食品基质中不同金黄色葡萄球菌肠毒素基因型表达差异研究

目的 比较研究食品基质中不同金黄色葡萄球菌肠毒素(Staphylococcal enterotoxins ,SEs)基因型的蛋白表达差异,为预防金黄色葡萄球菌食物中毒提供参考依据。方法 采用特异聚合酶链反应方法(polymerase chain reaction,PCR)对食品中分离的金黄色葡萄球菌菌株进行肠毒素基因型检测;选择sea、seb、sec、sed等基因型阳性菌株,分别接种于胰酪大豆胨液体培养基(trypticase soy broth,TSB)、牛奶、鸡肉中,按照国家标准GB 4789.10—2016酶联免疫吸附试验法(enzyme linked immunosorbent assay,ELISA)定量检测TSB培养基、牛奶和鲜鸡肉中不同金黄色葡萄球菌肠毒素基因型的蛋白表达量。结果 30株金黄色葡萄球菌中检测到14株肠毒素基因型阳性菌株,所占比例为46.67%,其中sea基因携带率最高(16.67%),而seb、sec、sed、seh则各占6.67%。SEA 在TSB、牛奶、鸡肉3种基质中的平均表达量为7.37 ng/mL,高于SEB、SEC、SED;不同基质环境对肠毒素的表达具有一定影响,如SEA、SEB、SEC、SED在TSB中的表达水平最高,平均表达量为9.04 ng/mL,牛奶次之,鸡肉最低。结论 肠毒素基因型的表达与菌株自身的调控及环境作用密切相关,本研究对肠毒素的产生机制进行初步了解,有助于进一步降低食物中毒风险。     

食源性金黄色葡萄球菌肠毒素基因型分布研究

目的 采用PCR法扩增食源性金黄色葡萄球菌中肠毒素基因以了解该菌肠毒素基因携带情况,比较食物中毒和食品监测来源菌株中肠毒素基因检出率差异.方法 合成sea、seb、sec、sed和see五种肠毒素基因特异性引物,用常规PCR方法扩增食物中毒和食品监测来源菌株中各自肠毒素基因,同时采用mini-VIDAS检测食物中毒来源菌株中肠毒素.结果 110株菌株中有30株检出肠毒素基因,检出率为27.3％,肠毒素基因阳性菌株均只检出1种肠毒素基因.其中来自2起食物中毒的14株菌株均检出seb型肠毒素和相关基因,检出率为100％.来源于食品监测样本的96株菌株中有16株检出肠毒素基因,检出率为16.7％,包括sea型4株、seb型2株、sec型4株、sed型6株.结论 在宁波市食品监测中所分离的金黄色葡萄球菌所携带的肠毒素基因主要有sea、seb、sec和sed四型,而seb型肠毒素是引起金黄色葡萄球菌肠毒素所致食物中毒的主要因素.     

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。     

2003年～2015年金黄色葡萄球菌食物中毒事件特征分析

为了解2003年～2015年金黄色葡萄球菌引起的食物中毒事件发生规律,对中国知网收录的2003年～2015年金黄色葡萄球菌食物中毒暴发资料进行统计与分析。结果为共收集到发生在2003年～2015年间的金黄色葡萄球菌食物中毒事件86起,累计发病2 431人,死亡0人。肠毒素A（enterotoxin,sea）所引起的食物中毒事件最多,其次为肠毒素C（enterotoxin C,sec）。金黄色葡萄球菌所引起的食物中毒事件大多发生在夏、秋两季,然而食物中毒人数以春季最多,其次为夏季和秋季,冬季中毒人数最少。发生在集体食堂的食物中毒事件最多。肉及其制品引起的食物中毒事件数最多。因此,应针对重点人群、重点场所开展主动监测,指导合理加工或保藏食物,防止食物交叉污染或腐败变质,从而防止食物中毒事件的发生。     

