Attaching And Effacing Escherichia Coli Research Articles

Refining the pathovar paradigm via phylogenomics of the attaching and effacing Escherichia coli

The attaching and effacing Escherichia coli (AEEC) are characterized by the presence of a type III secretion system encoded by the locus of enterocyte effacement (LEE). Enterohemorrhagic E. coli (EHEC) are often identified as isolates that are LEE+ and carry the Shiga toxin (stx)-encoding phage, which are labeled Shiga toxin-producing E. coli; whereas enteropathogenic E. coli (EPEC) are LEE+ and often carry the EPEC adherence factor plasmid-encoded bundle-forming pilus (bfp) genes. All other LEE+/bfp-/stx- isolates have been historically designated atypical EPEC. These groups have been defined based on the presence or absence of a limited number of virulence factors, many of which are encoded on mobile elements. This study describes the comparative analysis of the genomes of 114 LEE+ E. coli isolates. Based on a whole-genome phylogeny and analysis of type III secretion system effectors, the AEEC are divided into five distinct genomic lineages. The LEE+/stx+/bfp- genomes were primarily divided into two genomic lineages, the O157/O55 EHEC1 and non-O157 EHEC2. The LEE+/bfp+/stx- AEEC isolates sequenced in this study separated into the EPEC1, EPEC2, and EPEC4 genomic lineages. A multiplex PCR assay for identification of each of these AEEC genomic lineages was developed. Of the 114 AEEC genomes analyzed, 31 LEE+ isolates were not in any of the known AEEC lineages and thus represent unclassified AEEC that in most cases are more similar to other E. coli pathovars than to text modification AEEC. Our findings demonstrate evolutionary relationships among diverse AEEC pathogens and the utility of phylogenomics for lineage-specific identification of AEEC clinical isolates.

Effect of dietary laminarin and fucoidan on selected microbiota, intestinal morphology and immune status of the newly weaned pig.

A 2 × 2 factorial experiment was conducted to investigate the interactions between laminarin (LAM; 0 and 300 parts per million (ppm)) and fucoidan (FUC; 0 and 240 ppm) levels on intestinal morphology, selected microbiota and inflammatory cytokine gene expression in the weaned pig. There was an interaction between LAM and FUC supplementation on the Enterobacteriaceae population (P< 0·05) and the abundance of attaching and effacing Escherichia coli (AEEC) strains (P< 0·05) in the colon. Pigs offered the FUC diet had a reduced Enterobacteriaceae population compared with pigs offered the basal diet. However, the effect of FUC on the Enterobacteriaceae population was not observed when combined with LAM. Pigs offered the LAM diet had reduced abundance of AEEC strains compared with pigs offered the basal diet. However, there was no effect of LAM on the abundance of AEEC strains when combined with FUC. There was an interaction between LAM and FUC supplementation on villous height (P< 0·01) and the villous height:crypt depth ratio (P< 0·01) in the duodenum. Pigs offered the LAM or FUC diet had an increased villous height and villous height:crypt depth ratio compared with pigs offered the basal diet. However, there was no effect of the LAM and FUC combination diet on intestinal morphology. Pigs offered the LAM-supplemented diets had a lower IL-6 (P< 0·05), IL-17A (P< 0·01) and IL-1β (P< 0·01) mRNA expression in the colon compared with pigs offered the diets without LAM. In conclusion, supplementation with either LAM or FUC alone modified intestinal morphology and selected intestinal microbiota, but these effects were lost when offered in combination.

Non-neutral evolution in non-LEE-encoded type III effectors of attaching and effacing Escherichia coli

Attaching and effacing Escherichia coli (AEEC) employ type III secretion system (T3SS) to secrete effector proteins into host cells and regulate their function. Here we have investigated T3SS genes of AEEC for non-neutral evolution. Our analysis revealed non-neutral evolution in three genes (nleE1, nleB2 and nleD) which encode effector proteins. These genes are located outside the locus of enterocyte effacement (LEE). In general, non-LEE effector genes show greater deviation from neutral evolution than LEE effector genes. These results suggest that effector genes located outside LEE are under greater selection pressure than those present in LEE.

