产肠毒素性大肠埃希氏菌的检验

为查明夏季一起员工食物中毒的病原菌,采用GB/T4789-2003规定的PCR检测试剂盒进行检测。结果表明:从5份食物中毒者的便样和2份呕吐物中检出产肠毒素(LT)大肠埃希氏菌,其它肠道致病菌未检出。可以认为这起食物中毒是由产肠毒素(LT)大肠埃希氏菌所致。     

2002-2005年北京市顺义区食品致病菌监测分析

目的了解北京市顺义区食品致病菌的污染状况。方法按照GB/T4789─2003食品微生物检验标准方法进行检验。结果2002-2005年共监测了6类食品（590份）中的4种致病菌（沙门菌、金黄色葡萄球菌、肠出血性大肠埃希菌O157：H7、单核细胞增生性李斯特菌）。检出致病菌57株,阳性率为9.7%,其中检出沙门菌2株,金黄色葡萄球菌26株,单核细胞增生性李斯特菌29株,未检出肠出血性大肠埃希菌O157：H7。6类食品中生牛奶、生畜（禽）肉类产品致病菌阳性率较高,分别为15.5%、14.5%;冰淇淋、水产品、散装熟食中也有检出,分别为5.0%、4.4%、1.9%;生食蔬菜中没有检出致病菌。结论生牛奶、生畜（禽）肉类产品是致病菌污染的主要食品,由此造成的二次污染是引起食物中毒的隐患,应该引起足够重视。     

椰毒假单胞菌酵米面亚种食物中毒的病原分离鉴定

目的通过对食物中毒病原菌的分离鉴定,为查明中毒原因提供科学依据。方法分离鉴定和毒性试验按照国家标准方法 WS/T 12—1996《椰毒假单胞菌酵米面亚种食物中毒诊断标准及处理原则》和GB/T 4789.29—2003《食品卫生微生物学检验椰毒假单胞菌酵米面亚种检验》进行。米酵菌酸检测按照GB/T 11675—2003《银耳卫生标准》执行,采用液相色谱-质谱联用法对样品开展检测。结果经VITEK 2 COMPACT全自动微生物生化鉴定仪和基因指纹鉴定仪进行鉴定,4份样品中3份鉴定结果为唐菖蒲伯克霍尔德菌,小鼠毒性试验阳性。4份样品均检测出米酵菌酸。结论本次食物中毒源于食源性椰毒假单胞菌酵米面亚种污染。     

2015~2016年安康市食物中毒检测分析

目的 回顾分析2015~2016安康市食物中毒事件, 总结安康市事物食物中毒特点。方法 收集安康市的中毒事件的纸质材料进行分析。参考GB 4789-2016《食品卫生微生物学检验》、WS271-2007《感染性腹泻诊断标准》和WS289-2008《霍乱诊断标准》等试验方法对食物中毒样本进行检测。结果 2015~2016年安康市发生食物中毒事件14起, 1起为有机磷中毒, 其余13起均为食源性致病菌中毒。检出致病菌4种16株, 分别是: 沙门氏菌4株; 金黄色葡萄球菌5株、蜡样芽胞杆菌3株; 致泻性大肠埃希氏菌EPEC-A、EHEC、EAEC、EIEC各1株。 结论 安康市食物中毒多以细菌性为主, 污染的致病菌种类多且致病性强。     

关于发布《食品微生物检验副溶血性弧菌检验》(GB4789.7-2013)等75项食品安全国家标准等的公告(2013年第7号)

<正>根据《中华人民共和国食品安全法》和《食品安全国家标准管理办法》规定,经食品安全国家标准审评委员会审查通过,现发布《食品微生物学检验副溶血性弧菌检验》(GB 4789.7-2013)等75项食品安全国家标准和《食品添加剂二丁基羧基甲苯(BHT)》(GB 1900-2010)第1号修改单。其编号和名称如下:GB 4789.7-2013食品微生物学检验副溶血性弧菌检验(代替GB/T 4789.7-2008)GB 4789.26-2013食品微生物学检验商业无菌检验(代替GB/T 4789.26-2003)GB 4789.28-2013食品微生物学检验培养基和试剂的质量要求(代替GB/T 4789.28-2003)GB 4789.31-2013食品微生物学检验沙门氏菌、志贺氏菌和致泻大肠埃希氏菌的肠杆菌科噬菌体诊断检验(代替GB/T 4789.31-2003)     