PCR检测食品中金黄色葡萄球菌肠毒素基因

目的:检测食品中金黄色葡萄球菌肠毒素基因谱携带情况,探讨与食物中毒发病相关的金黄色葡萄球菌基因类型。方法:取食品中检出的金黄色葡萄球菌进行分离培养鉴定,通过PCR技术检测样本18种不同型的肠毒素基因的携带情况,并进行统计学分析。结果:食品中检出的金黄色葡萄球菌肠毒素基因有较高的阳性检出率,主要检出sea、seb、sei、seg、sel、sem、sen等基因,其中sea基因检出率最高,达到77.27%;sei、seg、sem、sen基因具有相关性,检出率为9.09%;seb、sel基因的检出率为4.55%。结论:在食品中主要检出金黄色葡萄球菌肠毒素sea、sei、seg、sem、sen、seb、sel等基因,其中,sei、seg、sem、sen基因的相关性还有待进一步深入研究。     

SYBR Green Ⅰ荧光定量PCR检测乳中携带sea基因金黄色葡萄球菌的研究

金黄色葡萄球菌是引起食物中毒的一种常见的致病菌,而其产生的肠毒素A是乳及乳制品食物中毒事件的主要原因。本研究建立了SYBR Green Ⅰ荧光定量PCR方法对乳中携带sea基因的金黄色葡萄球菌进行检测。该方法能快速、稳定地在8h内完成对乳中金黄色葡萄球菌的检测,对人工污染乳中金黄色葡萄球菌检测的最低检出限为83CFU/ml。     

2006—2011年西安市食品及食物中毒中金黄色葡萄球菌毒素基因分布及分型研究

了解2006—2011年西安市污染食品及食物中毒中金黄色葡萄球菌(S.aureus)分离株肠毒素(SEs)、杀白细胞素(PVL)、表皮剥落毒素(ETs)、毒性休克综合征毒素-1(TSST-1)等毒素基因的分布状况,并比较两种分离株在基因分布及分型上的差异。方法 采用多重PCR 法检测61株S.aureus(包括40株食品分离株和21株食物中毒分离株)sea、seb、sec、sed、pvl、eta、etb、tsst-1基因,其中sea、seb、sec、sed基因引物加入同一反应体系,剩余4对基因引物加入另一反应体系。结果 40株食品分离株中17株检出毒素基因(42.50%);21株食物中毒分离株中18株检出毒素基因(85.71%),食物中毒分离株毒素基因的检出率明显高于食品分离株(P<0.01)。食品分离株中主要流行的毒素基因为sea(25%)、eta(12.5%),未检测到携带etb、tsst-1基因的菌株;同时得到8种毒素基因型,主要流行的基因型为sea(10.00%)、sea+eta(7.50%)。食物中毒分离株中主要流行的毒素基因为sea(76.19%)、sec(28.57%),未检测到携带pvl基因的菌株;同时得到6种毒素基因型,主要流行的基因型为sea(42.86%)、sea+sec+tsst-1(14.29%)。结论 S.aureus食品分离株和食物中毒分离株在毒素基因的分布及分型上存在较大差异。     

金黄色葡萄球菌肠毒素及移动基因元件研究进展

金黄色葡萄球菌肠毒素是一种热源性的超抗原。食用被肠毒素污染的食物能够引起食物中毒,导致恶心、呕吐、腹痛有时伴随腹泻。本文综述了肠毒素的分类命名、理化性质、以及编码不同肠毒素的基因在移动基因元件中的存在情况。这些移动基因元件在金黄色葡萄球菌毒力传播和进化过程中起着至关重要的作用,了解它们对于金黄色葡萄球菌流行病学溯源以及理解毒力机制具有一定的指导意义。     

