Shiga toxin-producing Escherichia coli and their bacteriophages as a model for the analysis of virulence and stress response of a food-borne pathogen

Enterohemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing Escherichia coli (STEC) that are able to cause serious food-borne intestinal diseases which can be followed in 5 to 15% by extraintestinal sequelae such as the hemolytic-uremic syndrome (HUS). One of the major pathogenicity factors of EHEC is the production of one or more Shiga toxins (Stx), which act as inhibitors of protein biosynthesis and have profound effects on the signal transduction and immunological response in eukaryotic cells. The stx genes are located in the genome of heterogeneous, lambdoid, functional or cryptic bacteriophages and are expressed during the phage life cycle. Due to the linkage between the phage life cycle and stx expression, STEC and their bacteriophages are useful as a model for the analysis of stress response and virulence of this food-borne pathogen.     

Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission

Enterohaemorrhagic Escherichia coli (EHEC) constitute a subset of serotypes (E. coli O157 and some other serogroups) of Shiga toxin (Stx)-producing E. coli (STEC) firmly associated with severe human illnesses like bloody diarrhoea and haemolytic uraemic syndrome. Stx production is essential but not sufficient for EHEC virulence. Most strains are capable of colonising the intestinal mucosa of the host with the "attaching and effacing" mechanism, genetically governed by a large pathogenicity island (PAI) defined as the Locus of Enterocyte Effacement. Other virulence factors carried by mobile genetic elements like PAI and plasmids have been recently described, and their role in the pathogenic process has not been fully elucidated. EHEC are zoonotic pathogens. They rarely cause disease in animals, and ruminants are recognised as their main natural reservoir. Cattle are considered to be the most important source of human infections with EHEC O157, and the ecology of the organism in cattle farming has been extensively studied. The organism has also been reported in sheep, goats, water buffalos, and deer. Pigs and poultry are not considered to be a source of EHEC and the sporadic reports may derive from accidental exposure to ruminant dejections. The epidemiology of EHEC infections has remarkably changed during the past ten years and an increasing number of unusual food vehicles have been associated with human infections. New routes of transmission have emerged, like contact with animals during farm visits and a wide variety of environment-related exposures. As for other zoonotic agents, having animals and raw products that are free from EHEC is not possible in practice. However, their occurrence can be minimised by applying high standards of hygiene in all the steps of the food production chain.     

Study on the Virulence Gene Distribution and Genetic Evolution of Cattle Shiga Toxin-producing Escherichia coli Isolates

This study was aimed to understand the relationship of virulence gene distribution and genetic evolution between cattle originated Shiga toxin-producing Escherichia coli (STEC) and human originated enterohaemorrhagic Escherichia coli (EHEC) O157. This experiment collected 18 strains STEC in a dairy farm from Jiangsu province and 9 STEC reference strains (human, sheep, swine and avian), according to the method of U.S. Centers for Disease Prevention and Control Center (PulseNet), using the XbaⅠ enzyme digestion and pulsed field gel electrophoresis (PFGE) analysis, virulence genes were detected in some STEC isolates. The virulence gene distribution of O157 from different origin was remarkably different. The cattle originated STEC O157 and the human originated EHEC O157:H7 (EDL933W) had the most similar virulence gene distribution. In contrast, virulence genes were lack in cattle STEC O18 and O26, even though the cattle STEC O18 and O26 had the similar genotype as human EHEC O157:H7 (EDL933W). PFGE of Xba Ⅰ digested chromosomal DNA from 27 isolates of STEC exhibited 22 profiles. In general,the Dice coefficients of different originated STEC ranged from 72% to 100%.Cattle STEC O157 had a high similarity with two strains of human originated EHEC O157, while a low similarity was demonstrated between cattle STEC O157 and STEC O157 of swine and avian. The Dice coefficients of the cattle STEC O157 and the two strains of human EHEC O157 ranged from 83% to 95%. The Dice coefficients of cattle STEC O26 (Ⅶ,Ⅷ) and the two strains of human EHEC O157 were more than 82%. Therefore, it was concluded that the cattle STEC O157 and human EHEC O157 had a closer relationship in terms of virulence gene distribution and in genetic evolution.     

