大肠杆菌O157多克隆抗体及食品中双抗ELISA测定方法的研究

本研究获得了抗肠出血性大肠杆菌O157:H7的多克隆抗体 ,建立了一种适宜食品样品检测的双抗ELISA检测方法。该方法对纯培养菌液检出限为 10 3 ～ 10 4 cfu ml;只对O157菌株有特异性反应 ,对非O157菌株无交叉反应 ;经过增菌 ,鸡肉与牛奶染菌样品中的大肠杆菌O157的检出限均为 0 1cfu g(cfu ml)。     

Expression and putative roles in attachment of outer membrane proteins of Escherichia coli O157 from planktonic and sessile culture

Many strains of Shiga toxigenic Escherichia coli (STEC), particularly the serotype O157:H7, are foodborne pathogens causing disease in many countries throughout the world. E. coli O157:H7 is able to attach and survive on various surfaces such as stainless steel (SS) found within the food processing environment. We examined the outer membrane protein (OMP) profiles of four E. coli O157 (three toxigenic O157:H7 and one nontoxigenic O157:HR) and one non-STEC strain (O1:H7), previously reported to have different abilities to attach to SS following growth in planktonic (nutrient broth) and sessile (nutrient agar) culture. The OMPs of the five E. coli strains grown in planktonic and sessile culture were extracted using N-lauroyl sarcosine and the OMP profiles were separated using two-dimensional (2D) gel electrophoresis. Qualitative and quantitative variations in the total number of OMPs expressed between planktonic and sessile cultures were found for all E. coli isolates tested. A number of differentially expressed protein spots were selected from 2D gels and were identified. FlgE was found to be expressed in planktonic culture but not sessile culture. MipA and OmpX had higher expression in sessile culture than planktonic culture, while expression of OmpA did not differ between E. coli strains or between the two modes of growth. Although differential expression of OMPs was found between isolates grown in planktonic and sessile culture, further investigations are required to determine a role of some of these identified proteins during growth of E. coli in planktonic and sessile culture and their influence during the attachment process.     

A one year study of Escherichia coli O157 in raw beef and lamb products

Between April 1996 and March 1997 we examined 5093 samples of raw beef and lamb products for the presence of E. coli O157. Samples were purchased from 81 small butchers' shops in south Yorkshire. In March 1997 we also examined five samples of dried mint for the presence of E. coli O157. Strains of E. coli O157 were isolated by enrichment culture in modified buffered peptone water followed by immunomagnetic separation and culture of magnetic beads onto cefixime tellurite sorbitol MacConkey agar. Strains were characterized by phage typing, toxin genotyping and plasmid analysis. Strains of E. coli O157 were isolated from 72 (1.4%) of 5093 samples; it was isolated from 36 (1.1%) of 3216 samples of beef products and from 29 (2.9%) samples of lamb products. The highest prevalence was found in lamb sausages and lamb burgers where E. coli O157 was isolated from 3 (4.1%) of 73 and 18 (3.7%) of 484 samples respectively. Strains of E. coli O157 were isolated most frequently during early summer. Strains of E. coli O157 were also isolated from 2 of 5 samples of dried mint although we did not determine how the mint had become contaminated. All isolates of E. coli O157 were Verocytotoxin-producing as determined by both Vero cell assay and DNA hybridization for the genes encoding Verocytotoxin and all were positive for the eaeA gene. A combination of phage typing, toxin genotyping and plasmid profile subdivided the 72 strains of E. coli isolated into 20 different subtypes, of which 18 were indistinguishable from strains isolated previously from cattle and sheep; of these 18 strains, 8 were indistinguishable from strains isolated from human cases of infection during the study period.     

大肠埃希菌O157:H7分离鉴定过程中赫尔曼埃希菌的鉴别

目的：鉴别大肠埃希菌O157：H7和赫尔曼埃希菌。方法：观察菌株在山梨醇麦康凯，营养琼脂及Chromagar O157培养基上菌落形态。生化试验检测菌株的生化特性，血清凝集试验检测菌株的O157和H7抗原，聚合酶链反应法检测O157和H7特异性基因。结果：在营养琼脂培养基上，5株赫尔曼埃希菌均产黄色色素，2株O157：H7菌不产色素；在Chromagar O157培养基上，2株O157：H7菌株呈紫红色，2株赫尔曼埃希菌株呈蓝色，其余3株赫尔曼埃希菌株呈黄绿色。O157：H7菌株KCN试验均为阴性，而赫尔曼埃希菌阳性。O157和H7抗血清坡片凝集试验，7析均为强凝集。O157：H7菌株与O157单克隆抗血清玻片凝集试验均为强凝集，而赫尔曼埃希菌均不凝集。O157：H7菌株O157和H7特异性基因均为阳性，赫尔曼埃希菌均为阴性。结论：赫尔曼埃希菌与O157多克隆抗血清有交叉反应，但单克隆抗血清能区分O157：H7和赫尔曼埃希菌。KCN试验和特异性基因检测亦能鉴别这两种菌。在营养琼脂和Chromagar O157培养基上的菌落形成也有助于两菌的区分。     

Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples.