近红外光谱建模法快速测定桔子精油中柠檬烯的含量

为了解日常检测分离株产肠毒素和耐药情况,探讨食源性金黄色葡萄球菌产毒素的类型及分布状况,研究其耐药特性,对本实验室2016年～2017年日常食品检测中的分离株,分别用全自动荧光酶联免疫法检测肠毒素总量和酶联免疫吸附法对肠毒素SEA～SEE进行分型,并用全自动微生物鉴定药敏分析系统进行药敏试验。结果表明,370份食品中分离到的29株金黄色葡萄球菌,产肠毒素的有16株,阳性率为55.2%,其中食物中毒分离株5株都为肠毒素阳性。产2种及以上肠毒素的菌株为12株,占41.4%。A型～E型常见肠毒素都有检出,其中产SEE的菌株最多,有12株,占41.4%,产SEA的菌株次之,为11株。29株食源性金黄色葡萄球菌对庆大霉素、妥布霉素、青霉素、安苄西林、苯唑西林、阿莫西林-克拉维酸、复方新诺名、克林霉素、红霉素、利福平、四环素均有不同程度的耐药,并出现多重耐药性,其中对青霉素和安苄西林的耐药率最高,均为82.8%,其次为红霉素44.8%。该研究的食品监测中分离到的金黄色葡萄球菌其产肠毒素率较高,主要的类型为SEE和SEA。而且分离得到的食源性金黄色葡萄球菌存在不同程度的耐药性和多重耐药现象,建议从各个环节加强监测,降低因耐药菌带来的食品安全风险。     

一起食物中毒事件中产独特肠毒素表型的金黄色葡萄球菌病原学分析

目的对一起疑似为金黄色葡萄球菌所导致的食物中毒事件进行葡萄球菌肠毒素检测,结合金黄色葡萄球菌病原学分析,为明确食物中毒诊断提供依据。方法根据流行病学调查,采用ELISA方法对可疑食物进行葡萄球菌肠毒素检测,同时对可疑食物和患者呕吐物进行金黄色葡萄球菌分离,运用Vitek2 Compact全自动细菌鉴定仪和血浆凝固酶试验鉴定为金黄色葡萄球菌,采用脉冲场凝胶电泳(PFGE)对病原菌进行同源性分析,以ELISA方法对检出的金黄色葡萄球菌菌株进行肠毒素检测,用PCR方法对肠毒素基因进行分型。结果食物和患者样品中分别分离出2和11株金黄色葡萄球菌,PCR方法及ELISA方法对肠毒素分型结果显示,其中12株同时存在SEA、SEB、SED、SEE 4种肠毒素及相关基因,PFGE聚类分析显示,其中12株产肠毒素金黄色葡萄球菌具有高度同源性。结论本起食物中毒事件为具有独特肠毒素表型的金黄色葡萄球菌导致,在金黄色葡萄球菌中毒实验室调查过程中,肠毒素检测结合病原菌溯源分析可以为相关公共卫生事件提供科学依据。     

Characteristics of enterotoxin H-producing Staphylococcus aureus isolated from clinical cases and properties of the enterotoxin productivity

Staphylococcal enterotoxin H (SEH) is predicted to be involved in staphylococcal food poisoning. To characterize SEH-producing Staphylococcus aureus isolates from staphylococcal food poisoning cases in Japan, we investigated the relationship between SEH production and coagulase serotype, which is an epidemiological marker, and compared the properties of SEH production with those of staphylococcal enterotoxins A (SEA) and B (SEB). SEH production was determined by a newly developed sandwich enzyme-linked immunosorbent assay. Eighty-six (59.7%) of 144 isolates from staphylococcal food poisoning cases produced SEH. Seventy-one of the SEH-producing isolates simultaneously produced SEA, SEB, or both. All SEH-producing isolates belonged to coagulase type VII, which was the predominant type, representing 99 (68.8%) of 144 isolates. The amount of SEH produced in brain heart infusion was almost the same as the amount of SEA and approximately 10-fold lower than that of SEB. SEH and SEA were produced mainly during the late exponential phase of growth, whereas SEB was produced mostly during the stationary phase. The production levels of SEH and SEA were gradually affected by decreases in water activity, but the production of SEB was greatly reduced under conditions of low water activity. These findings indicate that SEH-producing S. aureus isolates are of high prevalence in staphylococcal food poisoning cases. Given the unique epidemiological characteristic of these isolates, SEH and SEA probably are responsible for food poisoning.     