牛源产志贺毒素大肠杆菌分离株的毒力基因分布和遗传进化分析

为了探讨牛源产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli,STEC）分离株在毒力基因分布和遗传进化方面与人源EHEC O157菌株之间的关系,本试验选择收集来自江苏某奶牛场的STEC菌株18株以及人源、羊源、猪源、禽源STEC参考菌株9株,参照美国疾病预防控制中心PulseNet推荐的方法,运用XbaⅠ酶进行酶切并完成脉冲肠凝胶电泳（PFGE）分型和聚类分析;同时对部分STEC菌株进行毒力基因检测。结果表明,经毒力基因检测,不同来源的O157菌株毒力基因分布不尽相同,其中牛源STEC O157与参考株EHEC O157∶H7（EDL933W）的基因排谱最为相近;牛源STEC O18和O26的基因排谱与参考株EHEC O157∶H7（EDL933W）类似,但存在部分基因的缺失。对27株不同来源的STEC分离株进行PFGE,产生了22种不同的酶切图谱。总体来看,不同来源的STEC Dice相似性系数在72%~100%之间。牛源O157分离株与猪源及禽源O157菌株的相似度偏低,而与两株人源O157分离株的相似度偏高,Dice相似性系数在83%~95%之间,牛源O26（克隆群Ⅶ、Ⅷ）与人源O157的相似性系数 > 82%。显然,从牛群中分离到的部分STEC菌株与人源EHEC O157具有较近的遗传进化关系。     

Shiga/verocytotoxins and Shiga/verotoxigenic Escherichia coli in animals.

Vero/Shiga toxins (VT/Stx) have an A-B structure: the A subunit carries the enzymatic activity and the B subunit binds the toxin to the membrane receptor (Gb3 or Gb4). The VT/Stx inhibit protein synthesis in the target eucaryotic cells, mainly the endothelial cells of blood vessels. The VT/Stx are subdivided into two families. VT1/Stx1 is a homogeneous family of toxins identical to the Stx of Shigella dysenteriae. VT2/Stx2 is a more heterogeneous family of toxins more distantly related to this Stx toxin. The VT2/Stx2 variants can be distinguished by the polymerase chain reaction (PCR) and/or the reaction with monoclonal antibodies. The VT/Stx-producing Escherichia coli are also subdivided into two main groups on the basis of the presence or absence of additional properties: the enterohaemorrhagic E. coli (EHEC) induce the formation of attaching/effacing lesions and carry a 60 MD plasmid encoding a specific haemolysin (the enterohaemolysin); the vero/shiga-toxigenic E. coli (VTEC/STEC) do not show these properties. The EHEC are isolated from humans and ruminants, especially young calves. They are associated with haemorrhagic enterocolitis and its sequelae in humans, the haemolytic-uraemic syndrome (HUS). The VT/Stx play a role in the occurrence of blood in the faeces and in the HUS by their action on the endothelial cells of blood vessels in the intestinal submucosa and in the renal glomeruli, after resorption through the intestinal walls. The VTEC/STEC are isolated from piglets, calves and humans. In recently weaned piglets, they cause the oedema disease, an enterotoxaemia characterized by subcutaneous, mesenteric and cerebral oedemas, with nervous disorders as main clinical signs. The oedema disease is the consequence of the action of the VT/Stx on the endothelial cells of blood vessels in various organs. In calves and humans, the role in disease of VTEC/STEC is controversial, but they could be associated with some cases of diarrhoea and HUS. The case of the O157:H7 EHEC which are present in healthy cattle of various ages, but are highly virulent for humans is of special interest. The potential zoonotic aspect of VT/Stx-producing E. coli infections in animals is detailed chapter by chapter. Prophylaxis of these infections by vaccination is the subject of the discussion on the future of the research studies on these pathogenic bacteria.     

