DNA sequencing and identification of serogroup-specific genes in the Escherichia coli O118 O antigen gene cluster and demonstration of antigenic diversity but only minor variation in DNA sequence of the O antigen clusters of E. coli O118 and O151

The DNA sequence of the O antigen gene cluster of an Escherichia coli serogroup O118 strain was determined, and 13 open reading frames (ORFs) were identified, encoding genes required for O antigen sugar biosynthesis, transfer, and processing. Polymerase chain reaction (PCR) assays targeting the wzx (O antigen flippase) and wzy (O antigen polymerase) genes in the O antigen gene cluster of E. coli O118 were designed for identification of these serogroups. Specificity testing using strains belonging to E. coli O118 isolated from various sources, representative strains of 167 other E. coli O serogroups, and 20 non-E. coli bacteria revealed that the PCR assays were specific for E. coli O118. Thus, the PCR assays can be used for rapid identification of E. coli O118 as an alterative to typing using antisera. However, the PCR assays targeting the E. coli O118 wzx and wzy genes were also positive using E. coli serogroup O151 DNA. Therefore, the sequence of the O antigen gene cluster of E. coli O151 was determined, and it was very similar to that of E. coli O118, with only three nucleotide differences. Although the lipopolysaccharide profiles of O118 and O151 showed differences, multilocus sequence typing of E. coli O118 and O151 strains only revealed minor variation at the nucleotide level. Since E. coli O118 strains are more frequently isolated from humans, animals, and the environment than E. coli O151, serogroup O151 may likely be a minor variant of E. coli O118. Further studies are needed to elucidate this possibility.     

Prevalence and characteristics of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy cattle.

From February to July of 1994, 328 faecal samples from 32 herds were collected and verotoxin-producing Escherichia coli (VTEC) found on 84% of the farms. The proportion of animals infected varied from 0-63%. VTEC were recovered from 52 (20%) of 257 cows and from 16 (23%) of 71 calves. Although the VTEC belonged to 25 different serogroups, 7 (O8, O20, O22, O77, O113, O126 and O162) accounted for 46% of strains. Nearly 45% of the strains. Nearly 45% of the 83 bovine VTEC strains belonged to serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. However, only 2 (2%) of 83 VTEC strains isolated from cattle belonged to enterohaemorrhagic E. coli (EHEC) serotypes (O26:H11 and O157:H7), and only 8 (10%) were positive for the attaching and effacing E. coli (eae) gene sequence. Polymerase chain reaction (PCR) showed that 17 (20%) of VTEC strains carried VT1 genes, 43 (52%) possessed VT2 genes, and 23 (28%) carried both VT1 and VT2 genes. Characterization of VTEC isolates revelated a heterogeneous population in terms of serogroup and toxin type in the positive herds. This study confirms that healthy cattle are a reservoir of VTEC, but, the absence of eae genes in most bovine VTEC strains suggests that they may be less virulent for humans than eae-positive EHEC.     

Virulence properties of Escherichia coli strains belonging to serogroups O26, O55, O111 and O128 isolated in the United Kingdom in 1991 from patients with diarrhoea.

Some strains of Escherichia coli belonging to serogroups O26, O55, O111 or O128 produce Vero cytotoxin (VT). These serogroups are included in the range of enteropathogenic E. coli (EPEC) serogroups for which commercial antisera are available. In an attempt to obtain information on VT-producing strains other than those of serogroup O157, 122 strains belonging to these four serogroups and isolated in 1991 from patients with diarrhoea in the United Kingdom were tested for hybridization with VT probes. Only 18 of the 122 strains were VT-positive and these were O26 or O128. However 90 strains hybridized with the E. coli attaching and effacing (eae) probe (including 14 VT-positive strains) and 17 with the enteroaggregative E. coli (EAggEC) probe. For 78 eae-positive and 9 EAggEC-positive strains, tissue culture tests correlated with the probe results as the strains gave, respectively, either localized adhesion and a positive fluorescent-actin staining test or a characteristic aggregative attachment. A total of 111 of the 122 strains belonging to serogroups O26, O55, O111 or O128 possessed properties that may be associated with the ability to cause human diarrhoeal disease, and similar studies are needed on strains from the other classical EPEC serogroups.     

Ribotyping as an epidemiologic tool for Escherichia coli.

