌及其毒素污染食品而引起的细菌性食物中毒事件,用Real-time PCR检测方法可对肉毒梭菌食物中毒事件作出快速判定。     

我国首例由丁酸梭菌引起婴儿E型肉毒中毒实验室诊断研究

目的对一起疑似婴儿肉毒中毒进行实验室诊断研究。方法对1例疑似婴儿肉毒中毒病例的粪便、食用过的食品和生活环境涂抹共计30份标本/样品进行梭菌分离鉴定及肉毒毒素检测,对分离菌株进行产毒试验。结果将患儿粪便标本培养物上清液腹腔注射小鼠后可致小鼠出现典型肉毒毒素中毒表现(竖毛、呼吸困难并出现典型的蜂腰、四肢麻痹),继而死亡,且培养物上清液经胰酶处理后毒性增强,表现为小鼠出现中毒及死亡时间较未处理组明显缩短。但培养物上清液经100℃加热处理后再次染毒动物,小鼠未出现中毒和死亡。混合型肉毒毒素诊断血清及单价抗E型肉毒毒素诊断血清可对小鼠起到保护作用。从患儿的粪便标本中分离到G~+芽胞杆菌,该菌在哥伦比亚血平板上呈不规则半透明扁平菌落,边缘根状生长,并携带E型肉毒毒素产毒基因,16S rRNA将其鉴定为丁酸梭菌,产毒试验结果显示该菌株可产E型肉毒毒素。结论该起婴儿中毒事件是由感染产E型肉毒毒素的丁酸梭菌引起。     

Competitive inhibition between different Clostridium botulinum types and strains

Mixtures of proteolytic and nonproteolytic strains of toxigenic Clostridium botulinum types A, B, and F; nonproteolytic types B, E, and F; Clostridium sporogenes; and nontoxic E-like organisms resembling nonproteolytic C. botulinum were tested against each other for the purpose of selecting a mixture of compatible C. botulinum strains for inoculated pack studies on the basis of their sensitivity to bacteriophages and bacteriocin-like agents. All of the proteolytic strains produced bacteriocin-like agents that were inhibitory to three or more of the other proteolytic types and C. sporogenes. When selected strains of proteolytic types A and B were grown together, type A cultures produced neurotoxin, but type B toxin production was inhibited. Nonproteolytic strains of C. botulinum also produced bacteriocin-like agents against each other. Of these, type E strain EF4 produced bacteriocin-like agents against both proteolytic and nonproteolytic types of C. botulinum and C. sporogenes. EF4, however, was not inhibitory to the nontoxigenic E-like strains. When EF4 was grown with type A strain 62A, it had an inhibitory effect on type A toxin production. Strain 62A inactivated the type E toxin of EF4 after 7 to 21 days at 30 degrees C. On the basis of the production of these bacteriocin-like agents by different strains of C. botulinum and their potential effect on neurotoxin production, it is very important that compatible strains are used in mixtures for inoculated pack studies to determine the safety of a food process or product.     

EFFECT OF GLYCERYL MONOLAURATE ON TOXIN PRODUCTION BY CLOSTRIDIUM BOTULINUM IN MEAT SLURRY

Glyceryl monolaurate when used in the proportion of 5 g per kg of meat slurry (pH 6.0–6.2) inhibited toxin production by Clostridium botulinum type A (strain 73A), type B (strain OKRA) and type E (strain RIV 2). The concentration of glycerol monolaurate required for inhibition was not reduced by addition of lactic acid to the meat slurry until the pH was lowered to pH 5.2 or below. In meat slurry (pH 6.0–6.2), potassium sorbate inhibited production of type B toxin at 32 g/kg. The addition of butylated hydroxy-anisole to glyceryl monolaurate had no effect upon the concentration needed for inhibition of botulinum toxin production.     