Minced beef samples inoculated with Escherichia coli O157 were cultured in buffered peptone water supplemented with vancomycin, cefsulodin and cefixime (BPW-VCC) and subcultured to cefixime tellurite sorbitol MacConkey (CT-SMAC) agar both directly and after immunomagnetic separation (IMS) of the organism with magnetic beads coated with an antibody against E. coli O157 (Dynabeads anti-E. coli O157, Dynal, Oslo). E. coli O157 was recovered from initial inocula of 200 organisms/g by direct subculture and 2 organisms/g by IMS. Twelve strains of E. coli O157 of different combinations of phage type, H antigen and toxin genotype were all recovered from initial inocula of two organisms/g by IMS. Non-specific binding of other organisms to the magnetic beads could be reduced by washing of the beads in PBS with Tween-20 0.002-0.005% E. coli O157 was not bound by magnetic coated with an unrelated antibody. During investigation of a dairy herd that was possibly linked to a small outbreak of infection with E. coli O157, the organism was isolated from 2 of 279 forestream milk samples from individual cattle; both isolates were made only by the IMS technique. IMS is rapid, technically simple, and a specific method for isolation of E. coli O157 and will be useful in epidemiological studies.     

The serological relationship between Escherichia coli O157 and Yersinia enterocolitica O9 using sera from patients with brucellosis.

Sera from ten patients with positive brucella serology were used to investigate antibody cross-reactions between the O-antigens of Escherichia coli O157 and Yersinia enterocolitica O9. SDS-PAGE profiles of lipopolysaccharide (LPS), purified from strains of E. coli O157 and Y. enterocolitica O9, were reacted with sera by immunoblotting. All ten sera contained antibodies which bound to the LPS of E. coli O157, and five of these sera also contained antibodies which bound to the LPS of Y. enterocolitica O9. Absorption studies using these five cross-reacting sera indicated the existence of at least three epitopes exposed on the O-antigens of E. coli O157 and Y. enterocolitica O9. One antigen binding site appeared to be exposed on the LPS of both organisms, while one epitope was exposed on the LPS of E. coli O157 only, and another on the LPS of Y. enterocolitica O9 only.     

O157血清群大肠杆菌分子亚型的研究

目的：了解山东和福建两省不同来源的O157血清群大肠杆菌分子亚型和菌株间的相关性。方法：对1993－1996年间分离到菌株进行脉冲场电泳（PFGE），图象经计算机处理后进行聚类分析。结果：O157大肠杆菌染色体DNA用XbaI酶切后，经PFGE可生15－20条带，以90％的同源性作为界值，可将11株菌分成9个PFGE亚型，其中山东分离到的6株菌分成4个亚型，福建的5株菌分属5个亚型。结论：山东、福建的O157血清群大肠杆菌均存在多个克隆系。     

Isolation and molecular characterization of Escherichia coli O157 from broiler and human samples

There is a lack of information about the role of poultry, specifically chicken, in transmission of Escherichia coli (E. coli) O157 and subsequent human illnesses. This study was therefore aimed at investigating the presence of E. coli O157 and its virulence genes in various samples collected from broiler chickens and humans in Eastern Turkey by culture, immunomagnetic separation (IMS), and polymerase chain reaction (PCR). The genetic relationship between broiler and human isolates was also examined by pulsed-field gel electrophoresis (PFGE). In the PCR analysis of sorbitol-negative isolates, E. coli O157 was identified in 0.1% (1/1000) and 0.4% (4/1000) of the liver and cecum samples of broiler chickens, respectively. On the other hand, none of the carcass samples were determined to be positive for E. coli O157. Overall, the results indicated that 12% (3/25) of the flocks were positive for E. coli O157. The differences between the flocks in terms of the positivity were determined to be statistically significant (p<0.001). Ten (2.7%) of 367 human stool samples were also positive for E. coli O157 in the PCR examination. None of the broiler and human E. coli O157 isolates possessed H7, shigatoxins 1-2, or enterohemolysin genes, whereas all the broiler isolates and one of the human isolates were positive for intimin gene. In the PFGE analysis, a total of eight different profiles (four from broiler and four from human isolates) were observed. However, there were no genetic relationships between broiler and human E. coli O157 isolates. It can be concluded that more detailed studies are needed in poultry to better understand the role of these species in the epidemiology of E. coli 0157 infections in humans.     

