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。     

检测产志贺毒素大肠杆菌的菌落PCR方法

针对产志贺毒素大肠杆菌的毒力基因stx 设计特异性引物,并建立一种菌落PCR 方法。菌落PCR 模拟实验证实,该方法特异性强,能良好的扩增出O157 的stx1 和stx2 基因,而普通大肠杆菌、蜡样芽孢杆菌、金黄色葡萄球菌则无PCR 扩增产物。应用分子检测初筛、选择性培养、菌落PCR 相结合的方法,检测实际食品样品,分离检测到一株携带 stx1 的产志贺毒素大肠杆菌。本实验建立的菌落PCR 方法可应用于食品检验。     

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。     

食品中非O157大肠杆菌志贺毒素基因序列分析

对一株从食品中分离的非O157产志贺毒素1型大肠杆菌(EC6)进行研究。将该菌株所产志贺毒素Stx1用 PCR扩增stx1 基因全长并克隆测序,其stx1 基因与GenBank 数据库收录的stx1 基因最高同源性为99%,表明EC6 发生了一定程度的基因突变。采用邻位相连法构建进化树,结果表明EC6 为stx1 基因亚型。     

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。     

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。     

食品中产志贺毒素大肠杆菌的多重PCR检测

针对产志贺毒素大肠杆菌的stx2、wzy、hlyA的保守序列,设计特异性的引物,建立一种新的多重PCR检验方法.结果显示,该方法特异性强,扩增结果与各参考菌株基因型一致,并能良好的区分出O157菌株和非O157型产志贺毒素大肠杆菌.该方法能用于食品样品的检测和流行病学分离株的快速鉴定.     

牛源性大肠杆菌O157:H7的分离鉴定及其耐酸性比较研究

为比较大肠杆菌（Escherichia coli）O157:H7产毒菌株耐受盐酸和乳酸的差异性,首先采集309 份牛粪及牛肉样,进行菌株分离鉴定,接着采用多重聚合酶链式反应（polymerase chain reaction,PCR）方法检测分离株及其他收集菌株的4 种毒力基因（eae、hly、stx1、stx2）,进而对携带毒力基因的产毒菌株分别进行盐酸和乳酸应激实验。结果表明：共分离鉴定出8 株大肠杆菌O157菌株,样品阳性检出率为2.59%;毒力基因检测表明,8 株菌株均不携带stx1和stx2基因,其中6 株菌株携带eae及hly基因;所有产毒菌株耐酸性实验结果表明,盐酸或乳酸处理2 h后20 株产毒菌株存活菌数均显著下降（P＜0.05）,但下降程度呈现明显的菌株差异性,同一菌株对盐酸、乳酸呈现明显的耐受差异。     

伊犁地区肉牛源产志贺毒素大肠杆菌的血清型和ERIC-PCR基因型研究

目的:了解产志贺毒素大肠杆菌在伊犁地区肉牛养殖环境和加工环节中的污染状况及其遗传多样性,为产业链中食源性致病性大肠杆菌的风险监测和控制提供基础数据。方法:采用传统方法和PCR方法对养殖环节的饲草料和粪便及屠宰环节的553份样品进行产志贺毒素大肠杆菌的污染调查,对分离鉴定的产志贺毒素大肠杆菌进行7种常见血清型(O145、O157、O45、O103、O111、O26、O121)的PCR检测和ERIC-PCR的基因分型。结果:检测553份样品中有39株编码志贺毒素基因,产志贺毒素大肠杆菌(STEC)的检测率是7.1%。常见血清型PCR检测中血清型O111有2株菌,检出率是5.1%;O145有5株菌,检出率是12.8%。ERIC-PCR基因分型产志贺毒素大肠杆菌有10种基因亚型,分成3簇,A簇有23株菌,相似性在59%~100%,表明这些菌株之间的亲缘关系较近。结论:伊犁地区肉牛粪便是产志贺毒素大肠杆菌的污染源,这些菌株的亲缘关系较近。     

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。     

Short communication: Detection of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle and pigs in Lima, Peru

