Enteroaggregative Escherichia coli virulence factors are found to be associated with infantile diarrhea in Brazil

We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Brazilian infants. Most EAEC strains harbor a plasmid (pAA) from which a DNA fragment has been used as a probe (EAEC probe). To better understand the characteristics of EAEC in Brazil, 109 strains carrying and lacking the EAEC probe sequence were tested for the presence of pAA plasmid-borne and chromosomal factors. Common virulence factors of probe-positive and probe-negative isolates included the presence of the Pet, EAST-1, Shf, Irp2, ShET1/Pic, and Hly virulence markers. The presence of AggR or one other virulence factor (AAF/I, AAF/II, AAF/III, or Aap) was predominantly identified only in probe-positive strains. In EAEC probe-positive strains, the virulence marker Aap was found significantly more frequently (P = 0.023) in isolates from children with diarrhea (22%) than in isolates from controls (3%). EAST-1 and Shf were the markers most frequently detected (61%) in EAEC probe-negative strains and were found to be significantly associated with diarrhea (P = 0.003 and P = 0.020, respectively). Furthermore, our data suggest that AggR can be used as an important genetic marker for EAEC probe-positive strains.     

Typical enteroaggregative Escherichia coli is the most prevalent pathotype among E. coli strains causing diarrhea in Mongolian children

Diarrhea remains one of the main sources of morbidity and mortality in the world, and a large proportion is caused by diarrheagenic Escherichia coli. In Mongolia, the epidemiology of diarrheagenic E. coli has not been well studied. A total of 238 E. coli strains from children with sporadic diarrhea and 278 E. coli strains from healthy children were examined by PCR for 10 virulence genes: enteropathogenic E. coli (EPEC) eae, tir, and bfpA; enterotoxigenic E. coli (ETEC) lt and st; enteroinvasive E. coli (EIEC) ipaH; enterohemorragic E. coli stx1 and stx2; and enteroaggregative E. coli (EAEC) aggR and astA. EAEC strains without AggR were identified by the HEp-2 cell adherence test. The detection of EAEC, ETEC, EPEC, and EIEC was significantly associated with diarrhea. The incidence of EAEC (15.1%), defined by either a molecular or a phenotypic assay, was higher in the diarrheal group than any other category (0 to 6.0%). The incidence of AggR-positive EAEC in the diarrheal group was significantly higher than in the control group (8.0 versus 1.4%; P = 0.0004), while that of AggR-negative EAEC was not (7.1 versus 4.3%). Nineteen AggR-positive EAEC strains harbored other EAEC virulence genes-aggA, 2 (5.5%); aafA, 4 (11.1%); agg-3a, 5 (13.8%); aap, 8 (22.2%); aatA, 11 (30.5%); capU, 9 (25.0%); pet, 6 (16.6%); and set, 3 (8.3%)-and showed 15 genotypes. EAEC may be an important pathogen of sporadic diarrhea in Mongolian children. Genetic analysis showed the heterogeneity of EAEC but illustrated the importance of the AggR regulon (denoting typical EAEC) as a marker for virulent EAEC strains.     

Comparative virulence characterization of the Shiga toxin phage-cured Escherichia coli O104:H4 and enteroaggregative Escherichia coli