Identification of Escherichia coli O157 by using a novel colorimetric detection method with DNA microarrays

Shiga toxin-producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73?±?7.12 to 76.71?±?8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100-1000 CFU/mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains.     

Escherichia coli O157 cluster evaluation

We investigated a multistate cluster of Escherichia coli O157:H7 isolates; pulsed-field gel electrophoresis subtyping, using a single enzyme, suggested an epidemiologic association. An investigation and additional subtyping, however, did not support the association. Confirmating E. coli O157 clusters with two or more restriction endonucleases is necessary before public health resources are allocated to follow-up investigations.     

A 1-year study of Escherichia coli O157 in cattle,sheep, pigs and poultry.

Samples of rectal faeces were collected immediately after slaughter from 400 cattle each month for a 1-year period and from 1000 each of sheep, pigs and poultry over the same period. Samples were examined for Escherichia coli O157 by enrichment culture in buffered peptone water with vancomycin, cefixime and cefsulodin followed by immunomagnetic separation and culture of magnetic particles onto cefixime tellurite sorbitol MacConkey agar. E. coli O157 was isolated from 752 (15.7%) of 4800 cattle, 22 (2.2%) of 1000 sheep and from 4 (0.4%) of 1000 pigs, but not from any of 1000 chickens. Of the cattle sampled. 1840 (38.4%) were prime beef animals, 1661 (34.6%) were dairy animals being culled and the status could not be determined for the other 1299 (27%) animals. E. coli O157 was found in 246 (13.4%) of the 1840 beef cattle and 268 (16.1%) of the 1661 dairy cattle. The monthly prevalence of E. coli O157 in cattle was 4.8-36.8% and was at its highest in spring and late summer. Seventeen of the 22 isolates from sheep were also made over the summer period. All E. coli O157 isolates from sheep and 749 (99.6%) of the 752 E. coli O157 isolates from cattle were verocytotoxigenic as determined by Vero cell assay and DNA hybridization, eaeA gene positive, contained a 92 kb plasmid and were thus typical of strains causing infections in man. In contrast isolates from pigs were non-toxigenic, eaeA gene negative and did not contain a 92 kb plasmid and would, therefore, be unlikely to be a source of infection for man.     

Community-wide outbreak of Escherichia coli O157:H7 associated with consumption of frozen beef burgers

On 24-25 October 2005 a cluster of five haemolytic uraemic syndrome (HUS) cases was reported in southwest France. An investigation was undertaken to identify the outbreak source and implement control measures. Cases were defined as individuals with HUS or diarrhoea with isolation of Escherichia coli O157:H7 in stools or a positive antibody response to E. coli O157 lipopolysaccharide, resident in southwest France with symptom onset after 19 September 2005. Sixty-nine identified patients had symptom onset between 5 October and 3 November 2005, including 17 cases of HUS. One brand of frozen beef burgers produced on 22 August 2005 was consumed by all patients in the week before symptom onset. E. coli O157:H7 strains from patients, patients' burgers and the manufacturing plant were genetically related. This is the largest community-wide outbreak of E. coli O157:H7 in France to date and the first associated with consumption of contaminated frozen beef burgers.     

Antimicrobial susceptibility and mechanism of resistance to antibiotics for Escherichia coli O157 and verotoxin-producing E. coli isolated in northern Kyushu and Yamaguchi area

The purpose of this study was to investigate the biochemical characteristics and antimicrobial susceptibility of Escherichia (E.) coli O157 and verotoxin-producing E. coli isolates from the Northern Kyushu Island and Yamaguchi area of Japan. A total of 54 isolates- 50 E. coli O157, 3 verotoxin-producing E. coli O26 and 1 verotoxin-producing E. coli O111 - were used in this study. Regarding H antigen, H7 type in E. coli O157 accounted for 98% (49/50), and residual 1 strain of E. coli O157 was untypable H type. Two of 3 E. coli O26 isolates were H11 type, residual 1 strain of E. coli O26 was untypable H type, and E. coli O111 isolate was non-motile strain. All 54 isolates were susceptible to cephems, fosfomycin, kanamycin, amikacin and co-trimoxazole. Tetracycline-resistant isolates existed in 13 of all 54 isolates, 5 of those 13 isolates had tetA, and the other 7 isolates had tetB. Eight amoxicillin-resistant isolates had TEM-1 beta-lactamase. Four of the 5 isolates that had tetA also had TEM-1 beta-lactamase. Nalidixic acid and 6 fluoroquinolone used had no insensitive or resistant isolates. Kanamycin-resistant isolates, fosfomycin- and nalidixic acid-insensitive isolates have been reported, so we must notice the antibiogram of such strains. It is important that the surveillance of antimicrobial susceptibility of enterohemorragic E. coli should be continued after this.     