Coagulase-positive Staphylococci and Staphylococcus aureus in food products marketed in Italy

Staphylococcus aureus is a very common organism capable of producing several enterotoxins (SEs) that cause intoxication symptoms of varying intensity in humans when ingested through contaminated food. This paper reports the results of an investigation on the presence of Coagulase-Positive Staphylococci (CPS) and S. aureus in several food products marketed in Italy and on food contact surface swabs sampled from the food industry. A total of 11,384 samples were examined and 1971 of them (17.3%) were found to contain CPS. The assays performed on 541 CPS strains led to the identification of 537 S. aureus strains on which characterization of type A, B, C and D staphylococcal enterotoxins (SEA, SEB, SEC and SED) was performed. A total of 298 S. aureus strains (55.5%) produced one or more SEs: 33.9% of the strains produced SEC, 26.5% SEA, 20.5% SEA+SED, 13.4% SED, 2.7% SEB, 1.7% SEA+SEB, 0.7% SEC+SED and 0.3% produced SEA+SEC and SEB+SEC. The investigation highlighted that these organisms are very common and constitute a potential risk for consumers' health.     

食源性金黄色葡萄球菌肠毒素及其检测方法

金黄色葡萄球菌是一种重要的食源性致病菌,在自然界中广泛分布,其引起的食物中毒是世界性的公共卫生问题。金黄色葡萄球菌肠毒素是引起金黄色葡萄球菌食物中毒的主要致病因子。目前共发现22种金黄色葡萄球菌肠毒素或类肠毒素(SEA-SEE、SEG-SET、SElU、SElU_2和SElV),其中具有催吐活性的被定义为肠毒素,没有催吐活性或者尚待验证的被定义为类肠毒素(SEl)。传统肠毒素SEA~SEE被报道是引起金黄色葡萄球菌食物中毒的主要肠毒素类型,但是大多数新型肠毒素或类肠毒素与食物中毒的关系还没有被真正认识。本文对近几年关于金黄色葡萄球菌肠毒素与食物中毒的关系、肠毒素的表达调控以及肠毒素检测方法的研究进展进行了总结,以期更好地了解金黄色葡萄球菌新型肠毒素致病性,为今后金黄色葡萄球菌食物中毒的预防和控制提供依据。     

Enterotoxin production by Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is an important cause of mastitis in cows. The ability of S. aureus strains to produce one or more enterotoxins in milk and dairy products is linked to staphylococcal food poisoning. To determine whether staphylococci causing bovine mastitis could cause human foodborne intoxication, the production of staphylococcal enterotoxins A through D (SEA, SEB, SEC, and SED) by 160 S. aureus isolates was evaluated with the use of a reverse passive latex agglutination enterotoxin kit. All S. aureus strains were isolated over a 9-month period from 2,343 routine submissions of a composite quarter collection of individual mastitic cows at 18 dairy farms in the San Joaquin Valley in California. Prior to enterotoxin detection, isolates were grown by a method that enhances the in vitro synthesis of enterotoxin. Twenty-two of 160 S. aureus isolates produced enterotoxin. Seven produced SEC, 12 produced SED, and 3 produced both SEC and SED. None of the isolates produced SEA or SEB.     

Present state of knowledge on staphylococcal intoxication

Globally, staphylococcal intoxication remains a very common food poisoning. In this review, emphasis is being placed on epidemiological aspects of the problem and the effect of food environment on the survival and growth of staphylococci and production of enterotoxins. The high prevalence of staphylococci in raw foods of animal origin requires effective processing for safety. Man remains a major reservoir for post-process recontamination. The effect of the intrinsic characteristics of foods (pH, water activity, Eh, preservatives competing microbial flora, natural food) and extrinsic parameters of processing and storage (temperature, freezing, irradiation, dehydration, packaging, humidity) on staphylococcal survival, growth and enterotoxin production has been evaluated extensively. While staphylococci can be destroyed easily the enterotoxins can survive practically all food processing. While extreme levels of intrinsic variables can control enterotoxin production, yet the environment of most foods is conductive to staphylococcal growth. Rapid food cooling, refrigeration and consumer, food handler and processor education remain the key to staphylococcal food poisoning prevention.     