Restriction fragment length polymorphism of ribosomal RNA genes was analysed among 133 Escherichia coli strains predominantly from blood and urine, including 21 isolates from faeces of healthy persons. The strains had also been characterized for their O:K:H serotypes, for the presence of P, S and type 1C fimbriae, non-P, non-S mannose-resistant haemagglutinins and haemolysin production. Hind III-digested genomic DNA was subjected to Southern blot analysis with either plasmid pKK3535 containing E. coli rRNA operon or purified rRNA as a probe. Among the 133 strains 20 ribotypes were obtained. The distribution of strains into different ribotypes generally correlated with their O:K:H serotype. Ribotype variation within serotypes was mainly seen among strains with the K5 capsule. The origin of the strains or the presence of virulence-associated factors did not correlate with the ribotype. In conclusion, ribotyping appears to be a valuable method in epidemiologic studies especially when the serotyping methods are not available.     

Serotype-related enterotoxigenicity in Escherichia coli O6.H16 and O148.H28.

The ability of certain Escherichia coli strains to produce enterotoxin is determined by transmissible plasmids. It is therefore possible that any E. coli strain might be able to acquire such a plasmid and that the correlation between enterotoxigenicity and serotype might be random. However, recent studies show that the enterotoxigenic strains so far describe belong to a restricted range of serotypes. Enterotoxigenic strains of E. coli O6.H16 and E. coli O148.H28 have been associated with outbreaks of diarrhoea in several countries, therefor strains of E. coli belonging to these serotypes were selected for further study. Twenty-three strains of E. coli O6.H16 and 14 strains of E. coli O148.H28 were examined; 20 strains of E. coli O6.H16 and all 14 strains of E. coli O148.H28 were enterotoxigenic but strains of E. coli O6 wit flagellar antigens other than H16 and strains of E. coli O148 wit flagellar antigens other than H28 were not enterotoxigenic. The examination of single colony subcultures derived from the E. coli O6.H16 strains showed that in some strains loss of enterotoxigenicity had occurred in a proportional of colonies.     

Ribotypes of clinical Vibrio cholerae non-O1 non-O139 strains in relation to O-serotypes

The emergence of Vibrio cholerae O139 in 1992 and reports of an increasing number of other non-O1 serogroups being associated with diarrhoea, stimulated us to characterize V. cholerae non-O1 non-O139 strains received at the National Institute of Infectious Diseases, Japan for serotyping. Ribotyping with the restriction enzyme BglI of 103 epidemiological unrelated mainly clinical strains representing 10 O-serotypes yielded 67 different typing patterns. Ribotype similarity within each serotype was compared by using the Dice coefficient (Sd) and different levels of homogeneity were observed (serotypes O5, O41 and O17, Sd between 82 and 90%: serotypes O13 and O141 Sd of 72; and O2, O6, O7, O11, O24 Sd of 62-66%). By cluster analysis, the strains were divided into several clusters of low similarity suggesting a high level of genetic diversity. A low degree of similarity between serotypes and ribotypes was found as strains within a specific serotypes often did not cluster but clustered with strains from other serotypes. However, epidemiological unrelated O5 strains showed identical or closely related ribotypes suggesting that these strains have undergone few genetic changes and may correspond to a clonal line. Surprisingly, 10 of 16 O141 strains studied contained a cholera toxin (CT) gene, including 7 strains recovered from stool and water samples in the United States. This is to our knowledge the first report of CT-positive clinical O141 strains. The closely related ribotypes shown by eight CT-positive strains is disturbing and suggest that these strains may be of a clonal origin and have the potential to cause cholera-like disease. Despite the low degree of correlation found between ribotypes and serotypes, both methods appears to be valuable techniques in studying the epidemiology of emerging serotypes of V. cholerae.     

Clonal diffusion of EPEC-like Escherichia coli from rabbits as detected by ribotyping and random amplified polymorphic DNA assays.

The genetic diversity and clonal relationships among 77 Escherichia coli strains isolated in France from diarrhoeic rabbits and that belonged to seven O serogroups including the predominant O103 serogroup, were estimated by ribotyping and random amplified polymorphic DNA (RAPD) assays. Fifteen ribotypes were defined. Most of the highly pathogenic O103 strains could be assigned to two major groups. Non-pathogenic strains were clearly distinguished. RAPD assays generally matched ribotyping, or gave more precision for subdividing strains from the two main O103 groups. The results on strains isolated from different areas and over a 9-year period showed the relevance of the association of these two methods for the survey of the spread of strains in breeding flocks and illustrated clonal diffusion in rabbit production structures.     