Attaching and effacing Escherichia coli and Shiga toxin-producing E. coli in children with acute diarrhoea and controls in Teresina/PI, Brazil

This 3.5-year prospective study was conducted to ascertain the level of attaching and effacing Escherichia coli (AEEC) associated diarrhoea in children from Teresina, a northeastern state of Brazil. Passed faecal specimens from 400 patients (250 with and 150 without diarrhoea) up to 60 months of age attending from 2004 to 2007 at two public hospitals were investigated. Conventional microbiology methods and PCR were employed. Escherichia coli was isolated from 390 children, 240 of them with diarrhoea. A total of 117 AEEC strains were cultivated from specimens from 63 children, 37 with and 26 without diarrhoea. No association between AEEC and diarrhoea was observed. Atypical enteropathogenic E. coli (a-EPEC) (79.4%) was more commonly found than typical EPEC (t-EPEC). Association between EPEC and EPEC subtypes and diarrhoea was not detected. Mixed infection by t-EPEC and a-EPEC and infection by Shiga toxin-producing E. coli (STEC) were rare. Enteropathogenic E. coli was more common in males and in children aged less than 12 months. Correlation between serotyping and PCR results was 0.19. High resistance rates of AEEC to ampicillin, cephalotin, and trimethoprim-sulfamethoxazole were found. In conclusion, EPEC is very common in children with diarrhoea and controls in the population we studied, with a-EPEC predominating. This diarrhoeagenic E. coli (DEC) pathotype is more common in infant males and is resistant to drugs frequently used in clinical practice.

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.

Phenotypic and Genotypic Characterization of Antimicrobial Resistance in Enterohemorrhagic Escherichia Coli and Atypical Enteropathogenic E. Coli Strains from Ruminants

Two hundred and twenty-six attaching and effacing Escherichia coli (AEEC) strains (20 enterohemorrhagic E. coli and 206 atypical enteropathogenic E. coli) isolated from calves, lambs, and goat kids with diarrhea and from healthy cattle, sheep, and goats were tested for their resistance to 10 antimicrobial agents by the disc diffusion method. Resistant and intermediate strains were analyzed by polymerase chain reaction for the presence of the major resistance genes. The overall percentage of resistant strains to tetracycline, streptomycin, erythromycin, and sulfamethoxazole was very high (>65%). Moreover, a high level of resistance (approximately 30%) to ampicillin, chloramphenicol, trimethoprim, and trimethoprim-sulfamethoxazole was also detected. The AEEC strains were very susceptible (>90%) to gentamicin and colistin. Because AEEC from ruminants can cause diseases in human beings, the high frequency of antimicrobial resistance detected in the current study is a source of concern. For each antimicrobial agent, the predominant resistance genes in the resistant strains were ampicillin, bla(TEM) (97.1%); tetracycline, tetA (76.7%); gentamicin, aac(3)II (80%); streptomycin, strA/strB (76.7%) and aadA (71.7%); chloramphenicol, catI (85.1%); trimethoprim, dhfrI (76.3%); and sulfamethoxazole, sul1 (60%) and sul2 (63.3%). In the majority of cases, resistance to a given antimicrobial, except for streptomycin, was caused by a single gene. A negative association between tetA and tetB, between aac(3)II and aac(3)IV, and between dhfrI and dhfrV was observed. The present study gives baseline data on frequency and molecular basis of antimicrobial resistance in AEEC strains from ruminants.