Escherichia coli O104:H4 (E. coli O104:H4), which caused in 2011 a massive foodborne outbreak in Germany, is characterized by an unusual combination of virulence traits. E. coli O104:H4 contains a prophage-encoded Shiga toxin (Stx) gene, which is the cardinal virulence factor of enterohemorrhagic E. coli (EHEC). However, the outbreak strain shares highest DNA sequence similarity with enteroaggregative E. coli (EAEC) and displays the EAEC-characteristic tight adherence to epithelial cells. The virulence potential of the underlying EAEC background has not been investigated and it is therefore not clear whether E. coli O104:H4 displays distinct virulence characteristics in comparison to prototypical EAEC. In this study, we performed a detailed comparative phenotypic characterization of the Stx phage-cured E. coli O104:H4 strain C227-11φcu, the closely related EAEC strain 55989 and two other well-characterized EAEC strains 042 and 17-2 with focus on virulence traits. C227-11φcu displayed superior aggregative adherence phenotype to cultured HCT-8 epithelial cells, adhering with 3–6 times more bacteria per epithelial cells than the tested EAEC strains. Otherwise, C227-11φcu showed similar virulence characteristics to its closest relative 55989, i.e. strong acid resistance, good biofilm formation and cytotoxic culture supernatants. Furthermore, C227-11φcu was characterized by significantly weaker motility and pro-inflammatory properties than 55989 and 042, nevertheless stronger than 17-2. Taken together, C227-11φcu displayed mostly robust, but not outstanding virulence characteristics in comparison to the tested EAEC. Therefore, it appears likely that the combination of Stx production and EAEC characteristics in general, rather than an exceptionally potent EAEC background resulted in the unusual virulence of the E. coli O104:H4. Thus, the emergence of such hypervirulent strains in the future might be more likely than previously anticipated.     

DNA Sequencing and Analysis of the Low-Ca2+-Response Plasmid pCD1 of Yersinia pestis KIM5

The low-Ca²⁺-response (LCR) plasmid pCD1 of the plague agent Yersinia pestis KIM5 was sequenced and analyzed for its genetic structure. pCD1 (70,509 bp) has an IncFIIA-like replicon and a SopABC-like partition region. We have assigned 60 apparently intact open reading frames (ORFs) that are not contained within transposable elements. Of these, 47 are proven or possible members of the LCR, a major virulence property of human-pathogenic Yersinia spp., that had been identified previously in one or more of Y. pestis or the enteropathogenic yersiniae Yersinia enterocolitica and Yersinia pseudotuberculosis. Of these 47 LCR-related ORFs, 35 constitute a continuous LCR cluster. The other LCR-related ORFs are interspersed among three intact insertion sequence (IS) elements (IS100 and two new IS elements, IS1616 and IS1617) and numerous defective or partial transposable elements. Regional variations in percent GC content and among ORFs encoding effector proteins of the LCR are additional evidence of a complex history for this plasmid. Our analysis suggested the possible addition of a new Syc- and Yop-encoding operon to the LCR-related pCD1 genes and gave no support for the existence of YopL. YadA likely is not expressed, as was the case for Y. pestis EV76, and the gene for the lipoprotein YlpA found in Y. enterocolitica likely is a pseudogene in Y. pestis. The yopM gene is longer than previously thought (by a sequence encoding two leucine-rich repeats), the ORF upstream of ypkA-yopJ is discussed as a potential Syc gene, and a previously undescribed ORF downstream of yopE was identified as being potentially significant. Eight other ORFs not associated with IS elements were identified and deserve future investigation into their functions.     

Characterization of Escherichia coli Isolates from Hospital Inpatients or Outpatients with Urinary Tract Infection

Uropathogenic Escherichia coli (UPEC) is the most common cause of community- and hospital-acquired urinary tract infections (UTIs). Isolates from uncomplicated community-acquired UTIs express a variety of virulence traits that promote the efficient colonization of the urinary tract. In contrast, nosocomial UTIs can be caused by E. coli strains that differ in their virulence traits from the community-acquired UTI isolates. UPEC virulence markers are used to distinguish these facultative extraintestinal pathogens, which belong to the intestinal flora of many healthy individuals, from intestinal pathogenic E. coli (IPEC). IPEC is a diarrheagenic pathogen with a characteristic virulence gene set that is absent in UPEC. Here, we characterized 265 isolates from patients with UTIs during inpatient or outpatient treatment at a hospital regarding their phylogenies and IPEC or UPEC virulence traits. Interestingly, 28 of these isolates (10.6%) carried typical IPEC virulence genes that are characteristic of enteroaggregative E. coli (EAEC), Shiga toxin-producing E. coli (STEC), and atypical enteropathogenic E. coli (aEPEC), although IPEC is not considered a uropathogen. Twenty-three isolates harbored the astA gene coding for the EAEC heat-stable enterotoxin 1 (EAST1), and most of them carried virulence genes that are characteristic of UPEC and/or EAEC. Our results indicate that UPEC isolates from hospital patients differ from archetypal community-acquired isolates from uncomplicated UTIs by their spectrum of virulence traits. They represent a diverse group, including EAEC, as well as other IPEC pathotypes, which in addition contain typical UPEC virulence genes. The combination of typical extraintestinal pathogenic E. coli (ExPEC) and IPEC virulence determinants in some isolates demonstrates the marked genome plasticity of E. coli and calls for a reevaluation of the strict pathotype classification of EAEC.     

