Genetic procedures for identification of enterotoxigenic strains of Staphylococcus aureus from three food poisoning outbreaks

Three food poisoning restaurant outbreaks due to Staphylococcus aureus, occurring during June-October 2002 in the Principality of Asturias (PA), Spain, provided the basis for investigating some aspects of the molecular epidemiology of this organism. The methods applied to identify strains and lineages included multiplex-polymerase chain reaction (PCR) to detect nine enterotoxin (se) genes, and three DNA fingerprinting procedures: pulsed-field gel electrophoresis (PFGE) with SmaI, randomly amplified polymorphic DNA (RAPD) with two selected primers, and plasmid restriction analysis with HindIII. Thirty-two isolates were differentiated into three non-se and 12 se strains, which were outbreak-specific, except for one that was represented in two of the outbreaks. In outbreak 1, the 16 food isolates analyzed had sec, seg and sei genes and generated a distinctive DNA fingerprint, being assigned to a single strain. This strain could be categorized as endemic in the PA and associated to manually handled dairy products and nasal carriers. In outbreak 2, the four food isolates analyzed fell into three strains, each one displaying a different se-gene profile (sea, sec and seg-seh-sei) and a distinctive DNA fingerprint. In outbreak 3, the five food isolates tested fell into four seg-sei strains generating identical RAPD but different PFGE and plasmid profiles, and one sea strain also collected from two nasal carriers. This last strain had also been found in manually handled vegetables in outbreak 2, and it belongs to a not very frequently found sea lineage in the PA. Multiplex-PCR to detect se genes together with the three applied DNA fingerprint typing procedures proved therefore to be useful tools in subclassifying S. aureus for epidemiological purposes.     

北京市朝阳区食物中毒相关金黄色葡萄球菌病原学分析

目的 了解2017年北京市朝阳区11株食物中毒相关金黄色葡萄球耐药性、耐药基因、毒力基因及分子分型。方法 依据GB4789.10-2016对甲、乙饭店留样食品、餐具及厨师手涂抹样品进行金黄色葡萄球菌分离鉴定;荧光定量PCR法检测耐药基因mecA、vanA与毒力基因sea、seb、sec、sed、see;纸片法与微量肉汤稀释法进行药物敏感性试验;脉冲场凝胶电泳(PFGE)进行溯源分析。结果 甲、乙饭店留样食品、餐具涂抹及厨师手涂抹样品共检出11株金黄色葡萄球菌;耐药基因mecA、vanA与毒力基因sea、seb、sec、sed、see均未检出;青霉素100%耐药,四环素、氯霉素耐药率分别为27.3%、9.09%,未发现耐甲氧西林金黄色葡萄球菌株;PFGE分型显示：甲饭店餐具涂抹菌株、手涂抹菌株、乙饭店所有菌株为3种带型。 结论 甲乙饭店分别为两起由金黄色葡萄球菌引起的食物中毒事件,皆为携带菌株的厨师污染餐具及食品引起,菌株具有较高耐药性。     

金黄色葡萄球菌及其引起的食物中毒的研究进展

金黄色葡萄球菌广泛存在于自然环境中,致病力强,是常见食物中毒的病原菌。本文对金黄色葡萄球菌致病因素、污染状况、食物中毒、试验诊断、预防措施等方面进行概述,为今后金黄色葡萄球菌食物中毒的预防提供参考。     

一起由金黄色葡萄球菌引起食物中毒的实验室诊断

目的对一起食物中毒进行相关样品的实验室检测,针对分离病原菌进行分子分型溯源和耐药分析。方法对可疑食品(冰激凌、牛奶)样品、患者呕吐物和粪便标本使用国家标准或其他方法进行沙门菌、志贺菌、致泻大肠埃希菌、金黄色葡萄球菌、单核细胞增生李斯特菌、蜡样芽胞杆菌和诺如病毒检测。对检出的金黄色葡萄球菌进行肠毒素检测、脉冲场凝胶电泳(PFGE)分型和抗生素敏感性检测。结果从生产冰激凌剩余牛奶及冰激凌样品中检出金黄色葡萄球菌12株,患者呕吐物和粪便标本中检出4株,菌株均产生A+C+E混合型肠毒素,PFGE显示患者呕吐物、粪便、中毒同批冰激凌样品、生产冰激凌用牛奶中检出的金黄色葡萄球菌菌株同源性为100.0%;药敏结果显示,这16株菌株均对青霉素和红霉素耐药。结论本起事件是由冰激凌加工点使用了被金黄色葡萄球菌污染且产生大量肠毒素的牛奶生产冰激凌所致。建议监管部门加强对农村食品小作坊、牛奶供应站等生产经营场所的监管,预防类似事件的发生。     

Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary

Staphylococcus aureus is a major foodborne pathogen due to its capability to produce a wide range of heat-stable enterotoxins. The primary purpose of this research was to characterize S. aureus isolates recovered from mammary quarter milk of mastitic cows and from bulk tank milk produced on Hungarian dairy farms of different sizes. Macrorestriction analysis of chromosomal DNA from S. aureus isolates was performed using the restriction enzyme SmaI followed by pulsed-field gel electrophoresis (PFGE). The prevalence rates of nine S. aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and of the toxic shock syndrome toxin 1 gene (tst) were determined by multiplex polymerase chain reaction (PCR). The bulk tank milks of 14 out of 20 farms were contaminated with S. aureus at levels of up to 6.0x10(3 )CFU/ml. Farm size had no significant effect (P>0.05) on the S. aureus counts in bulk milk. The prevalence rates of penicillin resistance were 88.9% and 20.0% among the S. aureus recovered from mastitic quarter milk and bulk tank milk, respectively. After phenotypic characterization, a total of 59 S. aureus isolates were selected for genotyping. PFGE analysis revealed 22 distinct pulsotypes, including 14 main types and 8 subtypes, at a similarity level of 86%. Only one or two main types were observed on each of the farms tested, indicating a lack of genetic diversity among S. aureus isolates within farms, and there were only two pulsotypes which occurred on more than one farm. The PFGE patterns showed genetic relatedness between the S. aureus strains recovered from quarter milk and bulk milk on two large farms, implying that on farms having a high number of mastitic cows, S. aureus from infected udders may contaminate bulk milk and, subsequently, raw milk products. Sixteen (27.1%) of the S. aureus isolates tested by multiplex PCR were found to be positive for enterotoxin genes, with 15 of them carrying just one gene and one strain carrying two genes (seg and sei). The most commonly detected toxin genes were seb, sea, and sec, whereas none of our isolates possessed the see, seh, sej, or tst genes. On 75% of the dairy farms surveyed, no enterotoxigenic staphylococci were recovered from either mastitic quarter milk or bulk tank milk.     

一起由金黄色葡萄球菌所致学校食物中毒调查分析

目的 查明一起学校食物中毒发生的原因和可疑危险因素,提出有效控制措施防止事态蔓延,为类似食源性疾病的调查和预防提供参考.方法 利用现场流行病学、食品卫生学和实验室检测等方法,开展病例主动搜索、个案调查和现场卫生学调查,采集食品、外环境和病例的标本进行实验室检测,以判断本次事件的可疑餐次和食物.结果 通过开展主动搜索,共...     

An outbreak of food poisoning due to egg yolk reaction-negative Staphylococcus aureus

An outbreak of staphylococcal food poisoning due to an egg yolk (EY) reaction-negative strain occurred in Japan. Twenty-one of 53 dam construction workers who ate boxed lunches prepared at their company cafeteria became ill, and eight required hospital treatment. The outbreak showed a typical incubation time (1.5-4 h with a median time of 2.7 h) and symptoms (vomiting and diarrhea) of staphylococcal food poisoning. Staphylococcus aureus, which produces staphylococcal enterotoxin (SE) A, was isolated from four fecal specimens of eight patients tested. Scrambled egg in the boxed lunches contained 20-40 ng/g of SEA, and 3.0 x 10(9)/g of viable S. aureus cells that produced this toxin. All isolates from patients and the food were EY reaction-negative, coagulase type II, and showed the same restriction fragment length polymorphism (RFLP) pattern. We concluded that the outbreak was caused by scrambled egg contaminated with EY reaction-negative S. aureus. In Japan, outbreaks of staphylococcal food poisoning are mainly caused by EY reaction-positive S. aureus, and EY reaction-negative colonies grown on agar plates containing EY are usually not analyzed further for detection of S. aureus. The present outbreak suggested that EY reaction-negative isolates should be subjected to further analysis to detect the causative agents of staphylococcal food poisoning.     

