Estimation of human dose of staphylococcal enterotoxin A from a large outbreak of staphylococcal food poisoning involving chocolate milk

An outbreak of gastroenteritis in a school district in the United States was determined to be staphylococcal food poisoning due to 2% chocolate milk containing staphylococcal enterotoxin A (SEA). Twelve one-half pint (approx 0.28 l) cartons of the 2% chocolate milk from this outbreak were analyzed for the quantity of SEA present in the milk. The amount of SEA in the cartons varied from 94 to 184 ng with the average being 144 ng (mean = 139 +/- 45). The attack rate for vomiting among those who consumed more than one carton was greater (38.3%) than among those who consumed only one carton (31.5%) with the highest attack rate among those who consumed three or more cartons (44.4%).     

Comparison of four kits for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning.

Four commercial kits, three based on sandwich ELISA techniques and one on latex agglutination were compared for the detection of staphylococcal enterotoxins in foods from outbreaks of food poisoning. Enterotoxin was detected in 14 of 18 foods with the Swiss SET-EIA and in 9 or 10 with the Unipath SET-RPLA and two ELISAs from Transia. The advantages and disadvantages of the four methods are discussed.     

食物中毒事件中致病菌肠毒素检测和分子分型研究

目的 对一起食物中毒事件样本进行检测,查明食物中毒原因,并对分离菌株进行相关性分析。方法 参照国家标准方法GB4789.10-2016,对采集样本进行细菌学检验。应用酶联免疫吸附法(ELISA)检测葡萄球菌肠毒素。通过脉冲场凝胶电泳(PFGE)进行分子分型,分析菌株之间的相关性。结果 食物中毒事件中5名患者洗胃液、食物、分离菌株,均检测出E型葡萄球菌肠毒素。3株分离菌株PFGE分子分型提示来源不同克隆株,除了产生E型葡萄球菌肠毒素外,还有B、C、D型。结论 该起食物中毒由不同PFGE型别的产毒金黄色葡萄球菌污染食物引起,菌株产葡萄球菌肠毒素的型别不完全相同。金黄色葡萄球菌引起的食物中毒应关注多污染源及菌株肠毒素基因表达情况。     

Risk evaluation for staphylococcal food poisoning in processed milk produced with skim milk powder

The growth of S. aureus and the production of staphylococcal enterotoxin A (SEA) in skim milk concentrates stored at inappropriate temperatures in a recovery milk tank (tank for excess concentrated skim milk) used in the manufacture of skimmed milk powder were investigated. Also, it was estimated if a possible outbreak of food poisoning would occur if the contaminated skimmed milk powder was used in the manufacture of processed milk. Skim milk concentrates with milk solid content of 15, 25, and 35% were inoculated with S. aureus at 1-2 log CFU/ml and incubated at 15, 25, or 35 degrees C for 0 to 24 h with or without shaking. Bacterial growth and the level of SEA production were measured. At 35 degrees C with shaking, there was a significant difference (p<0.05) in one way layout analysis of variance, and it was demonstrated that the growth of S. aureus and SEA production could be milk solid content-dependent. Shaking accelerated the growth of S. aureus and SEA production at 35 degrees C. Generally, skim milk powder is produced by mixing a set percentage of skim milk concentrates (recovery milk) from the recovery milk tank into raw milk. If recovery milk contaminated with S. aureus at levels of 1-2 log CFU/ml is kept at 15 to 35 degrees C due to a power failure, it was estimated that processed milk consumption of 670-1200 ml, 420-1500 ml and 18-83 ml would trigger the onset of food poisoning symptoms when skim milk concentrates (recovery milk) are stored at 25 degrees C for 24 h, 35 degrees C for 10 h, and 35 degrees C for 24 h, respectively, during the production of the skim milk powder. Based on these consumption levels, it was concluded that, if recovery milk cannot be refrigerated and is stored at room temperature (25 to 35 degrees C), it must be used within 8 h and preferably within 6 h.     

Spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin B in milk

This work evaluates a newly developed wavelength modulation-based SPR biosensor for the detection of staphylococcal enterotoxin B (SEB) in milk. Two modes of operation of the SPR biosensor are described: direct detection of SEB and sandwich assay. In the sandwich assay detection mode, secondary antibodies are bound to the already captured toxin to amplify sensor response. Samples including SEB in buffer and SEB in milk were analyzed in this work. The SPR biosensor has been shown to be capable of directly detecting concentrations of SEB in buffer as low as 5 ng/ml. In sandwich detection mode, the lowest detection limit was determined to be 0.5 ng/ml for both buffer and milk samples. The reported wavelength modulation-based SPR sensor provides a generic platform which can be tailored for detection of various foodborne pathogens and agents for food analysis and testing.     

