Single multiplex assay to identify simultaneously enteropathogenic, enteroaggregative, enterotoxigenic, enteroinvasive and Shiga toxin-producing Escherichia coli strains in Brazilian children

A multiplex PCR to differentiate typical and atypical enteropathogenic Escherichia coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) strains was developed and evaluated. The targets selected for each group were eae and bfpA for EPEC, aggR for EAEC, elt and est for ETEC, ipaH for EIEC and stx for STEC isolates. This PCR was specific and sensitive for rapid detection of target isolates in stools. Among 79 children with acute diarrhea, this technique identified 13 (16.4%) with atypical EPEC, four (5%) with EAEC, three (3.8%) with typical EPEC, one (1.3%) with ETEC and one (1.3%) with EIEC.     

Enterovirulent <Emphasis Type="Italic">E. coli</Emphasis> in inflammatory and noninflammatory bowel diseases

We determined the incidence of enterovirulent E. coli (EVEC; which can to cause gastrointestinal infections) in strains isolated from patients with both of the major inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) and from patients with noninflammatory bowel diseases (nonIBD). Cell detachment E. coli (CDEC) were detected in 14 % of all strains. A significant difference in the presence of CDEC was found between the groups of strains isolated from UC (24.1 %), nonIBD (11.9 %) and CD (4.7 %). Enteroaggregative E. coli (EAggEC) were detected in 2.5 %, typical enteropathogenic strains (EPEC) in 1.3 % and enterotoxigenic ones (ETEC) in 1.5 %. Enteroinvasive (EIEC) and shigatoxin producing E. coli (STEC) were not detected. Some strains showed a high invasion level in gentamicin-protection assay. These strains could therefore belong to adherent-invasive E. coli (AIEC) because they are free of genes encoding invasins (ipaH, ial) and are equipped with fimA gene. However, complete characterization of these strains and their classification as AIEC will require further tests.     

Octaplex PCR and fluorescence-based capillary electrophoresis for identification of human diarrheagenic Escherichia coli and Shigella spp

A multiplex PCR assay, amplifying seven specific virulence genes and one internal control gene in a single reaction, was developed to identify the five main pathotypes of diarrheagenic Escherichia coli and Shigella spp. The virulence genes selected for each category were Stx1, Stx2, and eaeA for enterohemorrhagic E. coli (EHEC), eaeA for enteropathogenic E. coli (EPEC), STIb and LTI for enterotoxigenic E. coli (ETEC), ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., and aggR for enteroaggregative E. coli (EAEC). Each forward primer was labelled with a fluorochrome and the PCR products were separated by multicolour capillary electrophoresis on an ABI PRISM310 Genetic Analyzer (Applied Biosystems). If present, several gene variants of each virulence gene were identified. The internal control gene rrs, encoding 16S rRNA, was amplified in all 110 clinical strains analyzed. Virulence genes were demonstrated in 103 (94%) of these strains. In the majority of the cases (98/103, 95%), classification obtained by the novel multiplex PCR assay was in agreement with that previously determined by phenotypic assays combined with other molecular genetic approaches. Numerous multiplex PCR assays have been published, but only a few of them detect all five E. coli pathotypes within a single reaction, and none of them has used multicolour capillary electrophoresis to separate the PCR products. The octaplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the five main pathotypes of E. coli, and Shigella spp. This assay will replace the previous molecular genetic methods used in our laboratory and work as an important supplement to the more time-consuming phenotypic assays.     

Studies on diarrheagenic Escherichia coli isolated from children with diarrhea in Myanmar

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. coli strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified into five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients.     