一起致泻性大肠埃希菌食物中毒的病原学检测分析

本文对一起疑似食物中毒事件中所采集的样本进行病原学检测分析,希望能够为预防食物中毒提供科学依据。应用全自动医用PCR分析系统对22种常见胃肠道感染相关病原体进行快速筛检,以实时荧光定量PCR技术进行毒力基因检测,并利用传统方法对病原菌进行分离培养。检测数据显示,35份样本的22种常见胃肠道感染相关病原体检测结果为检出致病性大肠埃希菌(EPEC);10份样本经实时荧光定量PCR技术检出EPEC毒力基因escV和内参基因uidA;经传统培养分离方法,最终从6份粪便样本中分离出目标菌EPEC。因此得出结论,综合流行病学调查、临床症状和实验室检验结果,推断出这是一起由携带EPEC的食堂工作人员通过污染食物导致学生感染发病的突发公共卫生事件。建议各级卫生监督部门加强对餐饮行业及其从业人员的监督检查力度,避免类似食物中毒事件的发生,进而保障人民的身体健康。     

2019年福建省哨点医院腹泻患者中致泻大肠埃希菌感染状况及病原学特征分析

目的 了解2019年福建省哨点医院腹泻患者中致泻大肠埃希菌(DEC)感染状况、毒力基因携带、分子分型及耐药情况。方法 采集腹泻患者210份粪便标本按照GB 4789.6—2016《食品安全国家标准 食品微生物学检验 致泻大肠埃希氏菌检验》方法分离大肠埃希菌后进行荧光聚合酶链式反应(PCR)确认,并对分离出的DEC进行脉冲场凝胶电泳(PFGE)分子分型及药敏试验。结果 共检出阳性菌株32株,检出率为15.2%(32/210),其中肠道致病性大肠埃希菌(EPEC)占37.5%(12/32),肠道集聚性大肠埃希菌(EAEC)占37.5%(12/32),产肠毒素大肠埃希菌(ETEC)占25.0%(8/32)。药敏试验结果表明,32株菌对氨苄西林耐药程度最高,耐药率为78.1%(25/32),其次是四环素和甲氧苄啶/磺胺甲噁唑,耐药率分别为62.5%(20/32)和59.4%(19/32)。多重耐药(MDR)率为50.0%(16/32)。PFGE结果表明,32株菌共分为28种PFGE带型,其中12株EPEC共分为10种带型,12株EAEC共分为10种带型,8株ETEC共分为8种带型,其中EPEC聚类分析结果有两组菌株具有100.0%相似带型,EAEC有一组菌株具有100.0%相似带型,ETEC聚类分析没有完全一致的带型。结论 2019年福建省哨点医院腹泻患者DEC感染主要以EPEC、EAEC为主,菌株分型带型存在多样性,菌株耐药状况严重,且存在较高的多重耐药率。     