Antimicrobial activity of foodborne Paenibacillus and Bacillus spp. against Clostridium botulinum

The saprophytic Paenibacillus and Bacillus spp. found in cooked chilled foods may have an effect on the growth of Clostridium botulinum, a major microbiological hazard, especially for pasteurized vacuum-packaged products. Culture supernatants of 200 strains of Paenibacillus and Bacillus strains isolated from commercial cooked chilled foods containing vegetables were screened for activity against C. botulinum type A, proteolytic type B, and type E strains in a well diffusion assay. Nineteen strains were positive against C. botulinum. Among those, seven Paenibacillus polymyxa strains showed the highest antibotulinal activity and the largest antimicrobial spectrum against C. botulinum strains. The antibotulinal activity was evaluated throughout the growth of a representative strain of the positive P. polymyxa strains. The antimicrobial activity was detected in the culture supernatant from late-log/early stationary phase of the bacteria, which occurred after 7 to 10 days of incubation at 10 degrees C and after 2 to 3 days at 20 degrees C in nutrient broth and in vegetable purées under aerobic or anaerobic conditions. In co-cultures with the positive strain of P. polymyxa in nutrient broth and vegetable purées, a C. botulinum type E strain was inhibited whenever P. polymyxa reached stationary phase and produced its antimicrobial activity before C. botulinum began its exponential growth phase. The antimicrobial activity of P. polymyxa against C. botulinum was attributed to the production of antimicrobial peptides resistant to high temperature and acidity. Other gram-positive and -negative bacteria (Escherichia coli, Streptococcus mutans, Leuconostoc mesenteroides, and Bacillus subtilis) were also sensitive to these antimicrobial peptides.     

PLASMIDS OF CLOSTRIDIUM BOTULINUM TYPE A, B, E, F, AND OTHER CLOSTRIDIAL SPECIES

Plasmids from 46 strains of Clostridium botulinum types A, B, E, and F were isolated using a modification of the Minton and Morris procedure (1981). When these strains were examined, 54% contained one or more plasmids ranging in molecular weight from ∼ 69 to 3.4 megadaltons (MDa). Attempted curing of plasmids for two C. botulinum type A strains, 73A and Hall, using acridine orange and elevated growth temperatures (42 to 45°C) produced no changes in plasmid profiles or toxicity as determined by agarose gel electrophoresis and immunodiffusion tests, respectively. However, exposure of type E C. botulinum strain BL764 to 1000 Krad gamma irradiation resulted in loss of one plasmid (8.4 MDa) but toxicity was retained.     

THE EFFECT OF LYSOZYME ON THE APPARENT HEAT RESISTANCE OF NONPROTEOLYTIC TYPE B CLOSTRIDIUM BOTULINUM

Lysozyme has been shown to increase the recovery of heated spores of type E Clostridium botulinum, thereby increasing the measured heat resistance. This study evaluated the effect of lysozyme on the apparent heat resistance of spores of nonproteolytic type B strains of C. botulinum, which had heat resistances up to 30 times greater than type E strains. Adding lysozyme to the recovery medium increased the apparent heat resistance 16 to 160 times that of values previously established. This increase was comparable to that obtained with type E strain Saratoga (73 times). Lysozyme had no effect on the recovery of heated spores of a proteolytic B strain.     

Clostridium sporogenes PA 3679 and its uses in the derivation of thermal processing schedules for low-acid shelf-stable foods and as a research model for proteolytic Clostridium botulinum

The putrefactive anaerobe Clostridium sporogenes PA 3679 has been widely used as a nontoxigenic surrogate for proteolytic Clostridium botulinum in the validation of thermal processes for low-acid shelf-stable foods, as a target organism in the derivation of thermal processes that reduce the risk of spoilage of such foods to an acceptable level, and as a research model for proteolytic strains of C. botulinum. Despite the importance of this organism, our knowledge of it has remained fragmented. In this article we draw together the literature associated with PA 3679 and discuss the identity of this organism, the phylogenetic relationships that exist between PA 3679 and various strains of C. sporogenes and proteolytic C. botulinum, the heat resistance characteristics of PA 3679, the advantages and limitations associated with its use in the derivation of thermal processing schedules, and the knowledge gaps and opportunities that exist with regard to its use as a research model for proteolytic C. botulinum. Phylogenetic analysis reviewed here suggests that PA 3679 is more closely related to various strains of proteolytic C. botulinum than to selected strains, including the type strain, of C. sporogenes. Even though PA 3679 is demonstrably nontoxigenic, the genetic basis of this nontoxigenic status remains to be elucidated, and the genetic sequence of this microorganism appears to be the key knowledge gap remaining to be filled. Our comprehensive review of comparative heat resistance data gathered for PA 3679 and proteolytic strains of C. botulinum over the past 100 years supports the practice of using PA 3679 as a (typically fail-safe) thermal processing surrogate for proteolytic C. botulinum.     