Virulence and fitness gene patterns of Shiga toxin-encoding Escherichia coli isolated from pigs with edema disease or diarrhea in Germany

Fecal Escherichia coli isolates (n = 3,218) from piglets with edema disease or diarrhea were screened for the genes of Stx2 and Stx2 variants. A total of 283 E. coli isolates (8.8%) proved exclusively positive for Stx2e and most of these (85.1%) harbored genes for F18 fimbria. No recognized adhesins were detectable in 14.5% of the isolates. Genes for heat-stable or heat-labile E. coli enterotoxins were found in F18+ as well as F18 isolates (51.9% and 33.3%, respectively). Five isolates also harbored fyuA and irp2 genes which are indicative of a high pathogenicity island in E. coli. All Stx2e+ isolates lacked genes for intimin, EHEC hemolysin, STEC autoagglutinating adhesin, subtilase cytotoxin, serine protease Espl. The majority of Stx2e+ isolates belonged to phylogenetic groups A (59.3%) and D (38.9%) and only few isolates were classified as B1 and B2 (1.8%). The results suggest that Stx2e-producing E. coli strains are highly prevalent in diseased pigs in Germany. Despite their significant diversity, most strains possess all typical features (Stx2e, F18) of porcine edema disease E. coli. However, a considerable portion of porcine strains resembles published human Stx2e+ strains in that they lack any recognized pig-associated adhesin. Thus, a zoonotic potential cannot be excluded for these strains.     

Animals as a source of infections for humans--diseases caused by EHEC]

EHEC (enterohaemorrhagic E. coli) bacteria are new, only since 1982 recognized zoonotic pathogens. EHEC differ from E. coli intestinal commensales by the fact that they are lysogenic infected with bacteriophages, which carry the genetic information for the production of shigatoxins (Stx type 1 and/or 2). Due to the obligatory released Stx EHEC are classified also among the Shigatoxin producing E. coli (STEC). EHEC are capable of causing a Hemorrhagic Colitis and some sequelae of diseases such as the haemolytic uraemic syndrome. Due to their virulence factors they can be divided into typical and non-typical EHEC. Typical EHEC possess a pathogenicity island (Locus of Enterocyte Effacement) harboring genes, which apart from the characteristic necrotic activity of Stx enable the pathogens to closely attach to the epithelial cells of the intestinal mucosa and to destruct the microvilli. Additionally a so-called virulence plasmid codes for the production of a haemolysin, a peroxidase-katalase, an enterotoxin as well as a serine protease. EHEC are one of the world-wide most important causes of foodborne infections. Depending upon the country, most of the incidences in 1998 varied between 1 to 3 cases per 100,000 inhabitants. Since EHEC are only notifiable in a few countries, one must count however on substantially higher numbers. In Germany the estimated incidence is about 13 cases per 100,000 inhabitants. Since the first EHEC outbreaks were recognized in humans, studies investigating the prevalence of EHEC within animals were repeatedly performed. From the outset one assumed that cattle are a possible reservoir. Actually EHEC were isolated from fecal samples world-wide (typical and non-typical EHEC) from a large percentage of cattle (> 50%). Besides EHEC were isolated sporadically from fecal samples of other animals and healthy humans. The EHEC bacteria are shed by infected humans and animals, in particular by infected ruminants. They are spread over manure, slurry, sewage etc. Humans can get infected directly by contact with infected persons or animals or indirectly by contaminated food, water etc. The clinical outcome within humans appears as aqueous to bloody diarrhea. Beyond that approximately 5 to 10% of the patients develop the haemolytic uraemic syndrome. In contrast to humans, animals are mostly infected clinically inapparent. The therapy is based upon a symptomatic treatment. At present in man the control of EHEC infections concentrates on a particularly strict hand hygiene after the contact with infected humans and animals (above all ruminants). Since EHEC are heat sensitive, the prophylaxis by sufficient heating of risk food (raw milk, ground beef) is of special importance. In veterinary medicine above all EHEC infections must be controlled in ruminants, which are the primary reservoir. Due to the wide spread of EHEC in the ruminant population it is not realistic to demand an EHEC free cattle stock. Since EHEC are spread only via fecal excretion, at present it is most important to reduce the fecal shedding and to avoid fecal contamination of food of animal origin. In detail prophylactic hygienic measures concerning the farm management, the feeding hygiene, the food hygiene, the meat hygiene as well as the food hygiene are available.     