福建省2000年～2003年家庭食物中毒现状及分析

为掌握家庭食物中毒的规律，对福建省2000年～2003年家庭食物中毒的情况进行分析。2000年～2003年福建省共发生食物中毒180起，中毒人数共3267人，死亡31人。其中家庭发生食物中毒80起，中毒人数1071人，死亡19人，分别占全省食物中毒总数的44．4％、32．8％和61．3％，食物中毒原因查明率为87．5％。家庭食物中毒主要发生在农村，共73起，占91．3％，城市仅7起，占8．7％，城乡之间的家庭食物中毒差异有显著性（P=0．044）。每起食物中毒平均发病人数为13人。家庭食物中毒的主要致病因素依次为农药及化学物品（37．5％）、有毒动植物（27．5％）、微生物（22．5％）。其中最主要致病因素为有机磷，共发生20起，占家庭食物中毒的25％，中毒人数为175人，占16．3％，死亡人数2人，占8．3％。造成食物中毒死亡的最主要因素是河纯鱼，共死亡7人，占36．8％。有毒动植物食物中毒发生最多的是4～6月份，主要是由于河纯鱼、有毒贝类、毒蘑菇引起的食物中毒。微生物食物中毒集中发生在7～11月，主要致病因素是致病性大肠杆茵，其次为副溶血性弧茵，分别占微生物食物中毒的27．8％和22．2％。化学与农药食物中毒发生最多的是在7～9月份。较重大的食物中毒全部发生在农村。今后须加强农村食品卫生监督管理，大力开展预防食物中毒的宣传和培训。     

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。     

一起由沙门氏菌和金黄色葡萄球菌混合引起的食物中毒病原分析

目的对引起食物中毒相关样品进行病原菌的分离、鉴定和耐药分析。方法参照GB 4789系列标准的方法流程对采集的食物样本进行沙门氏菌、金黄色葡萄球菌、志贺氏菌、副溶血弧菌和单核细胞增生李斯特氏菌共5项致病菌指标检测,按照纸片扩散法(K-B法)进行药敏实验。结果从食物中毒样品中检出肠炎沙门氏菌、金黄色葡萄球菌,确定为二者混合感染引起的食物中毒。药敏结果显示肠炎沙门氏菌对多数抗生素敏感,金黄色葡萄球菌耐药性强,对链霉素、头孢噻肟、氯霉素、红霉素有一定的耐药性。结论本起事件是由肠炎沙门氏菌和金黄色葡萄球菌混合感染引起的食物中毒事件,建议食品卫生监管人员应加强对餐饮环节的监督引导,避免类似事件发生。     

Estimation of economic loss due to food poisoning outbreaks

Food poisoning outbreaks frequently occur in many countries resulting in economic loss. A formula for calculation of economic loss due to food poisoning outbreaks in Indonesia was developed. Estimation of the loss was developed based on experiences in several countries. Related data were also collected from local health facilities, food industry officials, and other sources to improve the accuracy of the formula. Calculation of the economic loss was performed based on cases of food poisoning outbreaks recorded in 2013. Economic loss was estimated based on direct health-care costs, direct non-health-care costs, and indirect non-health-care costs. The economic loss of food poisoning outbreaks in Indonesia in 2013 was approximately US$ 78 million. The high-cost of economic loss due to food poisoning outbreaks suggests a need for additional and serious efforts for controlling all aspects of food safety.

     

Genotype analysis of enterotoxin H-positive Staphylococcus aureus strains isolated from food samples in the Czech Republic

Twenty-eight enterotoxin H-positive Staphylococcus aureus strains isolated from food samples collected in eleven districts of the Czech Republic between 2000 and 2005 were genotypically characterized by pulsed-field gel electrophoresis (PFGE) profiling, spa gene polymorphism analysis, enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) fingerprinting and prophage carriage detection. These strains accounted for about 21% of the food-derived, staphylococcal enterotoxin (SE)-positive isolates. One strain, detected in feta cheese, was implicated in a case of enterotoxinosis. Sixteen of the twenty-eight isolates carried the seh gene alone. The remaining twelve strains harbored the seh gene in combination with other enterotoxin genes, most often the seg and sei genes, followed by the sea, seb, sec and sed genes. Comparison of various genomic profiles resulted in the determination of twenty genotypes designated G-1 to G-20. Two new, to date not defined, spa types (t2000 and t2002) were identified in one strain isolated from raw meat and two strains obtained from prepacked pizza. Evidence has been given that the seh-positive S. aureus isolates from foodstuffs did not originate from a single source or a common ancestor.     

