武汉肉类食品中大肠杆菌O157∶H7分离株stx亚型和毒力特征分析

从22 家市场销售的117 份肉类食品中分离出4 株大肠杆菌O157∶H7菌株,经PCR检测这4 株菌的stx、hly、 eae毒力基因均为阳性。采用聚合酶链式反应（polymerase chain reaction,PCR）法对这4 株菌的stx亚型进行鉴定。 3 株菌同时携带stx1、stx2基因,且均为stx1a、stx2a亚型。菌株EC5.11仅携带stx2基因,但在所有的stx2分型PCR反 应中都为阴性。用PCR扩增该菌株stx2基因全长并克隆测序,序列分析结果表明EC5.11志贺毒素基因为stx2c亚型。 采用Vero细胞毒性实验检测这4 株菌产志贺毒素的状况,结果显示这些菌株都具有一定的Vero细胞毒性。     

牛源性非O157大肠杆菌的分离与鉴定

目的:对牛粪及牛肉中的非O157大肠杆菌进行分离、鉴定和毒力基因携带情况进行检测,以了解非O157大肠杆菌的污染状况。方法:参考USDA检测方法,对样品进行选择性增菌,用多重聚合酶链式反应(polymerase chain reaction,PCR)方法进行初步筛选,检测O抗原(O157、O121、O111、O103、O26),阳性样本用选择性显色培养基rainbow agar分离纯化,可疑菌株用多重PCR方法鉴定O抗原,PCR-限制性片段长度多态性鉴定H抗原,并采用血清凝集实验进行验证,确认的阳性菌株采用多重PCR方法进行毒力基因(stx1、stx2、eae、hly)检测。结果:在153份牛粪和49份牛肉样本中,共40份样品检出1个或多个O血清型阳性,牛粪检出率高于牛肉,非O157阳性率为19.3%,O157的阳性率为0.50%;经分离纯化后,共鉴定出阳性菌株30株,其中O26最多占73.3%,O121、O103、O157分别占16.7%、6.7%、3.3%。毒力基因检测结果显示,分离自牛肉的2株O26:H11,一株stx1、hly阳性,另一株hly阳性,分离自牛粪的1株O26:H11携带hly基因,因此30株菌株中带毒菌株阳性率为10.0%,非O157产志贺毒素大肠杆菌阳性率为3.4%。结论:牛粪和牛肉中非O157大肠杆菌的污染率明显高于O157,尤其是O26:H11血清型最高,且检出含志贺毒素基因的大肠杆菌,这提示零售牛肉市场存在非O157大肠杆菌污染的安全风险,我国应该加强对非O157大肠杆菌的检测和监控。     

食源性产志贺毒素大肠杆菌的分离及菌株特征分析

了解不同食品中产志贺毒素大肠杆菌的流行情况、菌株特征及潜在致病性。方法 对我国不同地区采集的355份食品样品进行产志贺毒素大肠杆菌分离鉴定,对菌株进行stx1/stx2基因分型、eae等毒力基因检测,并对菌株进行多位点序列分型(MLST)分析。结果 355份样品中44份stx2基因阳性,共分离出11株非O157 产志贺毒素大肠杆菌,其中3株携带stx2a亚型,3株携带stx2e亚型,1株携带stx2b亚型,4株不能分型;5株携带ehxA、saa毒力基因,2株携带subA基因,1株携带katP基因;MLST将11株菌分为7个不同的ST型,存在与溶血性尿毒综合症患者肠出血性大肠杆菌分离株(HUS-associated enterohemorrhagic E.coli,HUSEC)及主要流行血清群产志贺毒素大肠杆菌亲缘关系较近的ST型别。结论 我国食品中存在一定程度的非O157产志贺毒素大肠杆菌污染,部分菌株具有潜在致病性,应加强对食品中STEC的监测。     

蔬菜中大肠杆菌O157的分离与鉴定

对武汉市售蔬菜(50份)进行大肠杆菌O157的检验,经过新生霉素-EC增菌液增菌、免疫磁珠富集、选择性平板培养和血清学鉴定,从一份生菜中筛出1株O157阳性菌株EC9.23。PCR鉴定该菌毒力基因,stx1、stx2、rfbO157基因均为阳性,hly、eae、fliCH7基因均为阴性。说明从武汉市售蔬菜中可检出携带志贺毒素基因的O157菌株。     