2019—2021年武汉市致泻大肠埃希菌分子分型与耐药性研究

目的 了解2019—2021年湖北省武汉市腹泻患者来源致泻大肠埃希菌的毒力基因分布、耐药情况以及种群多样性。方法 定期收集来自武汉市5家医疗机构的致泻大肠埃希菌菌株并通过生化反应和飞行时间质谱进行确认。对经确认的菌株进行药敏试验以及多重PCR检测毒力基因。采用脉冲场凝胶电泳技术和多位点序列分型技术对收集的菌株进行分子分型。通过聚类比对和构建最小生成树进行同源性分析。结果 收集到59株致泻大肠埃希菌，共检出3种基因型别。其中检出产肠毒素大肠埃希菌（ETEC）29株，占比最高为49.15%；肠集聚性大肠埃希菌（EAEC）、肠致病性大肠埃希菌（EPEC）各检出15株，占比均为25.42%。59株致泻性大肠埃希菌对除多黏菌素E外的11种抗生素表现出不同程度的耐药，对氨苄西林（67.80%）、萘啶酸（61.02%）、四环素（45.76%）、复方新诺明（37.29%）、头孢噻肟（30.51%）、氯霉素（13.56%）表现出较高的耐药性。EAEC和EPEC均存在14种不同的脉冲场凝胶电泳带型，均可被分为11种不同的ST型别。ETEC存在28种不同的脉冲场凝胶电泳带型，可被分为10种不同的ST型别，且超过50%的ETEC属于同一种优势克隆复合体CC-10。结论 武汉市食源性致泻大肠埃希菌的污染长期存在且耐药情况严重，其中EAEC和EPEC型别多样，ETEC的优势克隆复合体CC-10被广泛检出。     

养猪场源致泻大肠埃希菌毒力基因和致病型研究

目的 了解养猪场源致泻大肠埃希菌(DEC)毒力基因及致病型分布情况,为从猪肉生产源头防控该菌引发的食源性疾病提供参考数据。方法 采用多重荧光实时定量聚合酶链式反应(PCR)检测分离自12个养猪场的生猪、养殖环境和养殖工人的大肠埃希菌毒力基因,鉴定DEC的致病型。结果 985株分离自养猪场的大肠埃希菌中,DEC占比28.83%(284/985),其中肠道聚集性大肠埃希菌(EAEC)225株(22.84%,225/985)、产肠毒素大肠埃希菌(ETEC)14株(1.42%,14/985)、肠道致病性大肠埃希菌(EPEC)12株(1.22%,12/985)、肠道出血性大肠埃希菌(EHEC)10株(1.02%,10/985)、肠道侵袭性大肠埃希菌(EIEC)9株(0.91%,9/985)、EHEC-ETEC杂交型菌13株(1.32%,13/985)以及EPEC-ETEC杂交型菌1株(0.10%,1/985)。1株分离自养殖工人鼻拭子的大肠埃希菌为EHEC-ETEC杂交型菌株,同时在该养猪场的生猪鼻拭子和粪便中也检测到同样毒力基因型的菌株。猪育肥前期DEC污染率高于猪育肥后期,差异有统计学意义(χ²=1.10,P＜0.05)。结论 养猪场DEC污染率较高,提示应加强养猪场致病菌检测,从养殖源头防控DEC污染猪肉生产链,减少食源性疾病及人畜共患病的发生。     

云南省一例唐菖蒲伯克霍尔德氏菌(椰毒假单胞菌酵米面亚种)食物中毒事件调查分析

目的 调查分析云南省一例唐菖蒲伯克霍尔德氏菌(椰毒假单胞菌酵米面亚种)食物中毒事件。 方法 参照GB/T 4789.29-2003《食品卫生微生物学检验 椰毒假单胞菌酵米面亚种检验》对样品进行唐菖蒲伯克霍尔德氏菌检测。按照GB 5009.189-2016《食品安全国家标准 食品中米酵菌酸的测定》检测样品中的米酵菌酸, 采用液相色谱法对样品进行检测。用VITEK 2COMPACT 全自动微生物生化鉴定仪和飞行时间质谱进行微生物鉴定。结果 2份样品鉴定结果为唐菖蒲伯克霍尔德菌。2份样品均检测出米酵菌酸, 含量分别为18.0和24.2 mg/kg。 结论 本次食物中毒源于食源性唐菖蒲伯克霍尔德氏菌(椰毒假单胞菌酵米面亚种)污染。     

一起嗜水气单胞菌引起的食物中毒调查

目的 了解本次食物中毒中毒原因及可疑食物污染来源.方法 采用病例对照研究方法,并采集可疑食物及病例标本进行检测.结果 共发生病例48例,症状主要为腹痛(100％)、腹泻(85％)、发热(40％)等.潜伏 期中位数为25 h,进食白切鸡是危险因素(OR=6.0,95％ CI:2.0～17),且与发病呈剂量反应关系((x2趋势=4.5,P=0.03).白切鸡及病例粪便中检出嗜水气单胞菌.结论 进食被嗜水气单胞菌污染的白切鸡是本次食物中毒发生的主要原因,建议加强农村游厨的食品卫生安全知识培训,防止类似事件发生.     