New adhesin of enteroaggregative Escherichia coli related to the Afa/Dr/AAF family

Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea worldwide. We analyzed 17 Danish EAEC strains, isolated in the course of a case control study, for phenotypic and genotypic properties. The strains belonged to at least 14 different serotypes. Using PCR to investigate the prevalence of various putative virulence genes, we found that all but two strains were typical EAEC, as they harbored all or part of the previously described AggR regulon. The majority of the strains harbored genes encoding aggregative adherence fimbriae (AAF). The most common was AAF/I, found in nine strains; eight strains carried no known AAF-related genes. We utilized TnphoA mutagenesis to localize the aggregative adherence (AA) adhesin from one typical EAEC strain, C1010-00, which lacked a known AAF. We identified a TnphoA insertion in a hypothetical Dr-related pilin deposited in GenBank as HdaA. Four additional Danish strains harbored HdaA, and all but one displayed AA to HEp-2 cells. By using PCR primers derived from the pilins and ushers from the three AAF and Hda, we found that 16 of 17 strains exhibited evidence of one of these factors; importantly, the one negative strain also lacked the aggR gene. Cloning of the complete Hda gene cluster and expression in E. coli DH5alpha resulted in AA and complementation of the C1010-00 nonadherent mutant. Four related adhesins have now been found to confer AA in typical EAEC strains; our data suggest that, together, these variants may account for AA in the large majority of strains.     

Analysis of the Organization of Multicopy Linear- and Circular-Plasmid-Carried Open Reading Frames in Borrelia burgdorferi Sensu Lato Isolates

Plasmid cp8.3 of Borrelia afzelii IP21 carries several open reading frames (ORFs) and a 184-bp inverted repeat (IR) element. It has been speculated that this plasmid may encode factors involved in virulence or infectivity. In this report, we have characterized the distribution, molecular variability, and organization of ORFs 1, 2, and 4 and the IR elements among isolates of the Borrelia burgdorferi sensu lato complex. ORFs 1 and 2 are contained within a segment of cp8.3 that is bordered by the IR elements, while ORF 4 resides just outside of the IR-bordered region. By PCR, ORF 4 was amplified from most isolates while ORFs 1 and 2 were amplified from only some B. afzelii isolates. However, Southern hybridization analyses with ORF 1, 2, and 4 probes detected related sequences even in some isolates that were PCR negative. The ORF restriction fragment length polymorphism patterns varied widely even among isolates of the same species. Two-dimensional contour-clamped homogeneous electric field–pulsed-field gel electrophoresis and Southern hybridization detected ORF 1-, 2-, and 4-related sequences on linear and circular plasmids. In addition, an ORF 4-related sequence was detected on a previously uncharacterized, circular plasmid that is greater than 70 kb in size. The IR elements originally identified on plasmid cp8.3 of B. afzelii IP21 were also analyzed by Southern hybridization. Related sequences were detected in some but not all B. burgdorferi sensu lato isolates. These sequences are carried on plasmids in addition to cp8.3 in some isolates. Single-primer PCR analyses demonstrated that in some isolates these sequences exist with IR orientation. The data presented here demonstrate that the IR elements and the ORF 1-, 2-, and 4-related sequences are multicopy and are variable in organization and in genomic location among isolates of the B. burgdorferi sensu lato complex. These analyses provide additional evidence for the highly variable organization of the plasmid component of the B. burgdorferi sensu lato genome.     