Occurrence and virulence patterns of E. coli O26, O103, O111 and O145 in slaughter cattle

The study attempted to investigate the occurrence of non-O157 E. coli serogroups O26, O103, O111 and O145 in cattle at slaughter and to determine the virulence potential of these isolates. A total of 399 fecal samples were analyzed by selective plating and E. coli isolates were characterized by polymerase chain reaction (PCR) for the genes vtx1, vtx2, eae and EHEC hlyA. Immunomagnetic separation (IMS) is required to increase the efficiency of the isolation procedure. E. coli O26, O103, O111 and O145 were recovered from 24 (6%) fecal samples. E. coli O26 and O103 seemed to be more abundant in slaughter cattle than E. coli O111 and O145. Sixteen out of the 24 isolates harbored vtx genes. All vtx-positive isolates harbored one or more additional virulence factors. Six out of the 8 vtx-negative isolates harbored eae and/or EHEC hlyA, whereas 2 strains harbored none of the tested virulence genes.     

Detection and characterization of verocytoxin-producing Escherichia coli types (VTEC) from different sources]

Hemolytic uremic syndrome (HUS) is caused by enterohemorrhagic E. coli (EHEC) belonging to a few serovars embracing strains of O26, O103, O111, O118, O145 and O157 serogroup, respectively. In own investigations 3.791 food specimen of animal origin were investigated by use of an enzyme-immuno-assay (EIA) and the polymerase chain reaction (PCR). All E. coli isolates (n = 459) of food as well as isolates from cattle feces (n = 440), from HUS patients (n = 50) and asymptomatic human carriers (n = 16) were investigated by means of the PCR using primer pairs for verocytotoxin genes (vtx1, vtx2, vtx2c, vtx2d, vtx2e), the E. coli attaching und effacing gene (eae), the enterohemolysin-gene (ehlyA) and the vt2 transporter protein-gene (ile X tRNA). Differences were found in respect to the eae- and the ile X tRNA genes, which could be detected in significantly higher ratios in the isolates from patients and human carriers. Furthermore vtx2d strains were exclusively analyzed to each 25% in food and cattle strains. In six food samples pathogenic strains of serovar O157 were detected whereas some of the cattle strains were estimated to belong to EHEC serovars O26:H11, O118:H- and O157:H7. The own data support the thesis that the risk for human beings is affiliated to a great extent by direct contact with ruminants, followed by person-to-person transmission. Regarding the epidemiological data the thesis that each VTEC strain ist potentially an EHEC strain can not longer be substantiated.     

Pigeons as a possible reservoir of Shiga toxin 2f-producing Escherichia coli pathogenic to humans

Escherichia coli harboring stx2f which secrete the respective Shiga toxin (Stx) are frequently found in pigeons. In this report we describe the isolation of a stx2f-containing E. coli O128 strain from an 11-month old child with diarrhea and comparison of this strain with stx2f-positive E. coli isolates from droppings of pigeons. The human E. coli O128:NM (nonmotile) isolate had a fliC restriction fragment length polymorphism pattern identical to that in one of the pigeon isolates belonging to the serotype O128:H2. All isolates examined, including that from the patient and five from pigeons, contained the intimin-encoding eae gene in addition to stx2f and all of the strains possessed the gene encoding the major subunit of the long polar fimbriae in enterohemorrhagic E. coli (EHEC) 026. Plasmid-associated virulence genes such as EHEC-hlyA, as well as urease and tellurite resistance-encoding operons were absent from all the strains and this correlated with their lack of hemolytic activity and urease production and tellurite sensitivity. These features, together with the sorbitol fermentation phenotype of Stx2f-producing E. coli, hamper the laboratory diagnosis of these strains. Our data demonstrate that pigeons may be a reservoir of Stx2f-producing E. coli strains associated with human disease.     

Plasticity of bacterial genomes: pathogenicity islands and the locus of enterocyte effacement (LEE)