某屠宰场大肠杆菌O26的分离与鉴定

为了了解屠宰场大肠杆菌O26污染情况,参考美国农业部(united states department of agriculture,USDA)的检测方法,对样品进行选择性增菌,经免疫磁珠富集、选择性显色培养基mRainbow Agar分离纯化后,挑选可疑菌株采用PCR方法鉴定O抗原,进一步采用血清凝集试验进行验证,并对确认的阳性菌株采用多重PCR方法进行毒力基因(stx1、stx2、eae、hly)检测。结果表明,采集的120份样品中共分离到1株大肠杆菌O26,但上述4种毒力基因均为阴性。该屠宰场存在大肠杆菌O26的污染,但分离出的大肠杆菌O26没有携带毒力基因。     

肉类及蔬菜食品中EHEC O157污染分布及分型研究

为深入了解肉类及蔬菜中肠出血性大肠埃希氏菌(EHEC O157)的污染分布规律和遗传多样性,随机采集全国18个城市的食品样品,参考GB/T 4789.36-2008方法进行样品处理,并用rfbE/fliCH7双重PCR对菌株进行鉴定;分别采用单重PCR和ERIC-PCR对菌株进行毒力基因(eae、hlyA、stx1和stx2)检测和分子分型。结果表明,414份样品中检出18份EHEC O157阳性样品,总污染率4.35%,其中生鲜肉12份,速冻肉6份,蔬菜未检出;经过生化、血清鉴定和双重PCR检测,鉴定出52株EHEC O157,包括29株O157:H7,3株O157:NM(fliCH7+,无动力),2株O157:NM(fliCH7-,无动力)和18株O157:hund(未确定H型);毒力基因检测结果发现,52株菌株中有50株携带毒力基因,其中40株(76.92%)携带eae,31株(59.62%)携带hlyA,20株(38.46%)携带stx1,24株(46.15%)携带stx2。ERIC-PCR分型结果表明,在相似系数为0.80时,菌株可分为12个聚类簇,F型为其主要基因簇,分离株的基因型呈现多样性。     

基于免疫磁分离的7种产志贺毒素大肠埃希氏菌快速检测方法的评价

目的 研究基于免疫磁分离的七种产志贺毒素大肠埃希氏菌快速检测方法的灵敏度与特异性。方法 将大肠埃希氏菌O157：H7和大肠埃希氏菌O103不同稀释度的菌悬液,用免疫磁珠富集后,检测其携带毒力基因stx1、stx2 和黏附基因eae以及O157:H7和O103的抗原基因。同时,对菌悬液进行活菌计数,进行灵敏度研究。对8株携带stx1、stx2、eae基因的目标菌菌悬液,以及25株非目标菌的标准菌株及分离菌株的菌悬液,用免疫磁珠富集后,检测其携带毒力基因stx1、stx2 和黏附基因eae以及抗原基因,进行特异性研究。结果 本方法检测大肠埃希氏菌O157：H7的stx1、stx2、eae以及抗原基因的灵敏度为10_²CFU/mL,检测大肠埃希氏菌O103抗原基因的灵敏度为10_³CFU/mL. 8株目标菌检测结果与其携带的基因一致,没有假阴性,包容性达到100%。25株目标菌检测结果与其携带的基因一致,未发现有假阳性,排他性达到100%。结论 方法具有良好的灵敏性及特异性,适用于食品中七种产志贺毒素大肠埃希氏菌的快速检测。     

成都市武侯区肉类食品大肠杆菌污染状况及毒力基因调查

了解成都市武侯区肉类食品中大肠杆菌污染情况和分离菌株的毒力特征,以预防和控制食品污染,保证食品安全。2011~2013年间采集成都市武侯区超市和菜市场的肉类食品,经选择性培养与分离鉴定,采用PCR方法检测8种相关毒力基因(eae A、stx1A、stx2A、hly A、agg R、est、elt A和bfp A)的流行情况。结果表明生肉类中大肠杆菌的检出率为81.07%,熟肉类中大肠杆菌的检出率为40%;在分离的324株大肠杆菌中有49株检测到一种及以上毒力基因;主要流行的毒力基因是eae A和agg R,未检测到est和bfp A基因。通过本研究发现成都市武侯区肉类食品中大肠杆菌污染比较严重,大肠杆菌携带毒力基因以eae A和agg R为主,该结果反映本市肉类产品的卫生安全状况,可为成都地区食品源大肠杆菌控制及毒力基因流行提供科学数据。     