Serotypes and colonization factors of enterotoxigenic Escherichia coli isolated in various countries

One hundred and six enterotoxigenic E. coli (ETEC) isolated from many geographical areas were serotyped and investigated for the presence of colonization factor antigens CFA/I and CFA/II, the expression of mannose-resistant haemagglutination (MRHA) and the levels of surface hydrophobicity. CFA/I was found in 6 (17%) of 36 LT⁺STa⁺ strains and in 15 (54%) of 28 STa⁺ strains; CFA/II was found in 16 (44%) of 36 LT⁺STa⁺ strains. None of 42 LT⁺ strains showed CFA/I or CFA/II. CFA/I was found in ETEC of serotypes O63:K?:H?, O78:K80, O128:K67 and O153:K?:H45, whereas CFA/II was found in serotypes O6:H?, O6:K15:H16 and 06:K?:H40. Of the 69 CFA/I^? CFA/II^? ETEC strains, 9 (13%) showed MRHA with some of the seven erythrocyte species used and 21 (30%) were hydrophobic. Among the 21 hydrophobic strains CFA-negative we have detected: (i) 6 LT⁺ strains of serogroup O25 negative for MRHA, (ii) 5 strains O159 (4 LT⁺ and 1 LT⁺ STa⁺) also negative for MRHA, and (iii) 3 STa⁺ strains of serotype O27:K?:H7 that haemagglutinated calf and sheep erythrocytes when grown on Minca-Is. The 106 ETEC strains belonged to 20 different 0 serogroups. However, 77 (73%) were of one of nine serogroups (O6, O8, O25, O27, O78, O148, O153, O159 and O167). E. coli strains belonging to O6 and O153 groups predominated among ETEC isolated in Spain, O159 strains in the Central African Republic, O25 and O148 strains in Japan, and O15 and O78 strains in India.     

熔解曲线分析方法甄别肠出血性大肠杆菌O157:H7

目的:采用熔解曲线分析技术,建立一种快速、简便、准确地甄别肠出血性大肠杆菌O157:H7方法。方法:选取肠出血性大肠杆菌O157:H7编码脂多糖基因(rfbE)和编码鞭毛抗原基因(fliC)作为检测靶基因,建立荧光PCR反应体系。结果:通过多种标准菌株试验,结果显示所建立的熔解曲线分析法可特异性地甄别肠出血性大肠杆菌O157:H7;纯培养的灵敏度达到2.8×101cfu/ml;检测108例临床样本,其中18例肠出血性大肠杆菌O157:H7阳性,3例为肠出血性大肠杆菌O157:非H7阳性,与常规培养方法的符合率达到100%。结论:本方法可简便、准确地甄别肠出血性大肠杆菌O157:H7,结果可靠,通过进一步增加反应体系中引物的数量可同时对肠出血性大肠杆菌其他血清型进行鉴定。     

Human infections with non-O157 Shiga toxin-producing Escherichia coli, Switzerland, 2000-2009

We characterized 97 non-O157 Shiga toxin (stx)-producing Escherichia coli strains isolated from human patients during 2000-2009 from the national reference laboratory in Switzerland. These strains belonged to 40 O:H serotypes; 4 serotypes (O26:H11/H-, O103:H2, O121:H19, and O145:H28/H-) accounted for 46.4% of the strains. Nonbloody diarrhea was reported by 23.2% of the patients, bloody diarrhea by 56.8%. Hemolytic uremic syndrome developed in 40.0% of patients; serotype O26:H11/H- was most often associated with this syndrome. Forty-five (46.4%) strains carried stx2 genes only, 36 strains (37.1%) carried stx1, and 16 (16.5%) strains carried stx1 and stx2. Genes encoding enterohemolysin and intimin were detected in 75.3% and 70.1% of the strains, respectively. Resistance to ≥1 antimicrobial agent was present in 25 isolates. High genetic diversity within strains indicates that non-O157 stx-producing E. coli infections in Switzerland most often occurred as single cases.     

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。     

In vitro adherence patterns of Shigella serogroups to bovine recto-anal junction squamous epithelial (RSE) cells are similar to those of Escherichia coli O157

The aims of this study were to determine whether Shigella species, which are human gastrointestinal pathogens, can adhere to cattle recto-anal junction squamous epithelial (RSE) cells using a recently standardized in vitro adherence assay, and to compare their adherence patterns with that of Escherichia coli O157. Shigella dysenteriae (serogroup A), S. flexneri (serogroup B), S. boydii (serogroup C), and S. sonnei (serogroup D) were tested in adherence assays using both RSE and HEp-2 cells, in the presence or absence of D+mannose. Escherichia coli O157, which adheres to RSE cells in a Type I fimbriae-independent manner, was used as a positive control. Shigella serogroups A, B, D, but not C adhered to RSE cells with distinct adherence patterns in the presence of D+mannose. No such distinction could be made between the four Shigella serogroups based on the HEp-2 cell adherence patterns. Thus, this study provides evidence that certain Shigella serogroups adhere to RSE cells in a manner that is similar to the adherence pattern of E. coli O157. These unexpected observations of in vitro binding of these foodborne human pathogens to cells of the bovine gastrointestinal tract warrant evaluation of Shigella carriage by cattle using both experimental and observational studies, especially for serogroups B and D. Such studies are currently underway.     