Many bacterial virulence attributes, like toxins, adhesins, invasins, iron uptake systems, are encoded within specific regions of the bacterial genome. These in size varying regions are termed pathogenicity islands (PAIs) since they confer pathogenic properties to the respective micro-organism. Per definition PAIs are exclusively found in pathogenic strains and are often inserted near transfer-RNA genes. Nevertheless, non-pathogenic bacteria also possess foreign DNA elements that confer advantageous features, leading to improved fitness. These additional DNA elements as well as PAIs are termed genomic islands and were acquired during bacterial evolution. Significant G+C content deviation in pathogenicity islands with respect to the rest of the genome, the presence of direct repeat sequences at the flanking regions, the presence of integrase gene determinants as other mobility features,the particular insertion site (tRNA gene) as well as the observed genetic instability suggests that pathogenicity islands were acquired by horizontal gene transfer. PAIs are the fascinating proof of the plasticity of bacterial genomes. PAIs were originally described in human pathogenic Escherichia (E.) coli strains. In the meantime PAIs have been found in various pathogenic bacteria of humans, animals and even plants. The Locus of Enterocyte Effacement (LEE) is one particular widely distributed PAI of E coli. In addition, it also confers pathogenicity to the related species Citrobacter (C.) rodentium and Escherichia (E.) alvei. The LEE is an important virulence feature of several animal pathogens. It is an obligate PAI of all animal and human enteropathogenic E. coli (EPEC), and most enterohaemorrhegic E. coli (EHEC) also harbor the LEE. The LEE encodes a type III secretion system, an adhesion (intimin) that mediates the intimate contact between the bacterium and the epithelial cell, as well as various proteins which are secreted via the type III secretion system. The LEE encoded virulence features are responsible for the formation of so called attaching and effacing (AE) lesions in the intestinal epithelium. Due to its wide distribution in animal pathogens, LEE encoded antigens are suitable vaccine antigens. Acquisition and structure of the LEE pathogenicity island is the crucial point of numerous investigations. However, the evolution of the LEE, its origin and further spread in E. coli, are far from being resolved.     

O157:H7 and O104:H4 Vero/Shiga toxin-producing Escherichia coli outbreaks: respective role of cattle and humans

ABSTRACT: An enteroaggregative Verotoxin (Vtx)-producing Escherichia coli strain of serotype O104:H4 has recently been associated with an outbreak of haemolytic-uremic syndrome and bloody diarrhoea in humans mainly in Germany, but also in 14 other European countries, USA and Canada. This O104:H4 E. coli strain has often been described as an enterohaemorrhagic E. coli (EHEC), i.e. a Vtx-producing E. coli with attaching and effacing properties. Although both EHEC and the German O104:H4 E. coli strains indeed produce Vtx, they nevertheless differ in several other virulence traits, as well as in epidemiological characteristics. For instance, the primary sources and vehicles of typical EHEC infections in humans are ruminants, whereas no animal reservoir has been identified for enteroaggregative E. coli (EAggEC). The present article is introduced by a brief overview of the main characteristics of Vtx-producing E. coli and EAggEC. Thereafter, the O104:H4 E. coli outbreak is compared to typical EHEC outbreaks and the virulence factors and host specificity of EHEC and EAggEC are discussed. Finally, a renewed nomenclature of Vtx-producing E. coli is proposed to avoid more confusion in communication during future outbreaks and to replace the acronym EHEC that only refers to a clinical condition.     

Virulence profiles of Shiga toxin 2e-producing Escherichia coli isolated from healthy pig at slaughter

Recently, virulence patterns of Stx2e-producing Escherichia coli from pigs with edema disease and from humans were compared and strains from diseased pigs were reported to be unlikely human pathogens [Sonntag, A.K., Bielaszewska, M., Mellmann, A., Dierksen, N., Schierack, P., Wieler, L.H., Schmidt, M.A., Karch, H., 2005. Shiga toxin 2e-producing Escherichia coli isolates from humans and pigs differ in their virulence profiles and interactions with intestinal epithelial cells. Appl. Environ. Microbiol. 71, 8855-8863]. In the present study, 31 Shiga toxin-producing E. coli (STEC) strains harboring stx2e, which were previously isolated out of fecal samples from healthy pigs at slaughter [Kaufmann, M., Zweifel, C., Blanco, M., Blanco, J.E., Blanco, J., Beutin, L., Stephan, R., 2006. Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland. J. Food Prot. 69, 260-266], were characterized by phenotypic and genotypic traits. Nine of the thirty-one sorbitol-positive non-O157 STEC (stx2e) isolated from healthy pigs belonged to serotypes found in STEC isolated from humans, including two serotypes (O9:H-, O26:H-) reported in association with hemolytic-uremic syndrome. Otherwise, the serotypes were different from those isolated from cases of edema disease in pigs. The eae (intimin) gene, which is strongly correlated with severe human disease, was not detected. Moreover, all strains were lacking the genes for enterohemolysin (ehxA), porcine A/E associated protein (paa), STEC autoagglutinating adhesin (saa) and the serin protease EspI (espI). Nine strains tested positive for astA (EAST1), one O141:H17 strain for fedA (F18 fimbrial adhesin) and one O159:H- strain for terF (tellurite resistance). Similar to the Stx2e-producing E. coli isolated from humans, which are mainly lacking further virulence factors, genes of an iron uptake system on the high-pathogenicity island (irp2, fyuA) were detected in three ONT:H10 and ONT:H19 strains from healthy pigs. Consequently, although the isolated strains are unlikely to be associated with severe human diseases, healthy pigs cannot be excluded as a potential source of human infection with Stx2e-producing STEC.     