2007年浙江省衢州地区动物中产志贺毒素大肠埃希菌O157:H7感染状况

目的:了解2007年浙江省衢州地区产志贺毒素大肠埃希菌O157:H7在动物中的分布情况及其耐药性、PFGE分型及毒力基因携带状况。方法:按全国O157:H7监测方案于5~10月份肠道传染病高发季节,在衢州地区采集各种动物粪便/肛拭,用免疫磁珠富集后进行O157:H7分离培养、鉴定,可疑菌株以PCR法检测O、H抗原及志贺样毒素(SLT1和SLT2)、粘附抹平因子(eaeA)及溶血素(hly)4种毒力基因。用脉冲场凝胶电泳(pulse field gel electrophoresis,PFGE)方法进行同源性分析,同时选择14种抗生素进行药敏试验,分析分离所得菌株的耐药状况。结果:共监测动物粪便标本300份,分离得产志贺毒素大肠埃希菌O157:H7菌株16株,分离率为5.33%。16株O157:H7菌株,毒力基因Hly、eaeA、SLT2均阳性,SLT1均阴性。脉冲场凝胶电泳分型显示,16株O157:H7菌株可分2个PFGE基因型,型间差异较小。耐药性分析显示这些菌株对红霉素、利福平的耐药率最高,达100.0%,对其他受试抗生素均敏感。结论:该地区动物中产志贺毒素大肠埃希菌O157:H7带菌率较高,所分离菌株主要携带SLT2基因,因此推测该地区存在发生产志贺毒素大肠埃希菌O157:H7感染暴发或流行的潜在危险,需增加对动物源性O157:H7的监测力度。     

福建省O157大肠杆菌初查

１９９７年４－７月在莆田、福州、霞莆等地区抽抽查部分人群及家养畜禽粪便等，首次在猪、鸭、羊、鸡、鸽等５种动物中检出３种不同类型的Ｏ１５７大肠杆菌，其中Ｏ１５７：Ｈ７与Ｏ１５７：ＮＭ各８株，Ｏ１５７：Ｈ？５株，同时在１位腹泻患儿中分离到此菌。本文不这些Ｏ１５７分离菌株的生物学特性包括形态、生化，致病性以及药物敏感性等进行了实验，发现不同来源、不同类型的Ｏ１５７大肠杆菌都可致使小鼠发病死亡。在防治工作     

The use of sorbitol-MacConkey agar in conjunction with a specific antiserum for the detection of Vero cytotoxin-producing strains of Escherichia coli O 157

Using DNA probes specific for the genes encoding Vero cytotoxins 1 and 2 in hybridization experiments on faecal samples, Vero cytotoxin-producing Escherichia coli (VTEC) of serogroup O 157 were detected in 21 of 63 cases of haemorrhagic colitis, 9 of 31 cases of non-bloody diarrhoea and 14 of 68 cases of haemolytic uraemic syndrome. Compared with these results sorbitol-MacConkey agar in conjunction with a specific O 157 antiserum gave a sensitivity of 62% in haemorrhagic colitis, 56% in non-bloody diarrhoea and 57% in haemolytic uraemic syndrome. The specificity of this method was 100% in all three groups. This demonstrates that sorbitol-MacConkey agar is a useful screening method for the detection of VTEC of serogroup O 157 when used in conjunction with a specific homologous antiserum. However, this method does not detect VTEC belonging to other serogroups and such strains were found, particularly in cases of haemolytic uraemic syndrome.     

Extended phage-typing scheme for Escherichia coli O157:H7

In Canada, the number of human isolates of verotoxigenic (VT + ve) Escherichia coli O157:H7 from diarrhoeal cases and haemolytic uraemic syndrome and haemorrhagic colitis has increased from 25 in 1982 to 2384 in 1989. A total of 3273 VT + ve E. coli O157:H7 strains (3255 strains isolated in Canada and 18 isolates from other countries) were phage typed. The phage typing scheme has been extended from 14 to 62 phage types. Of these, five types occurred exclusively in other countries (type 47 in Japan; and types 49, 50, 51 and 52 in the U.K.). Thirty-five different phage types were identified in Canada; only nine of these (1, 2, 4, 8, 14, 21, 23, 31 and 32), each accounted for more than 1% of the cases from human sources. The same nine types were the only ones observed among the isolates from non-human sources (meat and slaughter houses) suggesting a food-borne transmission in most of the human cases. Phage types 1 (30.5%); 4 (21%); 8 (13.5%); 31 (8.9%) and 14 (8%) were encountered in varying frequencies in most of the provinces; infrequently occurring phage types also showed regional variation. Thirteen different phage types were identified among 151 outbreaks representing 556 isolates of E. coli O157:H7. More than one phage type were encountered in 12 outbreaks whereas in 141 outbreaks, all strains in each, had the same phage type.     