肠出血性大肠杆菌变种的RAPD分型研究

对4株肠出血性大肠杆菌(EHEC)进行随机扩增多态性(RAPD)分析,并结合主要毒力基因如eae和hly,以及志贺毒素滴度,探讨RAPD实验对大肠杆菌致病菌进行基因分型结果的可靠性。实验菌株中有两株变种携带stx基因但不能正常表达志贺毒素,其中一株变种EC169与另两株正常表达志贺毒素的O157菌株具有相似的扩增图谱,而另一株非O157变种EC130与其他菌株聚类明显不同。从20条随机引物中筛选出了重复性强且具有多态性的随机引物G2、G7、G8、G11、G12,可用于大肠杆菌致病菌快速鉴别和食物中毒溯源。     

产志贺毒素突变株的毒力分析

对2株食物中毒病人体内分离的产志贺毒素突变株EC130和EC169进行毒力分析。EC130和EC169携带stx基因但不能正常表达志贺毒素,具有eae基因和hly基因,仍具有一定毒力。初步探讨了产志贺毒素突变株不能正常表达志贺毒素的机理,高产志贺毒素的对照株携带Q933基因,而EC130和EC169不携带Q933基因。结果表明,单一采用志贺毒素作为检测靶标,容易造成产志贺毒素突变株漏检,今后在检测食品中产志贺毒素株时应增加检验eae基因和hly基因。     

An alternative approach for enumeration of Escherichia coli in foods

A study was carried out in France in collaboration with the meat industry to investigate the occurrence and characteristics of Shiga toxin-producing E. coli (STEC) and O157 E. coli in a population of healthy bovines representative of French livestock. A total of 851 animals belonging to three bovine classes (106 young bulls, 374 dairy cows and 371 meat cows) were included in the study. Samples of feces and of the corresponding carcasses were collected from March 97 to August 97 in seven abattoirs spread throughout the national territory. STEC cultures from the 1702 samples were screened using PCR for the presence of stx genes. Positive samples were further subjected to colony blot hybridization and to O157-specific immunomagnetic separation. Probe-positive colonies and O157 colonies were then analyzed for the presence of virulence genes and phenotypic characters (serotype, Stx production). In 154 (18.1%) feces and 91 (10.7%) carcass samples stx genes were detected. Two hundred and twenty-two STEC colonies were isolated from 67 (7.9%) feces and 16 (1.9%) carcass samples, with 183 STEC isolated from feces and 39 from carcasses. Only eight O157 isolates were collected from feces samples. None of these O157 E. coli isolates presented stx genes and thus could not be considered as pathogenic regarding hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). In 3.2% of STEC isolated from feces and in 10.2% of STEC from carcasses eae genes were detected. In 17% of STEC from feces and in 30.7% from carcasses ehx genes were detected. Using these data, the 222 STEC colonies could be classified in 11 different 'virulence patterns' (presence/absence of stx1, stx2, eae and ehx genes), showing that more than 77% of isolates presented only one virulence factor. Only three STEC on 222 colonies (1.3%) presented the three virulence factors stx, eae and ehx in association, none of them reacting with antisera specific for enterohemorrhagic E. coli. (EHEC). These data, together with the fact that only five isolates on the 222 (2.2%) reacted with such antisera (three O111 and two O26 isolates) demonstrated that the natural bacterial populations isolated during this study were clearly distinct from EHEC.     