Development of a live oral attaching and effacing Escherichia coli vaccine candidate using Vibrio cholerae CVD 103-HgR as antigen vector

Attaching and effacing Escherichia coli (AEEC) share the ability to induce pedestal formation and intimate adherence of the bacteria to the intestinal epithelial cell and effacement of microvilli of epithelial tissue. The Locus of Enterocyte Effacement (LEE) pathogenicity island encodes the ability to induce attaching and effacing (A/E) lesions and contains the gene eae, which encodes intimin, an outer membrane protein that is an adhesin for A/E lesion formation. Here we show the utility of using intimin as a vaccine to protect rabbits from challenge with rabbit Enteropathogenic E. coli (REPEC), a member of the AEEC family. The C-terminal portion of intimin was delivered by the attenuated Vibrio cholerae vaccine strain CVD 103-HgR. To export intimin, a fusion was engineered with ClyA, a secreted protein from Salmonella enterica serovar Typhi. After immunization, antibodies specific to intimin from serum and bile samples were detected and moderate protection against challenge with a virulent REPEC strain was observed. Compared to animals immunized with vector alone, intimin-immunized rabbits exhibited reduced fecal bacterial shedding, milder diarrheal symptoms, lower weight loss, and reduced colonization of REPEC in the cecum. V. cholerae CVD 103-HgR shows promise as a vector to deliver antigens and confer protection against AEEC pathogens.

Characterization of attaching and effacing Escherichia coli (AEEC) isolated from pigs and sheep

BackgroundAttaching and effacing Escherichia coli (AEEC) are characterized by their ability to cause attaching-and-effacing (A/E) lesions in the gut mucosa of human and animal hosts leading to diarrhoea. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports data on the occurrence of eae positive E. coli carried by healthy pigs and sheep at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated AEEC strains.ResultsFaecal samples from 198 finished pigs and 279 sheep were examined at slaughter. The proportion of eae positive samples was 89% for pigs and 55% for sheep. By colony dot-blot hybridization, AEEC were isolated from 50 and 53 randomly selected porcine and ovine samples and further characterized. Strains of the serotypes O2:H40, O3:H8 and O26:H11 were found in both pigs and sheep. In pigs O2:H40, O2:H49, O108:H9, O145:H28 and in sheep O2:H40, O26:H11, O70:H40, O146:H21 were the most prevalent serotypes among typable strains. Eleven different intimin types were detected, whereas γ2/θ was the most frequent, followed by β1, ε and γ1. All but two ovine strains tested negative for the genes encoding Shiga toxins. All strains tested negative for the bfpA gene and the EAF plasmid. EAST1 (astA) was present in 18 of the isolated strains.ConclusionOur data show that pigs and sheep are a source of serologically and genetically diverse intimin-harbouring E. coli strains. Most of the strains show characteristics of atypical enteropathogenic E. coli. Nevertheless, there are stx-negative AEEC strains belonging to serotypes and intimin types that are associated with classical enterohaemorrhagic E. coli strains (O26:H11, β1; O145:H28, γ1).

Shiga Toxin–Negative Attaching and EffacingEscherichia coli:Distinct Clinical Associations with Bacterial Phylogeny and Virulence Traits and Inferred In‐Host Pathogen Evolution

Attaching and effacing Escherichia coli (AEEC) that lack Shiga toxin genes (stx) and the enteropathogenic E. coli adherence factor (EAF) plasmid (stx-/EAF-) are classified as atypical enteropathogenic E. coli and cause diarrhea worldwide. However, it is unknown whether there are bacterial lineage-specific human disease phenotypes. We compared stx-/EAF- AEEC recovered from patients (mostly children) with bloody and nonbloody diarrhea. stx-/EAF- AEEC were isolated using eae colony blot hybridization and were serotyped, tested (by polymerase chain reaction) for putative virulence genes, and analyzed for phylogenetic relationships by use of multilocus sequence typing. During the period 1995-2007, stx-/EAF- AEEC were isolated as the only bacterial pathogens from stool specimens obtained from 18 (15.3%) of 118 patients with bloody diarrhea and from 141 (1.3%) of 10,550 patients with nonbloody diarrhea (P<.001). All but 1 of 18 strains recovered from patients with bloody diarrhea resembled enterohemorrhagic E. coli (EHEC) on the basis of serotypes, non-stx virulence profiles, and multilocus sequence types. In contrast, most (75.9%) of 141 stx-/EAF- AEEC recovered from patients with nonbloody diarrhea belonged to other serotypes and differed from the former strains phylogenetically and with regard to virulence genes. Three of 18 patients with bloody diarrhea and none of 141 patients with nonbloody diarrhea who shedded stx-/EAF- AEEC developed hemolytic uremic syndrome. Most stx-/EAF- AEEC associated with bloody diarrhea are plausibly EHEC that lost Shiga toxin during infection (EHEC-LST). To prevent serious complications of such infections, Shiga toxin-independent diagnostic strategies to accurately and rapidly identify such patients should be developed and applied. Multilocus sequence typing has potential to distinguish EHEC-LST from less pathogenic stx-/EAF- AEEC.