Development of an enzyme-labeled oligonucleotide probe for detecting the Escherichia coli attaching and effacing A gene.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) can produce attaching and effacing (AE) lesions on intestinal epithelium in vitro and in vivo. A gene necessary to cause the AE lesion has been identified and designated Escherichia coli attaching and effacing A (eaeA) gene. In this study, an alkaline phosphatase (ALP)-conjugated oligonucleotide probe for the eaeA gene was developed and used to detect the eaeA gene among 163 strains of classical EPEC and 25 strains of EHEC O157. The prevalence rates of eaeA gene in the strains of classical EPEC and EHEC O157 were 51.5 and 100%, respectively. The eaeA-positive rate (60.0%) in strains of class I EPEC serogroups (O26, O55, O86, O111, O119, O125, O126, O127, O128ab, and O142) was significantly higher than that (22.9%) in strains of the class II EPEC serogroups (O18, O44, O114) (P<0.01). A total of 109 eaeA-positive classical EPEC and EHEC O157 were positive for fluorescent actin staining (FAS) assay, whereas 79 eaeA-negative classical EPEC were negative. Both the sensitivity and specificity of the eaeA probe versus the FAS assay positivity were 100%. Thus, use of the ALP-conjugated oligonucleotide probe for the eaeA gene would be specific and reliable in identifying the adherence capability of EPEC and EHEC.     

Investigation of diarrhoeic faecal samples for enterotoxigenic, Shiga toxin-producing and typical or atypical enteropathogenic Escherichia coli in Kashmir, India

Three hundred and twenty-six Escherichia coli isolates recovered from 326 human faecal specimens from sporadic cases of diarrhoea in Kashmir valley, India, were investigated for the presence of stx(1), stx(2), eaeA, hlyA and lt virulence genes. None of the samples was positive for stx genes or Shiga toxins by PCR or enzyme-linked immunosorbent assay. Twenty-three E. coli isolates showed the presence of the eaeA gene, whereas three isolates harboured the lt gene. Enteropathogenic E. coli (EPEC) belonged to 10 different serogroups. Out of 23 EPEC isolates, the majority (78.26%) were atypical while five (21.73%) were typical. Only one of the typical EPEC harboured the EAF plasmid. Subtyping of the eaeA gene showed the presence of eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta in one, two, four and two isolates, respectively. None of the E. coli isolates possessed eaeA-delta, eaeA-epsilon and eaeA-zeta. This study further upholds the opinion that Shiga toxin-producing E. coli do not pose a major threat to human health in India and eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta could be common EPEC subtypes prevalent in humans with diarrhoea in India. The present study appears to be the first report of subtype analysis of the eaeA gene from India and also records the isolation of EPEC with the eaeA-xi gene from humans.     

Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties

AIMS: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. METHODS AND RESULTS: A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. CONCLUSIONS: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.     

多重PCR方法快速检测4种主要致腹泻性大肠埃希菌

肠毒素性大肠埃希菌(ETEC)、肠致病性大肠埃希菌(EPEC)、肠出血性大肠埃希菌(EHEC)和侵袭性大肠埃希菌(EIEC)是引起腹泻的主要大肠埃希菌,威胁着食品安全和人类健康,建立同时检测4种致腹泻性大肠埃希菌的方法具有重要意义。基于ETEC LT肠毒素基因、EPEC bfpA基因、EHECO抗原基因和EIEC侵袭性质粒特异性基因,设计了4对特异性引物,通过对单一PCR反应条件的优化建立了快速检测4种主要致腹泻性大肠埃希菌的多重PCR方法,彼此之间无交叉反应。该多重PCR方法具有良好的特异性和灵敏性,对24株致病菌进行检测,所试4株致腹泻性大肠埃希菌均为PCR阳性,其他菌株则为阴性。实践证明,利用所建立的多重PCR方法对124份肉类、奶类制品及人工污染样品等进行检测,检出15份阳性,与国标(GB4789.6-1994)检测结果相同。结果表明本文建立的多重PCR方法可用于ETEC、EPEC、EHEC和EIEC的单一或混合感染的鉴别诊断及食品安全风险评估,具有良好的实用性。     

Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil

We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hybridization with the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1%) followed by ETEC (7.5%), and EAEC (4.2%). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O:H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.     

Rapid categorization of pathogenic Escherichia coli by multiplex PCR

A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli(EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli(EPEC); invE for enteroinvasive E. coli(EIEC); elt, estp, and esth for enterotoxigenic E. coli(ETEC); CVD432 and aggR for enteroaggregative E. coli(EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.     