Comparative genomic sequence analysis of novel <Emphasis Type="Italic">Helicoverpa</Emphasis> <Emphasis Type="Italic">armigera</Emphasis> nucleopolyhedrovirus (NPV) isolated from Kenya and three other previously sequenced <Emphasis Type="Italic">Helicoverpa</Emphasis> spp. NPVs

A newly cloned Helicoverpa armigera nucleopolyhedrovirus (HearNPV) from Kenya, HearNPV-NNg1, has a higher insecticidal activity than HearNPV-G4, which also exhibits lower insecticidal activity than HearNPV-C1. In the search for genes and/or nucleotide sequences that might be involved in the observed virulence differences among Helicoverpa spp. NPVs, the entire genome of NNg1 was sequenced and compared with previously sequenced genomes of G4, C1 and Helicoverpa zea single-nucleocapsid NPV (Hz). The NNg1 genome was 132,425 bp in length, with a total of 143 putative open reading frames (ORFs), and shared high levels of overall amino acid and nucleotide sequence identities with G4, C1 and Hz. Three NNg1 ORFs, ORF5, ORF100 and ORF124, which were shared with C1, were absent in G4 and Hz, while NNg1 and C1 were missing a homologue of G4/Hz ORF5. Another three ORFs, ORF60 (bro-b), ORF119 and ORF120, and one direct repeat sequence (dr) were unique to NNg1. Relative to the overall nucleotide sequence identity, lower sequence identities were observed between NNg1 hrs and the homologous hrs in the other three Helicoverpa spp. NPVs, despite containing the same number of hrs located at essentially the same positions on the genomes. Differences were also observed between NNg1 and each of the other three Helicoverpa spp. NPVs in the diversity of bro genes encoded on the genomes. These results indicate several putative genes and nucleotide sequences that may be responsible for the virulence differences observed among Helicoverpa spp., yet the specific genes and/or nucleotide sequences responsible have not been identified.     

The Escherichia coli efflux pump TolC promotes aggregation of enteroaggregative E. coli 042

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa. We have reported that the novel protein AatA, which is encoded on the EAEC virulence plasmid pAA2, localizes to the outer membrane and facilitates export of the dispersin Aap across the outer membrane. Because AatA is an E. coli efflux pump TolC homolog, we investigated the role of TolC in the virulence of EAEC. No difference in Aap secretion was observed between the wild type and its tolC mutant (042tolC). However, characteristic aggregation in high-glucose Dulbecco's minimal essential medium for the wild type was diminished for 042tolC. In a microtiter plate assay, there were significantly more planktonic cells for 042tolC than for the wild type, while there were significantly fewer spontaneously precipitated cells on the substratum for 042tolC than for the wild type. In a HEp-2 cell adherence test, 042tolC showed less aggregative adherence than did the wild type. The strong aggregation and aggregative adherence were restored in the complement strain with tolC. In a transwell assay, planktonic cells of 042tolC decreased when cocultured with the wild type or the complement, while precipitated cells of 042tolC increased when cocultured with them. These results suggest that TolC promotes the aggregation and adhesion of EAEC 042 by secreting an assumed humoral factor.     

Identification of proteins determining the virulence of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> using bioinformation analysis

The postgenomic stage of biotechnology allows bioinformation approaches to be used for revealing previously unknown factors participating in different processes of the vital the functions of organisms. In this work, bioinformation approaches have been applied to the analysis of factors involved in the virulence of grampositive bacterium Listeria monocytogenes. Several open reading frames (ORF) have been identified in the L. monocytogenes genome, which encode proteins with a high level of homology to bacterial peptidases and meet the developed criteria. The effect of ORFs on virulence has been proved experimentally by intravenous infection of mice with the constructed L. monocytogenes strains carrying site-specific insertion mutations in the studied genes. The obtained strains have been characterized by growth rate, morphology, and production of surface proteins.     