Immunomagnetic flow cytometric detection of staphylococcal enterotoxin B in raw and dry milk

A rapid and sensitive method for detection of staphylococcal enterotoxin B (SEB) in raw and dry milk samples with the use of antibody-based immunomagnetic separation (IMS) in conjunction with flow cytometry (FCM) was developed. Sheep anti-SEB immunoglobulin G (IgG) was immobilized on Dynabeads M-280. The SEB initially binds to the capturing antibody, which is bound on the magnetic beads. The rabbit anti-SEB IgG binds to the captured toxin and is further labeled with a Cy5-labeled goat anti-rabbit IgG antibody. The percentage of the beads that were fluorescent was measured by FCM. FCM was carried out for 1 min, and the data obtained were expressed as histograms for particle size (forward light scatter) and histograms for fluorescence intensity. A peak corresponding to the magnetic beads was clearly distinguished from a peak derived from contaminating particles in the sample solution. In the absence of SEB, about 10% of the beads emitted fluorescence. The percentage of fluorescent beads and the fluorescence intensity increased with increasing SEB concentrations. For this IMS-FCM assay, the lower limits of detection for SEB were estimated to be 0.01 and 0.25 ng/ml for buffer and milk samples, respectively.     

A simple and rapid antibody-capture ELISA for the detection of staphylococcal enterotoxin A in food including a simple extraction step

A sensitive and precise ELISA for the detection of staphylococcal enterotoxin A in food has been developed, using specific IgG anti-toxin antibodies purified from immunized rabbits. an antibody capture format was found to be the most useful following cross-reaction and sensitivity studies. the ELISA was used to investigate concentration of staphylococcal enterotoxin A from food samples by polyethylene glycol dialysis. the procedure was shown to be inefficient and highly variable, as well as time-consuming. Direct analysis of aqueous food extracts without a concentration step was found to be possible. With an assay time of 140 min, the ELISA detection limit was 12.5 pg of toxin (corresponding to a sensitivity of 0.5 ngg^-1 of food sample.     

一起由B群沙门菌污染聚餐食品所致食物中毒调查分析

目的调查一起农村婚宴聚餐引起食物中毒的致病因子、致病食品及其污染来源,采取有效措施控制事件蔓延,预防今后类似事件的发生。方法开展现场流行病学调查,制定病例定义并主动搜索病例,采用描述流行病学方法分析本次事件病例的流行病学特征。通过个案调查访谈,对参加聚餐的108人开展病例对照研究,分析可能的致病食品。现场勘查和访谈厨师,了解婚宴菜品的制作过程、原料来源和所用水源等,并采集病例肛拭子、婚宴剩余食品、饮用水样进行实验室检测。结果本次食物中毒罹患率为21.6%(138/640),临床表现以腹泻、腹痛、发热为主。单因素分析结果显示94.4%(51/54)的病例和66.7%(36/54)的对照食用过由灌肚、里脊肉片、炸排骨、萝卜丝四种食品组成的凉菜拼盘,食用凉菜拼盘增加发病风险(比值比=8.50, 95%置信区间:2.32～31.02)。叉生分析结果显示同时食用拼盘中两种以上凉菜将增加发病风险(比值比=9.25, 95%置信区间:2.46～34.82)。4份病例肛拭子和1份凉菜拼盘中灌肚均检出B群沙门菌。5株检出的沙门菌脉冲场凝胶电泳(PFGE)指纹图谱为同一带型,提示病例和食品分离株在分子水平具有高度的同源性,为同一暴露源。结论本次事件是一起由B群沙门菌污染聚餐食品导致的食物中毒。凉菜拼盘是主要的致病食品,食品加工卫生环境与制作过程不规范操作是导致本次食物中毒发生的危险因素。当前农村自办宴席仍存在诸多食源性疾病发生的风险环节,应加强监管。     

Real time biosensor analysis of staphylococcal enterotoxin A in food.

Currently there is no 'real-time' detection system to identify food borne toxins. In order to develop such a system, we have used a evanescent wave biosensor for real time detection of staphylococcal enterotoxin A (SEA) in foods. The approach used here is sandwich biosensor, a method utilizing two antibodies. The toxin binds initially to a capturing antibody which is bound covalently on the surface of the biosensor detector. The second antibody binds to the captured toxin. We were able to measure SEA in foods with little or no background interference, demonstrating that biosensor-based measurement of SEA was possible not only with purified SEA but also in complex food matrices such as hot dogs, potato salad, milk and mushrooms. Autoclaved samples of SEA did not evoke a positive response. With both purified SEA and SEA-spiked foods, the assay sensitivity is 10-100 ng/g depending on the material tested and the assay is rapid ( <4 min) when a single antibody is used.     

Detection of staphylococcal enterotoxin B in spiked food samples

Contamination of food with infectious agents, intentional or not, is a global concern with far-reaching economic and social impact. Staphylococcal enterotoxins are a major cause of food poisoning, but most methods for the identification of these agents in food require extensive pretreatment or concentration of the sample prior to analysis. The array biosensor was developed as a portable device for the simultaneous analysis of multiple complex samples for multiple targets with minimal sample preparation. In this study, we use an array biosensor to expand and improve on a staphylococcal enterotoxin B (SEB) assay with the ultimate intent of incorporating testing for SEB into a battery of sensitive and convenient assays for food safety validation. In addition to buffer studies, six different types of food samples, including beverages, homogenates of fruit and meat, and carcass washings, were spiked with SEB, incubated for at least 2 h to permit antigen sequestration, and assayed. For all samples, there were differences in fluorescence intensity, but 0.5 ng of SEB per ml could be detected in <20 min with little if any pretreatment and no sample preconcentration.     