Isolation and characterization of Escherichia coli O157:H7 from retail meats in Argentina

Between February and May 2000, 279 meat samples were collected from 136 retail stores in Gualeguaychú City, Argentina. Samples were assayed for Escherichia coli O157:H7 by selective enrichment in modified EC broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto both sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and a chromogenic medium. Eleven E. coli O157:H7 isolates were detected in 6 (3.8%) of 160 ground beef samples, in 4 (4.8%) of 83 fresh sausages, and in 1 (3.3%) of 30 dry sausages. E. coli O157:H7 was not isolated from five hamburger patties or one barbecue-type fresh sausage assayed. The isolates were tested for virulence-related genes. Ten additional Shiga toxin-producing E. coli (STEC) O157:H7 isolates of food origin, recovered from different locations in Argentina, were included for comparison purposes. All 21 isolates harbored both eae and EHEC-hlyA genes, and 12 (57.1%) encoded stx2/stx2vh-a. The isolates were of phage types 87 (seven strains), 14 (four strains), 4 (three strains), and 26 (one strain). Six strains were nontypable by phage typing. Pulsed-field gel electrophoresis (PFGE) revealed 19 XbaI-PFGE profiles. Fifteen (71%) strains were grouped in four clusters, which shared more than 80% of DNA restriction fragments. The enrichment culture method with IMS was a sensitive procedure to detect E. coli O157:H7 strains in retail meats. Some of the isolates from different stores presented a high clonal relatedness, as determined by XhaI-PFGE and phage typing, and harbored the virulence factors associated with human illness.     

Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.     

Prevalence, serotypes and virulence genes of Shiga toxin-producing Escherichia coli isolated from ovine and caprine milk and other dairy products in Spain

The aim of this study was to determinate the prevalence, serotypes and virulence genes of Shiga toxin-producing Escherichia coli (STEC) strains isolated from different dairy products (DP) in Spain with the purpose of determining whether DP represent a potential source of STEC pathogenic for humans. A total of 502 DP were examined from 64 different ovine and caprine flocks and 6 dairy plants in Extremadura (Western Spain). Samples were collected monthly between March 2003 and June 2004 and included 360 unpasteurised milk obtained from the bulk tank, 103 fresh cheese curds and 39 cheeses. Samples obtained were examined for STEC using genotypic (PCR) methods. STEC strains were detected from 39 (10.8%) bulk tank, 4 (3.9%) fresh cheese curds and 2 (5%) cheese, whereas O157:H7 serotype were isolated from one (0.3%) bulk tank. A total of 9 STEC strains (O27:H18, O45:H38, O76:H19, O91:H28, O157:H7, ONT:H7, ONT:H9 and ONT:H21) were identified in this study. One of them, the serotype O27:H18, has not been reported previously as STEC. PCR showed that 3 strains carried stx1 genes, 5 possessed stx2 genes and 1 both stx1 and stx2. Whereas all STEC caprine isolates showed ehxA genes, only O157:H7 serotype showed eae virulence genes. The strain O157:H7 isolated possessed intimin type gamma1 and belonged to phage type 31. This study confirms that dairy product is an important reservoir of STEC pathogenic for humans.     

Occurrence of Shiga toxin-producing Escherichia coli in a Spanish raw ewe's milk cheese

The aim of the present study was to investigate the occurrence of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in 'Castellano' cheese, a non-cooked and hard or semi-hard Spanish cheese made from ewe's milk. A total of 83 raw milk cheese samples with different ripening times (2.5, 6 and 12 months) were taken at 30 cheese factories. Samples were examined for the presence of STEC using in the first stage the Association of Official Analytical Chemists (AOAC) official method number 997.11, and then, in the second stage, isolates were tested for virulence genes using genotypic (PCR) methods. Three STEC strains were detected in two samples (2.4%) of 'Castellano' cheese, one with 2.5 and the other one with 12 month-ripening period. From those STEC isolates, two were identified as E. coli O14 and the third presented an O-specific polysaccharide not-groupable serologically (ONG). PCR showed that all isolates were characterized by harbouring the Shiga toxin (stx) stx1 gene and by the absence of the genes for stx2, eaeA, and ehxA virulence factors. This study revealed the potential of STEC to survive in long-ripened-hard cheeses.     