Duplex real-time PCR assays for rapid detection of virulence genes in E. coli isolated from post-weaning pigs and calves with diarrhoea

Duplex real-time PCR assays were used as modules to cover partially automated detection of 12 genes encoding adhesins, enterotoxins and Shiga toxins in faecal E. coli isolates. For this a total of 194 E. coli isolates from pigs suffering from post-weaning diarrhoea (PWD), including 65 isolates with haemolytic activity, and 83 isolates from calves with diarrhoea were examined. Data obtained by PCR were compared with O-typing and with haemolytic activity as indirect virulence markers. E. coli O-types O139:K82, O141:K85, and O149:K91 accounted for 43.8% (n = 85) of all porcine strains and for 55.4% (n = 36) of the porcine strains, which exhibited haemolytic activity. These strains carried virulence genes by 65.9% (n = 56) and 80.6% (haemolytic E. coli, n = 29), respectively. The E. coli O-types O139:K82 and O141:K85 were significantly associated with the adhesin gene F18, and O149:K81 with the F4 gene. In this context, detection of the gene encoding F18 was coupled predominantly with the genes responsible for the production of the toxins ST-I, ST-II and Stx2, and the F4 gene with those of the enterotoxins ST-I, ST-II and LT. Both virulence patterns were detected more pronounced in E. coli strains with haemolytic activity. Fifty-six of a total of 83 E. coli isolates originating from calves were O-typed as O101 (O101:K28, O101:K30, O101:K32; n = 29), O78:K80 (n = 23), and O9:K35 (n = 4). Most of the E. coli O78:K80 strains carried the F17 gene (69.6%, n = 16). Virulence genes encoding for F4, F5 or ST-I were detected only in single cases. Intimin and Shiga toxin genes that are present in enterohaemorrhagic E. coli (EHEC) were not detected.     

The possible influence of LuxS in the in vivo virulence of rabbit enteropathogenic Escherichia coli

Attaching and effacing (A/E) organisms, such as rabbit enteropathogenic Escherichia coli (EPEC), human EPEC or enterohemorrhagic E. coli (EHEC) share attaching and effacing phenotype and LEE pathogenicity island responsible for A/E. The present study was undertaken to investigate the impact of the LuxS quorum sensing (QS) signaling system in vitro and in vivo pathogenicity of A/E organisms using rabbit EPEC (rEPEC) strain E22 (O103:H2). Analysis of the bioluminescence indicated abolished production of the QS signal AI-2 by luxS mutant (E22DeltaluxS). Strain E22Deltalux also exhibited impaired expression of several normally secreted proteins and reduced adherence to cultured HeLa cells. Complementation of the intact luxS gene to E22DeltaluxS restored secreted protein expression comparable to the WT type but not adherence to HeLa cells. In experimentally infected rabbits, the isogenic luxS mutant induced clinical illness and intimate adherence to the intestinal mucosa, albeit to a less extent, comparable to that seen with the parent virulent strain. It is worth noting that reduced fecal bacterial shedding, mucosal adherence and improved cumulative weight gain were seen for the mutant strain complemented with luxS when compared to the WT. It appears that the luxS gene is not essential for in vivo pathogenicity by rEPEC where exogenous QS signals are present in the gut. The impact of AI-2 provided by multicopy plasmid on bacterial virulence is discussed.     