Effects of mastic resin and its essential oil on the growth of proteolytic Clostridium botulinum

Studies were done to determine the effect of mastic resin and its essential oil, alone and in conjunction with ethanol, on the growth of proteolytic strains of Clostridium botulinum in media, and on neurotoxin production in challenge studies with English-style crumpets. Preliminary studies, using a spot-on-the-lawn method, indicated that high levels of mastic resin in ethanol ( approximately 8% w/w) were required for complete inhibition of all strains of C. botulinum tested, but mastic resin in ethanol had a greater anti-botulinal effect than ethanol alone. However, only low levels of mastic oil ( approximately 0.3% v/v) were required for inhibition of proteolytic strains of C. botulinum. Both studies showed a strain specific inhibition, with C. botulinum type A strains being more sensitive to mastic resin and its essential oil than type B strains. However, mastic resin in ethanol proved to be more effective when used as a vapor phase inhibitor applied to cotton pads and placed inside inoculated plates than when added directly to media. While both mastic resin and its essential oil inhibited the growth of proteolytic strains of C. botulinum in vitro, they failed to inhibit neurotoxin production in challenge studies with C. botulinum in English-style crumpets.     

The incidence of Clostridium botulinum in Danish trout farms: I. Distribution in fish and their environment

Examination of four Danish trout farms demonstrated the presence of Clostridium botulinum in trout, the wet-fish feed, farm waters, pond muds and invertebrates and in the soil of the area surrounding the farm. The incidence in 530 trout examined varied from 5-100% in winter to 85–100% in late summer. Clostridium botulinum type E was the predominant type found.     

Inhibition of nonproteolytic Clostridium botulinum with lactic acid bacteria and their bacteriocins at refrigeration temperatures

Nonproteolytic Clostridium botulinum (strains 17B, Beluga, and 202F) was found to be inhibited by Lactobacillus, Lactococcus, Streptococcus, and Pediococcus species in tests by the spot-on-the-lawn simultaneous-antagonism method at 10, 15, and 25 degrees C. C. botulinum 17B was the most resistant strain. Inhibition zone size increased with decreasing incubation temperature. Six strains of Lactobacillus acidophilus and seven strains of bifidobacteria failed to produce an inhibition zone on buffered reinforced clostridium Prussian blue agar seeded with spores of any of the selected C. botulinum strains. C. botulinum 17B was sensitive to 50 to 100 IU of nisin per ml and to 10 to 20 AU of pediocin A per ml.     

Differentiation between types and strains of Clostridium botulinum by riboprinting

The ability of automated ribotyping to differentiate between major types and individual strains of Clostridium botulinum was tested using the Qualicon Riboprinter Microbial Characterization System. Pure spores of C. botulinum type A, proteolytic type B, nonproteolytic type B, and type E strains were inoculated onto modified anaerobic egg yolk agar and incubated 24 h at 35 degrees C. Plates were rinsed with buffer (2 mM Tris + 20 mM EDTA) to remove vegetative cells that were heated for 10 min at 80 degrees C, treated with a lysing agent, and ribotyped in the Qualicon Riboprinter utilizing the enzyme EcoRI. Riboprint patterns were obtained for 30 strains of the four major types of C. botulinum most commonly involved in human foodborne botulism. Proteolytic strains yielded the best and most consistent results. Fifteen ribogroups were identified among the 31 strains tested. Interestingly, in two cases, a single ribogroup contained patterns from isolates belonging to evolutionarily distinct Clostridium lineages. This degree of differentiation between strains of C. botulinum may be useful in hazard analysis and identification, hazard analysis and critical control point monitoring and validation, environmental monitoring, and in inoculation studies.