Detection and quantitation of enterohemorrhagic Escherichia coli O157, O111, and O26 in beef and bovine feces by real-time polymerase chain reaction

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and certain non-O157 EHEC serotypes (such as O26:H11, O26: NM, O11:H8, and O111:NM) have emerged as significant causes of human disease throughout the world. Important virulence attributes of EHEC are the intimin protein (encoded by the eae gene) and Shiga toxins 1 and 2 (encoded by the stx1 and stx2 genes, respectively). Two sets of real-time polymerase chain reaction (R-PCR) assays were developed for the simultaneous detection and quantitation of EHEC through the monitoring of the presence of the eae and stx genes, and these assays were evaluated. In the eaeR-PCR assay, three sets of primers and TaqMan probes were designed for the amplification and real-time detection of a portion of the eae gene specific to the EHEC O26, O111, and O157 serotypes. In the stxR-PCR assay, two sets of primers and TaqMan probes were used to amplify and detect the stx1 and stx2 genes. DNA prepared from 67 bacterial strains carrying known virulence markers was tested to determine the specificities of the two assays. In the eaeR-PCR assay, eaeO157- and eaeO111-specific primer-probe sets identified only EHEC O157 and O111 strains, respectively. The eaeO26-specific primer-probe set identified all EHEC 026 isolates and some Shiga toxin-negative serotypes of enteropathogenic E. coli and rabbit diarrheagenic E. coli. The stxR-PCR assay was able to identify only those strains carrying either or both of the Shiga toxin-encoding genes. The detection range of both R-PCR assays was linear over DNA concentrations corresponding to 10(3) to 10(8) CFU/ml of an EHEC strain. Both assays were able to detect and quantify very low levels (1 to 10 CFU/g of food or feces) of EHEC in feces and ground beef enriched for 16 h in a modified Trypticase soy broth. In conclusion, eae- and stx-based R-PCR assays are reliable and sensitive methods for the rapid screening and specific and quantitative detection of important serotypes of EHEC in cattle and in foods of bovine origin.     

Isolation and characterization of Shiga toxin-producing Escherichia coli O157:H7 and non-O157 from beef carcasses at a slaughter plant in Mexico

The contamination of beef carcasses with Shiga toxin-producing O157:H7 and non-O157 Escherichia coli (STEC) obtained from a slaughter plant in Guadalajara, Mexico was investigated. A total of 258 beef carcasses were sampled during a 12-month period. All samples were assayed for STEC by selective enrichment in modified tryptone soy broth supplemented with cefixime, cefsulodin and vancomycin, followed by plating on Sorbitol MacConkey Agar supplemented with cefixime and tellurite (CT-SMAC). Simultaneously, all samples were assayed by immunomagnetic separation (IMS) and plated on CT-SMAC and CHROMagar. The presence of the stx1, stx2, eaeA and hly933 genes, recognized as major virulence factors of STEC, was tested for O157:H7 and non-O157 E. coli isolates by multiplex polymerase chain reaction (PCR). STEC was detected in two (0.8%) samples. One of these STEC isolates corresponded to the serotype O157:H7 showing stx2, eaeA and hyl933 genes. The other isolate corresponded to non-O157 STEC and only had the stx1 gene. Thirteen carcasses (5%) were positive for nonmotile E. coli O157 and 7 (2.7%) were positive for E. coli O157:H7. The presence of O157:H7 and non-O157 STEC on beef carcasses in this slaughter plant in Guadalajara, Mexico, emphasizes the importance of implementing the Hazard Analysis and Critical Control Point (HACCP) system, as well as the need for implementing, evaluating, and validating antimicrobial interventions to reduce the presence of potential pathogenic microorganisms.     

Testing of swine feces obtained through the National Animal Health Monitoring System's Swine 2000 study for the presence of Escherichia coli O157:H7

Fecal samples collected from healthy pigs from 13 of the top 17 swine-producing states were tested for Escherichia coli O157:H7 as part of the National Animal Health Monitoring System Swine 2000 study. Serogroup O157 strains were isolated from 106 of 2,526 fecal samples. None of the isolates were positive by PCR for the fliCh7 (H7 flagellin) gene or for the hly933 (hemolysin) gene; however, one isolate was positive for the stxl gene (Shiga toxin 1), an additional four isolates were positive for the stx2 gene (Shiga toxin 2), and three isolates possessed the eae gene (intimin).