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions

Abstract A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli , Hafnia alvei , a strain of Citrobacter freundii , and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa. These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene. This gene encodes intimin, an outer membrane protein required for production of AE lesions. RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains. The RDEC-1 example of E. coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE. In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced. The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C. freundii biotype 4280, EHEC O157:H7, and EPEC O127:H6, respectively. The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C. freundii , EHEC O157:H7, and EPEC O127:H6 intimins, respectively. The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C. freundii , EHEC O157:H7, and EPEC O127:H6, respectively. The high degree of sequence homology of the ORF and the eaeA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.     

occurrence of mannose- resistant adhesins MRHA in E.Coli strains isolated from children with diarrhea]

The study was aimed at determination of the frequency of occurrence of mannose-resistant adhesins in E. coli strains isolated from children with diarrhoea. It was also of interest whether their presence is associated with the serological type or other virulence factors. The material used in this study consisted of 1022 strains of E. coli (EPEC, ETEC and EIEC) and 3431 isolates from sick children and 960 from healthy children (non-EPEC-ETEC-EIEC). Enterotoxigenicity and entero-invasiveness of strains was evaluated by biological tests performed on animals and in tissue culture. Production of MRHA adhesins was determined by the test of mannose-resistant active hemagglutination, and of colonization factors antigens CFA by application of agglutination and agar gel immunodiffusion tests. Most frequently MRHA adhesins were produced by ETEC strains-80% of strains. All of them appeared to be a colonization factor antigen CFA/I. EPEC strains produced various MRHA adhesins only by 12.6% of strains. Production of MRHA adhesins by EIEC strains was not detected. Frequency of occurrence of MRHA adhesins in E. coli strains which were non-EPEC-ETEC-EIEC was dependent from the isolation source. MRHA adhesins were most frequently found in strains isolated from sporadic cases of light diarrhoea in ambulatory treated children (49%), much less among isolates from children hospitalized because of severe diarrhoea (33%), and from healthy children in 9% of isolates only. These results may indicate the potential role of MRHA adhesins in pathogenesis of diarrhoea in children.     

Binding of basement-membrane laminin by Escherichia coli

An invasive Escherichia coli (EIEC) isolate was found to bind basement-membrane laminin in a saturable and time-dependent manner. Excess of unlabelled laminin inhibited the binding of the radioactively labelled protein. Non-invasive E. coli K-12 exhibited only low-level laminin binding but introduction of the virulence-associated plasmid from the EIEC isolate led to high-level binding. Expression of a receptor for laminin on the bacteria was therefore associated with the presence of the virulence plasmid. Scatchard plot analysis indicated approximately 1000 receptors per bacterial cell, and a Kd of high-affinity binding of 0.5 pM. A laminin-binding protein which correlated with the presence of the plasmid was isolated and characterized. Its sequence of the eight amino-terminal amino acids was identical to that of the LamB protein of E. coli, although the molecular mass of the two in sodium dodecyl sulphate/polyacrylamide gel (SDS-PAGE) appeared to be slightly different. Both proteins reacted in immunoblot assays with polyclonal antisera raised against either protein, and both proteins bound laminin. Southern-blot hybridization analysis established that both the EIEC strain and the K-12 strains with or without the virulence plasmid contained one lamB gene only, and no laminin-binding protein appeared when the virulence plasmid was introduced into bacteria deleted for the lamB gene. On the basis of these results we suggest that native LamB protein of E. coli or a modified variant of it serves as a major receptor for laminin binding and is present at an increased level in invasive E. coli containing the virulence plasmid.     

Characterization of two major groups of diarrheagenic Escherichia coli O26 strains which are globally spread in human patients and domestic animals of different species

Twenty-three Escherichia coli O26 strains from humans, cattle, sheep, pigs and chicken were investigated for virulence markers and for genetic similarity by pulsed field gel electrophoresis and multi locus sequence typing. Two groups of genetically closely related O26 strains were defined. One group is formed by enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli strains, which do not ferment rhamnose and dulcitol and most of these carry a plasmid encoding enterohemolysin. The other group consists of rhamnose and dulcitol fermenting EPEC strains, which carry plasmids encoding alpha-hemolysin. Multiple species of domestic animals were shown to serve as a reservoir for human pathogenic O26 EPEC and EHEC strains.     