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     

Occurrence of FIme plasmids in multiply antimicrobial-resistant Escherichia coli isolated from urinary tract infection.

Plasmids belonging to the FIme incompatibility group were found in seven different serogroups of multiply antimicrobial-resistant Escherichia coli isolated from patients with urinary tract infection (UTI) and living in south-east London. Although widespread in Salmonella spp., FIme plasmids have only previously been described in E. coli in a strain of serogroup O15 K52 H1 responsible for an extensive and protracted outbreak of invasive community-acquired infection in south-east London in 1986. Our findings suggest either a wider background occurrence of FIme plasmids in E. coli associated with UTI than previously reported or alternatively, the dissemination and subsequent molecular diversification of the FIme plasmid associated with the epidemic strain of serogroup O15 K52 H1.     

Properties of Vero cytotoxin-producing Escherichia coli of human and animal origin belonging to serotypes other than O157:H7

Eight non-O157:H7 Vero cytotoxin (VT)-producing Escherichia coli (VTEC) strains isolated from ill persons and nine bovine and lamb strains of serogroups matching the human strains, were characterized for various properties known to be associated with E. coli virulence. Five different serogroups were represented: O5, O55, O103, O111 and O153. The bovine and lamb strains produced VT1, while 3 human strains produced VT1, 3 produced VT2 and 2 were positive for both VT1 and VT2. The strains were non-haemolytic on horse blood agar, did not produce either heat stable toxin A (STA) or heat labile toxin (LT), and were noninvasive. The CVD419 probe which has been proposed to identify enterohaemorrhagic E. coli (EHEC) hybridized with all of the O5 and O103 strains, none of the O55 and O153 strains, and 3 of the 4 O111 strains. The strains carried several different sized plasmids and hybridization of Southern blots with the CVD419 probe identified plasmids ranging in size from 42 x 10(6) to 90 x 10(6). The strains did not hybridize with the enteroadherence factor (EAF) probe derived from an enteropathogenic strain and associated with the ability to give localized adherence to HEp-2 cells. Nevertheless five of the strains adhered in a localized pattern to HEp-2 cells and Intestine 407 cells. Adhesion to either HEp-2 or Intestine 407 cells did not correlate with hybridization with the CVD419 probe or haemagglutinating properties.     

Shiga Toxin Subtypes Associated with Shiga Toxin-Producing Escherichia coli Strains Isolated from Red Deer, Roe Deer, Chamois, and Ibex

Abstract A total of 52 Shiga toxin-producing Escherichia coli (STEC) strains, isolated from fecal samples of six ibex, 12 chamois, 15 roe deer, and 19 red deer were further characterized by subtyping the stx genes, examining strains for the top nine serogroups and testing for the presence of eae and ehxA. Eleven of the 52 strains belonged to one of the top nine STEC O groups (O26, O45, O91, O103, O111, O113, O121, O145, and O157). Eight STEC strains were of serogroup O145, two strains of serogroup O113, and one strain of serogroup O157. None of the strains harbored stx2a, stx2e, or stx2f. Stx2b (24 strains) and stx1c (21 strains) were the most frequently detected stx subtypes, occurring alone or in combination with another stx subtype. Eight strains harbored stx2g, five strains stx2d, three strains stx1a, two strains stx2c, and one strain stx1d. Stx2g and stx1d were detected in strains not harboring any other stx subtype. The eae and ehxA genes were detected in two and 24 STEC strains, respectively. Considering both, the serogroups and the virulence factors, the majority of the STEC strains isolated from red deer, roe deer, chamois, and ibex do not show the typical patterns of highly pathogenic STEC strains. To assess the potential pathogenicity of STEC for humans, strain isolation and characterization is therefore of central importance.     