一起金黄色葡萄球菌污染奶制品导致食物中毒的调查分析

目的查明一起食物中毒事件的原因。方法采用现场流行病学调查方法结合实验室检验进行分析。结果一起由于饮用受金黄色葡萄球菌污染的高钙牛奶饮品引起的食物中毒事件。结论食品安全监管部门应加强生产环节的监管,尤其是加大供应学生食品的监管力度。要加强食品从业人员卫生知识培训,提高卫生意识,防止类似中毒事件的再次发生。     

肠致病性大肠杆菌适配体筛选研究

目的食源性致病菌引起的食源性疾病已成为全球食品安全面临的巨大威胁,其中由大肠杆菌引起的食源性疾病最为常见。因此发展快速、准确检测大肠杆菌的方法对于保障食品安全、保护国民健康具有重要意义。方法应用Whole bacteria-SELEX(指数富集的配体系统进化技术),以肠致病性大肠杆菌为靶标,经过15轮的筛选富集,将所得产物进行克隆测序,并结合荧光分析考察所得序列的亲和力和特异性,根据解离常数值分析比较序列的亲和力和特异性,最终得到2条(Seq.1和Seq.28)与肠致病性大肠杆菌高特异性结合的适配体。结果适配体Seq.1和Seq.28均对肠致病性大肠杆菌表现出了相对良好的亲和力和特异性(对其他菌的相对亲和力均低于15%),解离常数分别为45.06±6.797和32.31±6.002 nmol/L。结论本文利用SELEX技术筛选特异性识别肠致病性大肠杆菌适配体,具有稳定性高、合成方便、易标记等特点,可进一步应用于食品中肠致病性大肠杆菌的快速检测。     

乳品中致病大肠杆菌荧光双重PCR技术建立

建立乳及乳制品中致病性大肠杆菌(EPEC)的快检方法。以EPEC特异的微绒毛粘连基因(eae gene)和茵毛束形成编码基因(bfp gene)为模板,设计两对引物,荧光探针对EPEC进行特异性扩增。该方法快速,特异性好,对EPEC的最低检出量为1 cfu/μL(纯菌),检出时间为3 h,同一样品重复检测3次ct值的变异系数均小于5%。建立可快速特异检测乳及乳制品中EPEC的双重荧光PCR方法。     

Shedding of escherichia coli O157:H7 by cattle fed diets containing monensin or tylosin

Monensin and tylosin have activity against gram-positive bacteria, and it has been theorized that their effects on the intestinal environment may promote proliferation of gram-negative bacteria such as Escherichia coli. Effects of these antibiotics on the shedding of E. coli O157:H7 were studied in a feedlot environment, using 32 finishing steers. A diet containing 85% barley grain, 10% barley silage, and 5% supplement was amended with 33 ppm monensin, 11 ppm tylosin, both of these additives, or no additives (control). All steers were orally inoculated with 10(10) CFU of a mixture of four strains of nalidixic acid-resistant E. coli O157:H7. Fecal (grab), oral (mouth swab) and water, water-water bowl interface, feed, and pen floor fecal pat samples were collected weekly for 12 weeks. Prevalence of E. coli O157:H7-positive fecal grab samples did not differ (P = 0.26) among treatments, nor did the rate (P = 0.81) or duration (P = 0.85) of shedding of the organism. Fecal grab samples were positive for E. coli O157:H7 more frequently (P < 0.001) than were oral swabs. More (P = 0.02) E. coli O157:H7-positive oral swabs were recovered from the tylosin group than from controls. E. coli O157:H7 was not detected in any of 47 water samples, but was present in 1 of 47 water bowl swabs, 7 of 48 feed samples, and 36 of 48 fecal pats. Pulsed-field gel electrophoresis suggested that differences existed among inoculated strains in their ability to persist in animals and in the environment. However, this study revealed no evidence that dietary inclusion of monensin or tylosin, alone or in combination, increased fecal shedding of E. coli O157:H7 or its persistence in the environment.     