仔猪腹泻致病性大肠杆菌分型鉴定及耐药性分析

为了解贵州省规模化养猪场腹泻仔猪致病性大肠杆菌流行情况及耐药性变化,本研究运用凝集试验、PCR和药敏纸片琼脂扩散法等方法对分离的78株致病性大肠杆菌进行血清型、毒力基因及耐药性分析。结果显示,78株致病性大肠杆菌以O138、O87血清型为主,占定型菌株的60.8%;其中62株致病性大肠杆菌检出毒力基因,检出率为79.5%,可分为8种毒力基因类型,分属肠致病性大肠杆菌（EPEC）、肠产毒性大肠杆菌（ETEC）和肠聚集性大肠杆菌（EAEC）,毒力基因eaeA、elt和escV检出率较高,分别为38.5%、28.2%和21.8%;分离到的致病性大肠杆菌对β-内酰胺类药物高度耐药,均为多重耐药株,耐药种类可达8种以上。结果表明,当前贵州省规模化养猪场腹泻仔猪致病性大肠杆菌的毒力基因检出率较高且基因型复杂,耐药性严重。本试验结果可为规模化养猪场防控仔猪腹泻提供基础资料及理论依据。     

Detection of Serotypes and Virulence Factors of Escherichia coli from Piglets in Beijing

To investigate the cause of piglets diarrhea, and the distribution of the serotype and virulence factors of swine Escherichia coli in Beijing, 400 diarrhea samples were collected. TSA serum agar culture method was used to isolate Escherichia coli, serotype identification test and virulence factor genes test were used to verify the presence of O-antigen. 64 strains of E.coli were isolated from 400 diarrhea samples, among which 42 strains of E.coli were classified as 8 serotypes:O101(18.7%),O64(12.5%),O8(10.9%),O20(10.9%),O45(4.7%),O149(4.7%),O2(1.6%) and O89(1.6%), and the major virulence factors were STa, Stx2e, astA and eaeA. There were 8 mainly serotypes that caused piglets diarrhea in Beijing area, among which O101 serotype accounted for the highest proportion. The major virulence factors were astA and eaeA, accounted for more than 50% of all strains, STa and Stx2e accounted for more than 30% of all strains. These data would provide effective data to support the prevention and control of piglet diarrhea in Beijing.     

北京地区仔猪大肠杆菌血清型鉴定及其毒力因子检测

本试验旨在调查北京地区仔猪腹泻的原因及猪源大肠杆菌血清型、毒力因子的分布情况。收集北京京郊地区仔猪腹泻样本400份,应用含2%血清的TSA培养基分离大肠杆菌,通过血清型鉴定试验和毒力因子检测验证O抗原。结果显示,400份样本中分离到64株大肠杆菌,定型42株。其主要为O101型（18.7%）、O64型（12.5%）、O8型（10.9%）、O20型（10.9%）、O45型（4.7%）、O149型（4.7%）、O2型（1.6%）、O89型（1.6%）8种血清型,其携带的毒力因子为STa、Stx2e、astA和eaeA等。结果表明,引起京郊仔猪腹泻的大肠杆菌主要有8个血清型,其中O101型发病比例最高。其携带的毒力因子主要为astA和eaeA,占全部菌株的50%以上,STa和Stx2e因子占全部菌株的30%以上。本试验结果将为今后京郊地区仔猪腹泻病的防控提供有效的数据支持。     

Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains

The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50-70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.     

Occurrence of enterohaemorrhagic E. coli (EHEC) in domestic animals

Among the verocytotoxin producing E. coli strains (VTEC) the enterohemorrhagic group (EHEC) have emerged as important source of serious disease in human, e.g. the haemolytic uremic syndrome (HUS). VTEC strains possess different virulence profiles where by virulence traits can be provided by the chromosome, by plasmids and, in the case of verocytotoxins (except: VT2e) by bacteriophages. The original and main reservoir are ruminants. In Germany, VTEC strains were isolated in ruminant stocks regularly. In part, the prevalence was estimated up to 100%. However, strains of important EHEC serovar groups, e.g. O157, O26, O111, O103 and O145 as main source of human infections are isolated rarly. This is even the case for food originated from those animals. The hygienic management to avoid fecal contamination of carcasses during the slaughter process is of crucial importance. Future preventive strategies in the field of primary production may be the development of vaccination programs and/or the feeding management to reduce the shedding of acid resistant VTEC. Slowly recognized environmental sources of infection and contamination are biotic (e.g. flys, rodents) and abiotic factors (e.g. pasture, water, feed). In an own study that investigated the prevalence of VTEC positive animals in free range cows during sojourn on pasture a significant increase was estimated. Even asymptomatic human carriers can serve as source of infection or contamination.