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and lambs with diarrhoea in India

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in calves and lambs with diarrhoea in India. METHODS AND RESULTS: Faecal samples originating from 391 calves and 101 lambs which had diarrhoea were screened for presence of E. coli. A total number of 309 (249 bovine and 60 ovine) E. coli strains were isolated. A total of 113 bovine and 15 ovine strains were subjected to multiplex polymerase chain reaction (m-PCR) for detection of stx1, stx2, eaeA and EHEC hlyA genes. STEC and EPEC belonging to different serogpoups were detected in 9.73% of calves studied. Six per cent and 26.66% of lambs studied were carrying STEC and EPEC, respectively. Majority of the STEC serogroups isolated in this study did not belong to those which have been identified earlier to be associated mainly with diarrhoea and enteritis in cattle and sheep outside India. The most frequent serogroup among bovine and ovine EPEC was O26 (40%). One of the most important STEC serogroup O157, known for certain life-threatening infections in humans, was isolated from both bovine and ovine faecal samples. CONCLUSIONS: A high percentage of STEC and EPEC belonging to different serogroups are prevalent in calves and lambs with diarrhoea in India and could be the cause of disease in them. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports, for the first time, the isolation and characterization of STEC and EPEC serogroups associated with diarrhoea in calves and lambs in India. Many STEC and EPEC strains belonged to serogoups known for certain life-threatening diseases in humans.     

Characterization of tccP2 carried by atypical enteropathogenic Escherichia coli

Atypical enteropathogenic Escherichia coli (EPEC) comprise an important group of paediatric pathogens. Atypical EPEC have reservoirs in farm and domestic animals where they can be either commensal or pathogenic; serogroup O26 is dominant in humans and animals. Central to intestinal colonization by EPEC is the translocation of the type III secretion system effector Tir into enterocytes, which following phosphorylation (Tir-Yp) recruits Nck to activate the N-WASP actin signalling cascade. The authors have recently shown that typical EPEC strains, belonging to the EPEC-2 lineage, carry a tir gene encoding Tir-Yp and can also use the alternative TccP2 actin-signalling cascade. The aim of this study was to determine if tccP2 is found in atypical EPEC isolated from human and farm animals. tccP2 was found at a frequency of 41% in non-O26 EPEC isolates and in 82.3% of the O26 strains. TccP2 of human and animal strains show high level of sequence identity. It is shown that most strains carry a tir gene encoding Tir-Yp. In addition the authors identified two new variants of tir genes in EPEC O104:H12 and NT:H19 strains.     

Phenotypic and molecular characteristics of Escherichia coli isolated from aquatic environment of Bangladesh

Pathogenic Escherichia coli remains important etiological agent of infantile diarrhea in Bangladesh. Previous studies have focused mostly on clinical strains, but very little is known about their presence in aquatic environments. The present study was designed to characterize potentially pathogenic E. coli isolated between November 2001 and December 2003 from aquatic environments of 13 districts of Bangladesh. Serotyping of 96 randomly selected strains revealed O161 to be the predominant serotype (19%), followed by O55 and O44 (12% each), and 11% untypable. Serotype-based pathotyping of the E. coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid pro-filing showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat-stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat-labile enterotoxin (LT), serotype-based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype-based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic.     

Characterization of the α-haemolysin determinant from the human enteropathogenic Escherichia coli O26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding α-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire α- hly CABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited α- hly determinants. The α- hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli α-hly plasmid pHly152, in particular, the structural α- hly CABD genes (99.2% identity) and the regulatory hly R regions (98.8% identity). pEO5 and α-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural α- hly CABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS 2 element upstream of the hly C gene in pHly152. The presence of transposon-like structures at both ends of the α- hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons.

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a "common" plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with > 80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.