Molecular epidemiology of microorganisms isolated from food workers and enteral feeding of public hospitals

This study aimed to compare strains of Staphylococcus aureus and E. coli isolated from food workers and enteral diet samples obtained from 2 public hospitals (H1/H2) in Goiania, Goias, Brazil, by the means of antibiogram and pulsed field gel electrophoresis (PFGE). In the H1, strains of S. aureus were present in 2 enteral diet samples and in 13 food worker swabs. Strains of E. coli were found in an enteral diet sample from H1 and in 2 enteral diet samples from H2 and in 6 food worker swabs in the H1 and in 12 food worker swabs from H2. According to the antibiogram, the 6 susceptibility profiles (A to F) of 15 S. aureus strains colonizing personnel and enteral feeding did not allow the identification of the probable source of diet contamination. All 20 E. coli strains isolated from the H1 and H2 were grouped in 4 phenotypic profiles (A to D). The phenotypes A (H1) and C (H2) showed the same profile for microorganisms isolated from handlers and diets, suggesting more phenotypic similarity among these samples. PFGE genotyping showed that S. aureus isolates from diets were related to a single strain isolated from a food worker suggesting that in this case the reason for the diet contamination may be a result of food handling. The food worker appears to be the most probable source of E. coli contamination for enteral feeding from H2. This fact emphasizes on the food workers as a risk of bacterial transmission for the diets and that the diet chain production must be controlled. PRACTICAL APPLICATION: The study emphasizes the importance of monitoring the enteral diet microbiological quality and the factors associated to its contamination. The study highlights the use of molecular biology as an instrument to correlate strains to determine the origin of the final product contamination.     

An evaluation of sampling methods for the detection of Escherichia coli and Salmonella on Turkey carcasses

The efficacy of rinse, excision, and swab methods for the microbiological analysis of prechill turkey carcasses was investigated. Aerobic plate counts from a 50-cm2 area of the breast sampled by excision and by swabbing were compared. Escherichia coli and Salmonella recoveries were determined from turkeys sampled by a carcass rinse (CR), a modified rinse with the carcass supported in a swing (MCR), a two-site swab of 50 cm2 at the back and thigh (2S), a one-site swab of 50 cm2 beneath the wing (1S), a whole-carcass swab of the inner and outer carcass surface (WS), and excision of 25 g of neck skin tissue (NE). The effect of diluent volume (25, 50, and 100 ml) on E. coli counts from swab samples was also assessed. The aerobic plate count from breast tissue sampled by excision was greater than that by swabbing (P < 0.05). E. coli recoveries by the MCR method were similar to those by CR. E. coli counts from IS and WS samples were higher when swabs were stomached in 50 rather than 25 ml of diluent (P < 0.05). For swabs stomached in 50 ml of diluent, E. coli recoveries by the MCR, 2S, 1S, and WS methods were similar. For swabs stomached in 50 ml of diluent, Salmonella recoveries by the WS and MCR methods were higher than those by the 2S and 1S methods. Excision was more effective than swabbing for obtaining total bacterial counts from reduced turkey carcass areas. Whole-carcass sampling by rinsing or swabbing is necessary for optimum Salmonella recovery. Sampling a reduced area of the carcass is sufficient for E. coli analysis.     