Genomic sequence analysis of Helicoverpa armigera nucleopolyhedrovirus isolated from Australia

The complete genomic sequence of Helicoverpa armigera nucleopolyhedrovirus from Australia, HearNPV-Au, was determined and analyzed. The HearNPV-Au genome was 130,992 bp in size with a G + C content of 39 mol% and contained 134 predicted open reading frames (ORFs) consisting of more than 150 nucleotides. HearNPV-Au shared 94 ORFs with AcMNPV, HearSNPV-G4 and SeMNPV, and was most closely related to HearSNPV-G4. The nucleotide sequence identity between HearNPV-Au and HearSNPV-G4 genome was 99 %. The major differences were found in homologous regions (hrs) and baculovirus repeat ORFs (bro) genes. Five hrs and two bro genes were identified in the HearNPV-Au genome. All of the 134 ORFs identified in HearNPV-Au were also found in HearSNPV-G4, except the homologue of ORF59 (bro) in HearSNPV-G4. The sequence data strongly suggested that HearNPV-Au and HearSNPV-G4 belong to the same virus species.     

Identification of enteroaggregative Escherichia coli in infants with acute diarrhea based on biofilm production in Manipal, south India

Background: Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes persistent diarrhea among infants, both in developing and industrialized countries. The EAEC strains adhere to epithelial cell surface, to the glass substratum and to each other in a distinctive stacked brick-formation. Thus, gold standard for identification of EAEC remains the HEp-2 cell adherence test, which is time consuming and requires specialized facilities. Aim: To evaluate the usefulness of quantitative biofilm assay to screen for EAEC from children with acute diarrhea. Materials and Methods: A total of 100 E. coli strains were collected from acute diarrheal cases from December 2005 to November 2006. The strains were screened for biofilm production using microtiter plate method. The biofilm in the microtiter plate was visualized after staining with crystal violet and was quantified using enzyme immunosorbent assay plate reader. The Aggregative plasmid and Heat stable toxin genes were evaluated by a multiplex polymerase chain reaction. The strains were identified as EAEC with an optical density at 570 nm (OD 570 ) > 0.2. Results: Of the total 100 Escherichia coli strains, 28 were positive by Polymerase Chain Reaction for two genes, AggR and EAST. Of the 28 PCR-positive strains screened for biofilm, 25 (89.2%) showed positive results by microtiter plate method. Conclusion: The quantitative biofilm assay using microtiter plate is convenient and economical and can be used as a screening method to screen E. coli isolates from acute diarrheal cases. The best use of this test is to screen large number of isolates quickly, and if positive this can be confirmed by multiplex PCR for AggR and EAST genes. This assay may contribute to demonstrating the true incidence of EAEC with and without AggR among clinically isolated E. coli strains, which can cause acute diarrhea.     

Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from different origins and sources

Sixty-two Escherichia coli strains carrying the wzx_O104-gene from different sources, origins and time periods were analyzed for their serotypes, virulence genes and compared for genomic similarity by pulsed-field gel-electrophoresis (PFGE). The O104 antigen was present in 55 strains and the structurally and genetically related capsular antigen K9 in five strains. The presence of 49 genes associated with enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC) was investigated. Fifty-four strains of serotypes O104:H2 (n = 1), O104:H4 (n = 37), O104:H7 (n = 5) and O104:H21 (n = 11) produced Shiga-toxins (Stx). Among STEC O104, a close association between serotype, virulence gene profile and genomic similarity was found. EAEC virulence genes were only present in STEC O104:H4 strains. EHEC-O157 plasmid-encoded genes were only found in STEC O104:H2, O104:H7 and O104:H21 strains. None of the 62 O104 or K9 strains carried an eae-gene involved in the attaching and effacing phenotype.     

Analysis of the nucleotide sequence of five genes at the left end of the unique short region of the equine herpesvirus 4 genome

Summary Eco RI fragment G of equine herpesvirus 4 strain 405/76 (EHV 4.405/76) is located at the left end of the unique short region close to or extending into the internal repeat region of the prototypic arrangement of the genome. The nucleotide sequence of two subclones designated HS and G 19, contiguous withinEco RI fragment G, was determined for each strand by obtaining a nested set of deletion clones of these double-stranded DNA plasmids. Analysis of the nucleotide sequence revealed that the two subclones contain 5449 base pairs with four complete open reading frames (ORFs) and part of a fifth ORF. Comparison of the predicted amino acid sequences of these reading frames showed that they correspond to ORFs 67, 68, 69, 70, and 71 of equine herpesvirus type 1 (EHV 1) [41], of which ORFs 68, 69, and 70 are homologous to human herpes simplex virus (HSV) genes in the unique short (US) region, i.e., US 2, US 3, and US 4. ORF 67 of EHV 4 and ORF 67 of EHV 1 are homologous (65.7%) but these genes have no homologue in HSV 1.     