Occurrence of toxigenic Staphylococcus aureus in ready-to-eat food in Korea

Toxigenic Staphylococcus aureus contamination in ready-to-eat (RTE) food is a leading cause of foodborne illness in Korea. To monitor food contamination by S. aureus, a total of 3332 RTE food samples were selected from nationwide wholesale marts between 2003 and 2004 and examined. A total of 285 (8.6%) of the overall samples were contaminated by S. aureus. According to the analysis, 31.6% of the tested cream-cakes, 19.8% of the raw fish, and 19.3% of the rice cakes with filling were contaminated with S. aureus. Forty-seven percent of the strains isolated from the contaminated food were enterotoxigenic S. aureus. The phenotypic result of the strain isolated from food showed that 48% of the strains produced one or more toxins, such as staphylococcal enterotoxins A, B, and C (SEA, SEB, and SEC). At least one SEA was produced by over 90% of the toxigenic strains. Other toxins, such as SEB, SEC, SED, SEA+SEC, and SEC+SED, were each detected. Toxic shock syndrome toxin 1 (TSST-1), a causative agent of toxic shock syndrome, was detected in 13 strains of the toxigenic isolates from the food. As the result of genotyping, 22 strains with a toxin gene that was not detected in the phenotypic analysis were also detected. Sixty-nine percent of the toxigenic strains had at least one sea gene, and the most prevalent genotype was sea+seh (34.4%), followed by sea (18.8%) and sea+seg+sei (15.6%). The tst gene encoding TSST-1 was found in 13 strains (13.5%). The genes (eta and etb) encoding exfoliative toxins A and B were not detected in any of the samples.     

A superantigen bioassay to detect staphylococcal enterotoxin A

Current detection methods for enterotoxins of Staphylococcus aureus are labor intensive and limited in sensitivity. Furthermore, these immunochemical protocols fail to adequately detect heat-treated enterotoxins. Staphylococcal enterotoxins cause severe gastrointestinal illness at relatively low concentrations and retain toxigenicity even after heat treatment. Presented here is a novel method to detect staphylococcal enterotoxin A (SEA). This method is a bioassay that exploits SEA's activity as a superantigen in that it induces in cytotoxic T lymphocytes a cytotoxic response against SEA-bound Raji cells. Target cell death is assayed colorimetrically with the CytoTox 96 cell lysis detection kit. In the experiments presented here, this bioassay was also able to detect heat-treated SEA, albeit with a slight compromise in sensitivity. This system detected SEA at picomolar concentrations. Because of the sensitivity of this assay, it is conceivable that it could be incorporated into current detection methods as a confirmatory test.     

Benefits of the Combined Use of Immunological- and PCR-Based Methods for Determination of Staphylococcal Enterotoxin Food Safety Criteria in Cheeses

The aim of this study, performed under official controls, was to assess the relationship between the content of staphylococcal enterotoxins SEA to SEE in cheese samples and the enterotoxigenic potential of Staphylococcus aureus isolates. The level of correlation may further underline the benefit of using complementary approaches based on immunological tests and the PCR method to improve the diagnosis for determination of staphylococcal enterotoxin food safety criteria in cheeses submitted to official controls and own checks.     

The influence of lactic acid bacteria and yeast on production of staphylococcal enterotoxins

Research of influence of 10 lactic acid bacteria cultures on staphylococcal growth and production of staphylococcal enterotoxins SEA and SEB has shown that lactic acid bacteria slow down SEA in 5,7 fold and SEB - 1,56 fold. All investigated cultures possessed inhibition action on SEA and SEB production. Has been established, what lactic acid bacteria slow down growth of staphylococcus in 1,48 fold. It has shown that appears, that the most effective inhibitors of SEA and SEB production are strains L. casei, Lactobacillus acidophilus 317/402.     

Dose-response Modelling of Staphylococcal Enterotoxins Using Outbreak Data

Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs). Yet, small amount of data are available for establishing a dose response. The objective of this work was to build a dose response relation based on the systematic investigations carried out during recent years in France. Over the period 2010-2014, more than 60 SFP outbreaks involving SEs, mainly from France, were microbiologically investigated. The enterotoxins were characterized as well as quantified. Attack rates, appearance times and natures of symptoms collected during epidemiological investigations were related to microbiological data. The outbreaks collected focused on enterotoxins SEA, SEB, SEC, and SED. Distribution of appearance times of symptoms and their natures were not influenced by the type of enterotoxins. The US EPA benchmark dose (BMD) methodology was then used to establish dose response. Attack rates of SFP outbreaks were modelled as a function of ingested doses and a BMD have been estimated for SEA.