Detection of Escherichia coli O157:H7 and Listeria monocytogenes in beef products by real-time polymerase chain reaction

Rapid methods for the detection of Escherichia coli O157:H7 and Listeria monocytogenes in food products are important to the food industry and for public health. Conventional microbiological methods and newly developed molecular-based techniques such as polymerase chain reaction (PCR)-based methods are time consuming. In this study, a faster method based on utilization of a hybridization probe with real-time PCR, was developed and applied for detection of E. coli O157:H7 and L. monocytogenes from artificially contaminated raw ground beef and fully cooked beef hotdogs. Target genes for E. coli O157:H7 and L. monocytogenes were rfbE and hylA, respectively. An analysis of 169 bacterial strains showed that the chosen primers and probes were specific for detection of E. coli O157:H7 and L. monocytogenes by real-time PCR. The assay was positive for nine of 10. E. coli O157:H7 strains, and all L. monocytogenes (7/7) strains evaluated. Bacterial strains lacking these genes were not detected by these assays. Detection limits of real-time PCR assays ranged from 10(3) to 10(8) colony forming units (CFU)/ml for E. coli O157:H7 in modified tryptic soy broth and 10(4) to 10(8) CFU/mL for L. monocytogenes in Fraser Broth. Detection sensitivity ranged from 10(3) to 10(4) CFU/g of raw ground beef or hotdog without enrichment for E. coli and L. monocytogenes. Approximately 1.4-2.2 CFU/g of E. coli O157:H7 in raw ground beef were detected following an enrichment step of 4 h. Approximately 1.2-6.0 CFU/g of L. monocytogenes in beef hotdogs were detected following an enrichment step of 30 h. The real-time PCR assays for detection of E. coli O157:H7 and L. monocytogenes in raw ground beef and beef hotdogs were specific, sensitive and rapid.     

12株疑似O157:H7大肠菌PCR鉴定研究

目的:对12株疑似O157:H7大肠菌采用PCR法进行鉴定。方法:利用单一PCR和多重聚合酶链反应(mPCR)检测不同来源菌株志贺样毒素(stx1和stx2)、溶血素(hly)、粘附抹平因子(eaeA)、β-葡糖醛酸糖苷酶(u idA)、O157抗原编码(rfbE)、H7鞭毛抗原编码(fliC)基因。结果:4株大肠菌rfbE和fliC基因检测为阳性,确认为EHEC O157:H7,其中1株菌株扩增出全部毒力基因,另3株菌株扩增出除stx1外其它全部毒力基因;2株大肠菌rfbE基因检测阳性,确认为O157:H7-大肠菌;其它均为非O157:H7其它大肠菌。结论:PCR技术的应用能对可疑O157:H7大肠菌进行有效鉴定与分析,应成为今后病原学鉴定的主要技术手段。     

Serum antibodies to secreted proteins in patients infected with Escherichia coli O157 and other VTEC.

Certain strains of verotoxigenic Escherichia coli (VTEC), and in particular those belonging to serogroup O157, cause attaching and effacing (AE) lesions of the host gut mucosa during pathogenesis. The mechanisms involved with bacterial attachment and the destruction of microvilli are determined by a cluster of genes within the LEE region, which also encode five secreted proteins. Sera from patients with antibodies to the lipopolysaccharide (LPS) of E. coli O157 and other VTEC were tested for antibodies to these secreted proteins. Twenty-one of 34 (62%) sera with antibodies to the lipopolysaccharide (LPS) of E. coli O157 also contained antibodies to one or more of the secreted proteins. Five of 12 sera containing antibodies to the LPS of a range of other VTEC serogroups also contained antibodies to 1 or more of the 5 secreted proteins, as did 16 of 70 (23%) sera from patients with haemolytic uraemic syndrome (HUS), haemorrhagic colitis (HC) or diarrhoea, but without bacteriological evidence of infection with VTEC and which did not contain antibodies to VTEC serogroups O5, O115, O145, O153 or O157. The detection of serum antibodies to secreted proteins may provide additional information for interpreting the results of established lipopolysaccharide-based VTEC serology.     

Investigation of human infections with verocytotoxin-producing strains of Escherichia coli (VTEC) belonging to serogroup O118 with evidence for zoonotic transmission

Twenty verocytotoxigenic Escherichia coli (VTEC) O118 strains isolated between 1996 and 1998 from human patients in Germany were analysed for their serotypes, their virulence markers and their epidemiological relatedness. Three strains were typed as O118:H12, these carried only the VT2d-Ount variant gene and were not associated with diarrhoea or haemolytic uraemic syndrome (HUS). Seventeen strains were serotyped as O118:H16 or O118:non-motile (NM). These carried all the genes for VTI, eae and EHEC-haemolysin. The O118:H16/NM strains were from diarrhoea (13 cases) and HUS (2 cases). Sixteen of the patients were young infants and most infections were associated with a rural environment. Evidence for zoonotic transmission from cattle to humans was found in two cases. The epidemiological relationship between the human and bovine O118:H16/NM isolates was indicated by homogeneous plasmid patterns and by very similar XbaI restriction patterns obtained by pulsed-field gel electrophoresis. VTEC O118:H16/NM are emerging pathogens in Germany and should be classified as new enterohaemorrhagic E. coli (EHEC) types.