Aureusvirus, a novel genus in the family Tombusviridae

Summary.  Aureusvirus is a new genus of plant viruses typified by pothos latent virus (PoLV) and comprising cucumber leaf spot virus (CLSV), previously classified as definitive species in the genus Carmovirus. Aureusviruses are soil-borne viruses readily transmitted by sap inoculation to a moderate range of hosts. Natural transmissions of CLSV is by the chytrid fungus Olpidium bornovanus, whereas PoLV infects the host without the apparent intervention of a vector. Aureusviruses have isometric particles with size (c. 30 nm) and structure similar to those of the family Tombusviridae, to which the genus belongs. The genome consists of a molecule of single-stranded, positive-sense RNA c. 4.4 kb in size comprising five ORFs. The structural organization (i.e. number and order of genes) is virtually identical to that of members of the genus Tombusvirus. However, the aureusvirus genome has a smaller size and shows distinct differences in the amino acid sequence of some of the ORFs. ORF 1 encodes a 25 kDa product and terminates with a leaky amber codon the readthrough of which results in a 84 kDa protein (ORF 2) with the conserved motifs of RNA dependent RNA polymerase. ORF 3 encodes the coat protein (40-41 kDa), ORF 4 the movement protein (27 kDa), and ORF 5 a 14-17 kDa product responsible for symptom severity. Virions accumulate in great quantity in the cytoplasm, often forming crystalline aggregates, and in bubble-like evaginations of the tonoplast protruding into the vacuole. Replication is likely to occur in the cytoplasm with a stategy based on direct expression of the 5' proximal ORF and expression of downstream ORFs through subgenomic RNAs.     

IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene. In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.     

Enteroaggregative Escherichia coli O78:H10, the Cause of an Outbreak of Urinary Tract Infection

In 1991, multiresistant Escherichia coli O78:H10 strains caused an outbreak of urinary tract infections in Copenhagen, Denmark. The phylogenetic origin, clonal background, and virulence characteristics of the outbreak isolates, and their relationship to nonoutbreak O78:H10 strains according to these traits and resistance profiles, are unknown. Accordingly, we extensively characterized 51 archived E. coli O78:H10 isolates (48 human isolates from seven countries, including 19 Copenhagen outbreak isolates, and 1 each of calf, avian, and unknown-source isolates), collected from 1956 through 2000. E. coli O78:H10 was clonally heterogeneous, comprising one dominant clonal group (61% of isolates, including all 19 outbreak isolates) from ST10 (phylogenetic group A) plus several minor clonal groups (phylogenetic groups A and D). All ST10 isolates, versus 25% of non-ST10 isolates, were identified by molecular methods as enteroaggregative E. coli (EAEC) (P < 0.001). Genes present in >90% of outbreak isolates included fimH (type 1 fimbriae; ubiquitous in E. coli); fyuA, traT, and iutA (associated with extraintestinal pathogenic E. coli [ExPEC]); and sat, pic, aatA, aggR, aggA, ORF61, aaiC, aap, and ORF3 (associated with EAEC). An outbreak isolate was lethal in a murine subcutaneous sepsis model and exhibited characteristic EAEC “stacked brick” adherence to cultured epithelial cells. Thus, the 1991 Copenhagen outbreak was caused by a tight, non-animal-associated subset within a broadly disseminated O78:H10 clonal group (ST10; phylogenetic group A), members of which exhibit both ExPEC and EAEC characteristics, whereas O78:H10 isolates overall are phylogenetically diverse. Whether ST10 O78:H10 EAEC strains are both uropathogenic and diarrheagenic warrants further investigation.