The aim of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in cattle and pigs as a possible STEC reservoir in Lima, Peru. One hundred and fourteen cattle and 112 pigs from 10 and 4 farms, respectively, were studied. Five E. coli colonies per culture were studied by a multiplex real-time PCR to identify Shiga toxin-producing (stx1, stx2, eaeA), enterotoxigenic (lt, st), enteropathogenic (eaeA), enteroinvasive (ipaH), enteroaggregative (aggR), and diffusely adherent E. coli (daaD). Shiga toxin-producing E. coli were isolated from 16 cattle (14%) but none from pigs. stx1 was found in all bovine isolates, 11 of which also carried eaeA genes (69%); only 1 sample had both stx1 and stx2. Thirteen stx-positive strains were classified as Shiga-toxigenic (81%) using an enzymatic immunoassay, 2 STEC strains were from serogroup O157 (13%), and 7 were sorbitol negative (44%). Enteropathogenic E. coli were detected more frequently in cattle (18%, 20/114) than in pigs (5%, 6/112). To our knowledge, this is the first study on the prevalence of STEC in farms animals in Peru using molecular methods. Further studies are needed in a large number of farms to determine the relevance of these findings and its consequences for public health.     

Diversity of virulence profiles of Shiga toxin-producing Escherichia coli serotypes in food-producing animals in Brazil

The prevalence, serotypes and virulence profiles of Shiga toxin-producing Escherichia coli (STEC) were investigated in 205 healthy beef and dairy cattle, and 106 goats reared in the southeastern region of Minas Gerais State, Brazil. The prevalence of STEC was 57.5% (61/106) in goats, 39.2%, (40/102) in beef cattle and 17.5% (18/103) in dairy cattle. Among the 514 STEC isolates, 40 different serotypes were found and some of them were identified in a specific host. STEC isolates harboring stx(1) corresponded to 15.6% (28/180), 26.7% (16/60) and 24.1% (66/274) in beef cattle, dairy cattle and goats, respectively. stx(2) was found in 30% (54/180), 53.3% (32/60) and 34.7% (95/274) of beef and dairy cattle, and goats. stx(1) plus stx(2) sequences were harbored by 54.4% (98/180), 20% (12/60) and 41.2% (113/274) of beef cattle, dairy cattle and goats, respectively. The eae sequence was found in 15% (9/60) and 0.6% (1/180) of STEC isolates from dairy and beef cattle, respectively, and the toxB gene was found only in one O157:H7 strain isolated from beef cattle. Strains with the genetic profiles stx(2) ehxA iha saa and stx(1) stx(2) ehxA iha saa were the most prevalent among STEC isolates from cattle. Profiles stx(1) stx(2) ehxA iha, stx(2), and stx(1) iha accounted for 75.5% (207 /274) of the STEC isolates from goats. While STEC strains carrying either stx(2) alone or associated with stx(1) were found more frequently in cattle, those harboring sequences stx(1c) and stx(2d) alone or associated with stx(1c) predominated in goats. Our data show a diversity of STEC strains in food-producing animals, most of them carrying genes linked to severe forms of human diseases.     

Shiga toxin-producing Escherichia coli in ground beef and lamb cuts: results of a one-year study

Shiga toxin-producing Escherichia coli (STEC) have been associated with a broad spectrum of diarrhoeal syndromes. Some of these cases have been attributed to foods of bovine origin or other foods cross-contaminated by beef products or cow manure. The purpose of this study was to determine the pattern of STEC distribution in selected red meats over time. Samples of ground beef and lamb cuts were collected over a 52-week period from 31 different outlets and 25 g portions were assayed for STEC. STEC were isolated from 46/285 (16%) ground beef and 111/275 (40%) lamb samples using an stx PCR screen followed by colony hybridisation. All isolates were tested by PCR for additional STEC virulence markers with 95% of ground beef isolates shown to possess stx(2) and 80% of lamb cutlet isolates shown to possess stx(1) and stx(2). The enterohaemolysin gene (ehxA) was detected in 65% and 53% of ground beef and lamb isolates respectively. Putative enterohaemorrhagic E. coli (EHEC), i.e. STEC possessing the E. coli attaching and effacing gene (eae) were not isolated. The STEC isolates comprised 18 and 15 different serotypes from ground beef and lamb respectively. STEC of serotypes O157, O111 and O26 (common enterohaemorrhagic E. coli serotypes) were not isolated. Serotypes O174 and O91 were the most common serotypes isolated from ground beef samples and O128 and O91 the most common from lamb cutlet samples. The presence of STEC in retail red meats highlights the need for a clearer understanding of STEC in food and human illness to interpret the public health significance of these findings.     