福建省O157大肠杆菌调查

目的 探索Ｏ１５７大肠杆菌在福建省人兽及食物中的存在情况,并了解这些检出菌的生物学特性。方法 １９９７～１９９８年选莆田、福州等地检查腹泻患粪便、畜食粪便标本及肉类标本２７２５份,以血清学方法检测Ｏ１５７大肠杆菌。结果 先从猪、鸽、牛、鸡、鸭等动物及腹泻中检出７６株Ｏ１５７大肠杆菌。据血清学及动力试验结果分类,３３株Ｏ１５７：Ｈ７,２１株Ｏ１５７：ＮＭ,２２株Ｏ１５７：Ｈ？,其中以猪检出率最高     

Multidrug resistance and plasmid patterns of Escherichia coli O157 and other E. coli Isolated from diarrhoeal stools and surface waters from some selected sources in Zaria, Nigeria

We have assessed the prevalence of Escherichia coli O157 in diarrhoeal patients and surface waters from some selected sources in Zaria (Nigeria), evaluating the antibiotic susceptibility and plasmid profiles of 184 E. coli isolates, obtained from 228 water samples and 112 diarrhoeal stool specimens (collected from children aged <15 years), using standard methods. The detection rate of E. coli O157 in surface waters was 2.2% and its prevalence in children with diarrhoea was 5.4%. The most active antibiotics were gentamicin, chloramphenicol and fluoroquinolones. Seventy-nine (42.9%) of 184 E. coli isolates were resistant to four or more antibiotics. Multidrug resistance (MDR) was higher amongst aquatic isolates than the clinical isolates. Out of 35 MDR isolates (20 of which were O157 strains), 22 (62.9%) harboured plasmids all of which were no less than 2.1 kb in size. Amongst the 20 E. coli O157 strains, only seven (35.0%) contained multiple plasmids. An aquatic O157 isolate containing two plasmids was resistant to seven drugs, including ampicillin, cefuroxime, ciprofloxacin, cotrimoxazole, nalidixic acid, nitrofurantoin and tetracycline. Loss of plasmid correlated with loss of resistance to antibiotics in cured (mutant) strains selected in tetracycline (50 μg/mL)-nutrient agar plates. Our findings revealed that plasmids were prevalent in both the aquatic and clinical isolates, and suggest that the observed MDR is plasmid-mediated. The occurrence of plasmid-mediated multidrug resistant E. coli O157 in surface waters used as sources for drinking, recreation and fresh produce irrigation heightens public health concern.     

Escherichia coli O157 in the rectoanal mucosal region of cattle

The rectoanal junction mucosal region is the site of colonization of Escherichia coli O157 in cattle. Our objective was to determine the genetic relatedness of E. coli O157 in the mucosa of the rectoanal junction to isolates from colon contents and feces. Colon contents and rectums were collected from cattle at harvest. Rectums were opened and feces were sampled with a cotton swab. The mucosa of the rectum was cleansed free of visible feces with water and saline. The region, 2 to 5 cm proximal to the rectoanal junction, was swabbed with a foam-tipped applicator and then incisions were made in this region and the submucosa was swabbed with an applicator. Isolation and identification of E. coli O157 was performed in accordance with well-documented methods. Prevalence of E. coli O157 in the colon contents, feces, rectal mucosa, and rectal submucosa was 21%, 29%, 54%, and 34%, respectively. Pulsed-field gel electrophoresis was used to compare clonal similarity among isolates from different sampling regions. Sixty-seven cattle had E. coli O157 isolated from the rectal mucosa swab and feces of which 82% were clonally similar (dice similarity >95%) within animal. Escherichia coli O157 isolates from feces and colon contents were similar in 76% of cattle, but E. coli O157 isolates from the rectoanal mucosal swab and colon contents were only similar in 61.4% of cattle. Our results suggest that E. coli O157 in the feces may be from two sources, colonized in the rectoanal mucosa or transient in the gastrointestinal tract.