同一肇事地不同餐次食物中毒现场流行病学调查及肠毒素基因鉴定

目的查明本次食物中毒发生的原因,确认致病危害因素及其来源,控制暴发的扩散和蔓延。方法采用现场流行病学调查方法,开展病例搜索和个案调查,并采集相关样本进行实验室检测及肠毒素基因鉴定。运用流行病学曲线推算潜伏期范围,确定中毒餐次和中毒食品。结果共搜索确诊中毒病例27例,主要临床特征为恶心、呕吐、腹泻,部分病例伴腹痛、头痛、头晕等。中毒餐次为2014年12月10日的午餐和晚餐,中毒食品为烤肉拌饭,致病因子为金黄色葡萄球菌A型肠毒素。烤肉中分离的金黄色葡萄球菌菌株间PFGE图谱相似度100%,具有同源性;RT-PCR鉴定毒素基因为A型肠毒素。结论发生在同一肇事地不同餐次由金黄色葡萄球菌A型肠毒素引起的食物中毒,交叉污染是金黄色葡萄球菌污染来源最重要的传播方式,应加强餐饮行业监管和关键环节控制,防止类似事件的再次发生。     

Chemiluminescent imaging detection of staphylococcal enterotoxin C1 in milk and water samples

A sensitive, simple and rapid technique for high throughput simultaneous detection of staphylococcal enterotoxin C₁ (SEC₁) has been developed. The proposed method has the advantage of showing the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of luminol-based enhanced chemiluminescence (ECL) assay, and high throughput of chemiluminescence (CL) imaging assay. It was based on a standard sandwich immunoassay format; 96-well ELISA plates were used as solid phase material. A commercial high-sensitivity cooled CCD camera has been applied to image the weak CL. Under the optimum conditions, the increased CL intensity was proportional with the concentration of SEC₁ in the range of 8.0–125.0 ng ml⁻¹ and the detection limit was 0.5 ng ml⁻¹ (3σ). The relative standard deviation (RSD) for eight parallel measurements of 25.0 ng ml⁻¹ SEC₁ was 0.06. The proposed method has been successfully applied to the determination of SEC₁ in milk and water samples. The results obtained compared well with those by ELISA.     

金黄色葡萄球菌肠毒素B检测方法的研究进展

金黄色葡萄球菌分泌的肠毒素B(staphylococcal enterotoxin B,SEB)作为与食品中毒相关的重要毒素之一而被广泛报道。同时由于其具有热稳定性、易制备、高毒及易传播等特点引起科研人员的高度重视,因此,建立SEB高效的检测方法非常重要。本文对SEB的检测方法进行了综述,主要讨论了生物学检测、免疫凝集实验、琼脂糖扩散法、酶联免疫检测技术、放射性免疫检测技术、免疫荧光检测技术、胶体金试纸条检测技术、基因探针、仪器分析、生物传感检测等检测方法的原理及相关国内外研究进展的应用和局限,这对提前预防金黄色葡萄球菌肠毒素B引起的食物中毒具有重要意义,同时也为今后开发新型检测方法提供理论参考和技术支持。     

Surface plasmon resonance analysis of staphylococcal enterotoxin B in food

Surface plasmon resonance (SPR) biosensors are electro-optical instruments used for analyzing real-time protein-protein interactions. This work evaluates an SPR biosensor (Biacore 3000) in the detection of staphylococcal enterotoxin B (SEB) in foods. A sandwich SPR immunosensor involving two antibodies was used. The capturing antibody, bound covalently to the surface of the biosensor chip, performs the initial binding of the antigen and a second antibody binds to the captured antigen. The second antibody makes antigen verification possible and amplifies the signal. Pure SEB as well as SEB in spiked foods (milk and meat) were detected with little interference from the food matrix. In the control experiments with uncontaminated food samples no significant signal was detected. The SPR biosensor assay detects SEB at approximately10 ng/ml rapidly, with initial binding within 2 min. The entire measurement cycle (including washing and chip regeneration) may take 5 min using one antibody or 8 min using two antibodies. These results suggest that the SPR biosensor may be a useful tool for real-time analysis of toxin in foods.     

Bacteriological investigation of an outbreak of Clostridium perfringens food poisoning caused by Japanese food without animal protein.

An outbreak of Clostridium perfringens food poisoning occurred in a senior citizen's home in Japan. Japanese food, spinach boiled with fried bean curd, was considered to be the causative food as a result of the detection of the C. perfringens enterotoxin gene by nested PCR. The number of enterotoxin-positive C. perfringens was enumerated as 4.3 x 10(5)/g in the causative food by the MPN method combined with nested PCR. By cultivation, enterotoxin-positive C. perfringens was isolated from all the fecal specimens of patients tested and the causative food. The isolates from patients were serotypable, heat-resistant and the majority produced enterotoxin, however most isolates from the causative food were nonserotypable, enterotoxin-negative and heat-sensitive.