Phylogenetic background of attaching and effacing Escherichia coli isolates from animals

Detection and distribution of eae gene in forty-four attaching and effacing Escherichia coli (AEEC) strains of animal origin were investigated. Association of distinct intimin alleles with phylogenetic background were assessed among strains in comparison with different serogroups. Phylogenetic analysis showed that 31 EHEC/eae+ STEC strains belong to groups A, B1 and E, 13 EPEC strains segregated in B1 and B2. Moreover, group A possessed the eae gamma2/theta type, group B1 the eae beta1, eae kappa, eae zeta, and eae epsilon types, group B2 the eae alpha1, eae alpha2 and eae iota types, while the group E possessed the eae gamma1 type. The presence of numerous eae-types show that EPEC and EHEC/eae+ STEC tested have a high genetic homology within each phylogenetic group.

Pathotypic Characterization of Enterocyte Effacement-related LEE Genes in EHEC and EPEC Isolated from Diarrheal Patients

Attaching and effacing Escherichia coli (AEEC) cause enteric infections in humans and animals. Attaching indicates the intimate attachment of bacteria to the enterocyte, and effacing relates to the localized effacement of brush border microvilli. Enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli (EHEC) infections are characterized by the formation of attaching and effacing (AE) lesion on the intestinal epithelial cells. Therefore, they are often grouped together as AEEC. Development of multiplex PCR allowed us to type five of the most important genes implicated in the formation of the AE lesion. A total of 60 AEEC strains isolated from diarrheal patients were investigated by multiplex PCR for the presence of the insertion site of locus of enterocyte effacement (LEE) and LEE-related (eae, tir, espA, espB, and espD) genes. Associating the results of LEE genes typing in the AEEC strains, three different pathotypes are determined: eae γ -tir γ -espA γ -espB γ -espD γ (O157:H7), eae β -tir β -espA β -espB β -espD β (O26:H11), and eae α -tir α -espA α -espB α -espD α (O55:H6). These results indicate that AEEC are a heterogenous groups of organisms.

Use of virulence factor-specific egg yolk-derived immunoglobulins as a promising alternative to antibiotics for prevention of attaching and effacingEscherichia coliinfections

Using a porcine ileal in vitro organ culture model, we have demonstrated that egg yolk-derived antibodies specific for the attaching and effacing Escherichia coli (AEEC) virulence factors intimin and translocated intimin receptor (Tir), but not those specific for the AEEC-secreted proteins EspA, EspB and EspD, significantly reduced the bacterial adherence of the porcine enteropathogenic E. coli strain ECL1001, formerly 86-1390. Moreover, antibodies specific for intimin and Tir also significantly reduced bacterial adherence of heterologous AEEC strains, including human, bovine and canine enteropathogenic E. coli strains, as well as of O157:H7 Shiga toxin-producing E. coli strains in this model. In addition, we demonstrated that the oral administration of these anti-intimin antibodies significantly reduced the extent of attaching and effacing lesions found in the small intestine of weaned pigs challenged with the porcine enteropathogenic E. coli strain ECL1001. Overall, our results underline the potential use of specific egg yolk-derived antibodies as a novel approach for the prevention of AEEC infections.

Bovine attaching and effacing Escherichia coli possess a pathogenesis island related to the LEE of the human enteropathogenic Escherichia coli strain E2348/69.