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.     

Isolation of shiga toxigenic Escherichia coli from raw beef in Palestine

Shiga toxigenic Escherichia coli (STEC) isolated from raw beef samples in northern Palestine during a 1-year period were characterized for virulence genes by a polymerase chain reaction (PCR) assay and screened for their antibiotic resistance. STEC was identified in 44 (14.7%) of 300 raw beef samples. Twelve (27.3%) of the STEC isolates were serotype O157. Nine of those were isolated during summer. The majority of STEC isolates (70.5%) harbored both stx1 and stx2 genes, while the others harbored either stx1 or stx2. High levels of resistance against different antimicrobial agents were detected. Resistance to at least three drugs was found in 55% of the isolates.     

Prevalence of Shiga toxin-producing Escherichia coli in food products of animal origin as determined by molecular methods

In this study we report on the prevalence and distribution of Shiga toxin-producing Escherichia coli (STEC) in food products of animal origin, collected in the Piedmont region of Italy, as determined by a combination of quantitative PCR (qPCR) protocols, applied directly to the samples, and of culture-dependent isolation and subsequent molecular identification and characterization of isolates. The qPCR protocols were developed and optimized in this study and targeted the rpoB gene (as a marker for total E. coli) and the stx?, stx? and eaeA genes (as markers for potentially virulent E.coli). They were then used to test for STEC in 101 food samples, before and after enrichment. A STEC prevalence of 42% (21/50) for dairy products and 70% (36/51) for meat products was obtained. A total of 54 STEC isolates were recovered from dairy and meat samples, resulting in a prevalence of 36% and 27% in dairy and meat products, respectively, by the culture method. A large number of strains carried the stx? gene (39 out of the 54 STEC strains) compared to strains that carried stx? (30 out of 54); only 11 out of 54 strains contained the eaeA gene, while 14 strains contained both stx? and stx?. Eight of the 54 isolates belonged to the O157 serogroup, and none belonged to serogroups O26, O145, O111 or O103. Strains isolated from meat products were diverse, as determined by Enterobacterial repetitive intergenic consensus-PCR (ERIC), while those isolated from dairy products were more similar and grouped together by cluster analysis. The results of the qPCR approach showed a high prevalence of STEC in dairy and meat based products, mainly fermented, indicating a possible safety risk for these types of food commodities.     

Shiga toxin-producing Escherichia coli in healthy young beef steers from Argentina: prevalence and virulence properties

Between July 1999 and December 2000, the prevalence of Shiga toxin-producing Escherichia coli (STEC) was established in 200 Argentine healthy young beef steers (14-16 months old) grown under local production systems with a feed grain period of 3-4 months, and the STEC strains isolated were examined in regard to their phenotypic and genotypic characteristics. Stool samples (n = 70) and rectal swabs (n = 130) were taken at the slaughterhouse level. By polymerase chain reaction (PCR), Shiga toxin (stx) gene sequences were detected in 69% of the samples. Eighty-six STEC strains were isolated from 39% of the animals. Serogroups identified, in order of frequency, were: O8 (16 strains), O113 (14), O103 (5), O91 (4), O171 (3), O174 (3), O25 (2), O112 (2), O145 (2), O2, O11, O104, O121, O128, O143, O146, O157. The most frequent serotype isolated was O8:H19 (12.9%). A total of 17 serotypes, including E. coli O157:H7 found in one animal (0.5%), have been previously associated with hemolytic uremic syndrome (HUS), bloody and non-bloody diarrhea in different countries, including Argentina. The prevalent genotype isolated was stx2 (51 of 86, 59.3%). Subtyping of stx2 variants showed the prevalence of stx2vh-b (25.6%) and stx2vh-a types (24.4%), and revealed the presence of an atypical stx2-v. Only 7.0% of STEC strains carried eae, and 33.7% harbored EHEC-hlyA gene. The full virulent genotype (stx/eae/EHEC-hlyA) was found to be present in 4 of the 86 (4.7%) STEC strains isolated. This research indicates that young steers from the main beef-producing area of Argentina are an important reservoir of STEC strains; however, its importance as agents of human diseases in our country has still to be established.     

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.