Characterization of Staphylococcus aureus strains isolated from raw milk utilized in small-scale artisan cheese production

Staphylococcus aureus is an important agent of bacterial mastitis in milking animals and of foodborne intoxication in humans. The purpose of this study was to examine the genetic and phenotypic diversity, enterotoxigenicity, and antimicrobial resistance of S. aureus strains isolated from raw milk used for the production of artisan cheese in Vermont. Cross-tabulations revealed that the 16 ribotypes identified among the 90 milk isolates examined were typically associated with a specific animal species and that more than half of these ribotypes were unique to individual farms. In general, specific EcoRI ribotypes were commonly associated with specific phenotypical characteristics, including staphylococcal enterotoxin production or the lack thereof. Limited antimicrobial resistance was observed among the isolates, with resistance to ampicillin (12.51%) or penicillin (17.04%) most common. Two isolates of the same ribotype obtained from the same farm were resistant to oxacillin with 2% NaCl. More than half (52.22%) of isolates produced toxin, and 31 of the 32 isolates solely produced staphylococcal enterotoxin type C. Although these data demonstrate that S. aureus strains found in raw milk intended for artisan cheese manufacture are capable of enterotoxin production, staphylococcal enterotoxin C is not typically linked to foodborne illness. Because S. aureus is a common contaminant of cheese, an understanding of the ecology of this pathogen and of the antimicrobial susceptibility and toxigenicity of various strains will ultimately contribute to the development of control practices needed to enhance the safety of artisan and farmstead cheese production.     

Isolation and characterization of heat resistant enterotoxigenic Staphylococcus aureus from a food poisoning outbreak in Indian subcontinent

Outbreaks of staphylococcal food poisoning (SFP) are very common across the world; however, there is hardly any report of SFP from the Indian subcontinent. An outbreak occurred in the state of Madhya Pradesh (India) after the consumption of a snack called "Bhalla" made up of potato balls fried in vegetable oil. More than 100 children and adults who ate the snack suffered from the typical symptoms of SFP and required hospitalization. Food and clinical samples were found to contain a large number of enterotoxigenic Staphylococcus aureus. All enterotoxigenic isolates produced a combination of SEB and SED enterotoxins and were sensitive to oxacillin and vancomycin. Isolates were characterized by molecular biology tools, viz., SDS-PAGE, amplified ribosomal DNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD) and nucleotide sequencing of seb, sed, and 16S rDNA genes. Results of these studies suggested that the isolates, irrespective of their isolation from food or clinical samples, were clonal in origin. Further, seb gene sequence of isolates showed nucleotide variations at multiple sites when compared with other sequences available in the database. Representative isolates, one each from food and clinical samples, were found to be highly heat resistant (D(60) approximately 15-16 min). Isolates obtained in the current outbreak need to be further studied to find out the impact on food safety guidelines with respect to thermal processing.     

食品中一株凝固酶异常的金黄色葡萄球菌的分离鉴定

目的 对餐饮环节采集的一份鸡排进行金黄色葡萄球菌检验时,发现一株血浆凝固酶试验异常的金黄色葡萄球菌,并对其分离、鉴定、分析和总结,避免因不典型菌株的漏检而带来的食品安全风险。方法 采用国标法检测金黄色葡萄球菌,血浆凝固酶试验时间延长至24h,同时用生化鉴定法和BAX System Q7快速检测法进行互相验证。结果 国标法凝固酶试验6h不凝固,12h出现部分凝固,24h完全凝固,生化鉴定法和BAX System Q7快速检测法结果都是检出金黄色葡萄球菌。结论 在食品中金黄色葡萄球菌检验过程中,选择性平板上菌落典型,而凝固酶试验异常的情况,应延长试验时间并用其他方法加以验证。     

Possible risk factors associated with penicillin-resistant strains of Staphylococcus aureus from bovine subclinical mastitis in early lactation.

A randomized controlled field study of selective dry cow therapy with 686 cows allocated to two control groups (sampling only or placebo) or two therapy groups was used to screen for possible factors associated with penicillin-resistant strains of Staphylococcus aureus after the dry period. Therapy was given either as a total dose of 400,000 IU of penicillin and 100 mg of neomycin per infected quarter as dry cow preparation or as a total dose of 1.2 million IU of penicillin and 1200 mg of dihydrostreptomycin per infected quarter as a lactation formula. Success cows had all quarters identified as being free of penicillin-resistant strains of Staphylococcus aureus both at calving and at 30 +/- 17 d after calving. Failure cows were those having penicillin-resistant strains of Staph. aureus in any quarter at both or one of these two samples after the dry period. Using logistic regression, four variables were found to be associated with penicillin-resistant strains of Staph. aureus after the dry period. These included the identification of penicillin-resistant strains of Staph. aureus either at 45 +/- 32 d before drying off and at drying off, treatment for acute clinical mastitis at least once during the previous lactation, the weighted SCC of all cows' milk by daily milk yield within the herd, and therapy in the lactation formula compared with the two control groups. Our finding that the use of lactation formula increases the risk of resistance development is contradictory to present arguments underlying Norwegian dry cow therapy strategy.     