Serotypes, virulence genes, and intimin types of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) isolated from calves in São Paulo, Brazil

Shiga toxin-producing Escherichia coli (STEC), is the most important recently emerged group of foodborne pathogens. Ruminants, especially cattle, have been implicated as a principal reservoir of STEC, undercooked ground beef and raw milk being the major vehicles of foodborne outbreaks. Enteropathogenic E. coli (EPEC) strains are defined as eae-harboring diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes. In order to determine the occurrence, serotypes and virulence markers of STEC and EPEC strains, 546 fecal samples from 264 diarrheic calves and 282 healthy calves in beef farms in S?o Paulo, Brazil, were screened by PCR. STEC and EPEC were isolated in 10% and 2.7% of the 546 animals, respectively. Although IMS test was used, the STEC serotype O157:H7 was not detected. The most frequent serotypes among STEC strains were O7:H10, O22:H16, O111:H(-), O119:H(-) and O174:H21, whereas O26:H11, O123:H11 and O177:H11 were the most prevalent among EPEC strains. In this study, serotypes not previously reported were found among STEC strains: O7:H7, O7:H10, O48:H7, O111:H19, O123:H2, O132:H51, O173:H(-), and O175:H49. The eae gene was detected in 25% of the STEC and 100% of EPEC strains. The intimin type theta/gamma2 was the most frequent among STEC, whereas the intimin beta1 was the most frequent intimin type among EPEC strains. To our knowledge, this is the first report of the occurrence of the new intimin muB in one strain of animal origin. This new intimin was detected in one atypical EPEC strain of serotype O123:H? isolated from diarrheic cattle. The enterohemolysin (ehxA) was detected in 51% of the STEC and 80% of the EPEC strains, whereas STEC autoagglutinating adhesin (saa) virulence gene was detected only in those STEC strains negative for eae gene. All 15 bovine EPEC strains isolated in this study were negative for both eaf and bfp genes. Our data shows that in Brazil cattle are not only a reservoir of STEC and atypical EPEC, but also a potential source of infection in humans, since the important STEC serotypes previously described and associated with severe diseases in humans, such as O111:H(-), O113:H21, O118:H16, and O174:H21 were isolated.     

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,共搜索到21例病例(疑似19例,确诊2例),学生罹患率为12.96%(21/162)。临床症状主要是呕吐(95.24%, 20/21)、腹痛(66.67%, 14/21)、恶心(66.67%, 14/21)、腹泻(57.14%, 12/21)。病例对照研究显示,可疑食物为10月16日晚餐的剩米饭(OR=18.18,95%CI: 2.05～161.38)。在1例病例的肛拭子和另外1例病例的呕吐物中分离出金黄色葡萄球菌和B型肠毒素。结论 本次食物中毒事件是由金黄色葡萄球菌产生B型肠毒素所致,可疑食物为10月16日晚餐中食堂提供的剩米饭。米饭在常温下放置5 h左右且食用前未彻底加热是本次事件发生的危险因素。学校食堂存在食品卫生安全隐患,应进一步加强对学校食堂的食品安全监管和从业人员的食品安全教育,提高食品安全意识,预防类似事件的发生。     

Diarrheagenic Escherichia coli detected by 16-plex PCR in raw meat and beef intestines sold at local markets in Ouagadougou, Burkina Faso

The study investigated the prevalence of five major Escherichia coli pathogroups in raw meats and beef intestines sold at the local markets in Ouagadougou, Burkina Faso. One hundred and twenty samples (36 beef, 36 beef intestine, 24 mutton and 24 chicken samples) were purchased from four markets between October 2008 and February 2009. Fifteen virulence genes specific for Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and enteroaggregative E. coli (EAEC) were examined using 16-plex PCR for mixed bacterial cultures derived from the samples. One or more diarrheagenic E. coli pathogroup was detected in 51 (43%) of all the 120 samples: in 16 (44%) beef, 19 (53%) beef intestine, 9 (38%) mutton and in 7 (29%) chicken samples. Thirty three (28%) samples were positive for stx(1) and/or stx(2) indicating presence of STEC. EPEC virulence markers (eae, escV and/or ent and/or bfp and/or EHEC-hlyA) were detected in 14 (12%) stx-negative samples. ETEC virulence markers (elt and/or estIb and/or estIa) were detected in 10 (8%) samples and EAEC virulence markers (pic or aggR) in 5 (4%) samples. No EIEC was detected. The results show that in Burkina Faso the microbiological quality of retail meat is alarmingly poor due to the common occurrence of diarrheagenic E. coli bacteria.