Enterotoxigenic and antibiotic resistance determination of Staphylococcus aureus strains isolated from food handlers in Gaborone, Botswana

The prevalence, antibiotic resistance, and enterotoxigenic potential of Staphylococcus aureus strains from different anatomical sites on food handlers in Gaborone, Botswana, were determined. Of a total of 200 food handlers tested, 115 (57.5%) were positive for S. aureus. Of the 204 S. aureus isolates, 63 (30.9%), 91 (44.6%), and 50 (24.5%) were isolated from the hand, nasal cavity, and face, respectively, and 43 (21%) of the isolates were enterotoxigenic. The most prevalent enterotoxin was type A, which accounted for 34.9% of all the enterotoxigenic strains, and enterotoxin D was produced by the fewest number of strains (9.3%). Resistance to methicillin was encountered in 33 (22.4%) of the penicillin G-resistant isolates, and 9 (27.3%) of these methicillin-resistant isolates also were resistant to vancomycin. Nineteen antibiotic resistance profiles were determined, and the nasal cavity had the highest diversity of resistance profiles. The nasal cavity also had the highest number of resistant strains, 77 (53%), whereas the hand and face had 49 (32%) and 24 (16.0%) resistant strains, respectively. To reduce the Staphylococcus carriage rate among food handlers, training coupled with a commitment to high standards of personal and environmental hygiene is recommended.     

食品中金黄色葡萄球菌PCR-ELISA检测技术建立

针对食品中金黄色葡萄球菌检测,选取金黄色葡萄球菌耐热核酸酶nuc基因作为靶标,利用地高辛标记引物扩增的PCR产物与生物素标记的探针杂交,然后与ELISA平板上的链霉素杂交,通过抗地高辛抗体显色建立PCR-ELISA检测技术。应用该方法检测人工污染牛奶中金黄色葡萄球菌,同时将大肠埃希氏菌、沙门氏菌、志贺氏菌、单增李斯特菌等常见食源性致病菌作为阴性对照。结果显示,金黄色葡萄球菌的纯培养物以及人工污染金黄色葡萄球菌牛奶,金黄色葡萄球菌为阳性,其他对照菌为阴性,两者PCR-ELISA检测敏感性均为10¹CFU/m L,比普通PCR检测的敏感性（10³CFU/m L）高100倍。因此,该研究建立的PCR-ELISA方法具有良好的特异性与敏感性,能够特异、敏感、准确地检测牛奶中的金黄色葡萄球菌,为食品中金黄色葡萄菌的快速鉴定、风险评估与有效监测提供重要技术手段。      

食品中金黄色葡萄球菌的污染状况及耐药性分析

为了解金黄色葡萄球菌对食品的污染情况 ,采集 6类 2 0 4件食品 ,进行金黄色葡萄球菌检测。采用GB 4 789— 1994食品卫生检验方法检测金黄色葡萄球菌 ,用minVIDAS全自动免疫分析仪测定肠毒素 ,用K B法进行药敏试验。在 2 0 4件食品中检出 4 4件金黄色葡萄球菌 ,总检出率2 1 5 6 % ,其中生奶的检出率最高 (39 39% ) ,生肉、水产品次之 (33 33% ) ,熟肉制品、乳制品检出率分别为 6 0 %和 5 0 % ,冰激凌中未检出金黄色葡萄球菌。 4 4株金黄色葡萄球菌有 2 7株产生肠毒素 ,占 6 1 36 %。在药敏试验中 4 4株金黄色葡萄球菌 10 0 %对苯唑西林耐药 ;93 18%对青霉素G耐药 ;86 4 0 %对阿莫西林耐药 ,对丁胺卡那、头孢三嗪、头孢唑啉、万古霉素敏感。金黄色葡萄球菌对奶、肉、水产品的污染比较严重 ,特别是产生肠毒素的菌株存在着引起食物中毒的可能性。