Attaching and effacing Escherichia coli (AEEC) has been described as a cause of diarrhea in calves. The molecular pathogenesis of AEEC was mainly studied in human enteropathogenic E. coli strain E2348/69 in which the virulence correlated with the presence of a 35.4 kb pathogenesis island called LEE. We showed that several strains isolated from calves with diarrhea were able to produce attaching and effacing lesions in a rabbit ileal loop model and that they possess a pathogenesis island related to the LEE. Moreover, we showed that the LEE from bovine strains was inserted mainly at a different position in the chromosome compared to the human enteropathogenic E. coli strain E2348/69.

Recombinant anti-EspA antibodies block Escherichia coli O157:H7-induced attaching and effacing lesions in vitro

Intimin and EspA proteins are virulence factors expressed by attaching and effacing Escherichia coli (AEEC) such as enteropathogenic and enterohaemorrhagic E. coli. The EspA protein makes up a filament structure forming part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Bacterial surface displayed intimin interacts with translocated intimin receptor in the host cell membrane leading to intimate attachment of the bacterium and subsequent attaching and effacing lesions. Here, we have assessed the use of recombinant monoclonal antibodies against E. coli O157:H7 EspA and intimin for the disruption of AEEC interaction with the host cell. Anti-γ intimin antibodies did not reduce either adhesion of E. coli O157:H7 to host cell mono-layers or subsequent host cell actin rearrangement. Anti-EspA antibodies similarly had no effect on bacterial adhesion however they had a marked effect upon E. coli O157:H7-induced host cell actin rearrangement, where both monoclonal and polyclonal antibodies completely blocked cytoskeletal changes within the host cell. Furthermore, these anti-EspA antibodies were shown to reduce actin rearrangement induced by some but not all other AEEC serotypes tested. Both polyclonal and monoclonal antibodies could be used to label E. coli O157 EspA filaments and these immunoreagents did not inhibit the formation of such filaments. This is the first report of monoclonal antibodies to EspA capable of disrupting the TTSS function of E. coli O157:H7.

Investigation of domestic animals and pets as a reservoir for intimin- ( eae) gene positive Escherichia coli types

Domestic animals belonging to seven different species (cattle, sheep, dogs, cats, pigs, chicken and goats) were investigated as natural reservoirs for attaching and effacing Escherichia coli (AEEC). For this, 2165 E. coli strains from faeces of 803 animals were examined for the presence of the intimin -( eae) gene as a characteristic of AEEC strains. Ten percent of the animals were found to excrete AEEC, most frequently found in sheep (19.2%) and pigs (17.6), followed by cattle (10.4%), dogs (7.2%), cats (6.5%) and poultry (2.3%). The 97 AEEC strains from animals were grouped into 44 serotypes. Only four E. coli serotypes (O2:H8, O26:[H11], O109:[H25] and O145:[H28] were found in more than one animal host species. AEEC O26:[H11] strains were most frequently isolated (13.4%) being present in cattle, poultry, pigs and sheep. A search for virulence markers associated with enterohemorrhagic E. coli (EHEC) revealed Shiga-toxin genes in three (3.1%) AEEC strains from sheep. Bundle forming pili genes as a trait of typical enteropathogenic E. coli (EPEC) were detected in four (4.1%) strains from dogs and cats. The remaining 90 AEEC strains were classified as atypical EPEC. Typing of intimin genes revealed intimin β being present in 51.5% of the strains, followed by intimins θ (23.7%), ɛ (6.2%), κ (5.2%), ζ (5.2%), α, η and ι (each 1.0%). Our data indicate that domestic animals and pets constitute an important natural reservoir of AEEC strains, and some of these (O26:[H11], O103:H2, O128:H2, O145:[H28] and O177:[H11]) are known to occur as pathogens in humans.

Typing of the eae and espB genes of attaching and effacing Escherichia coli isolates from ruminants

The types of the eae and espB genes of 178 attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic and healthy ruminants were investigated by PCR. Six types of the eae gene: β (beta), γ1 (gamma-1), γ2 (gamma-2), ε (epsilon), ζ (zeta) and ι (iota), and three types of the espB gene: α, β and γ were identified in the strains studied. Moreover, three strains were negative to all the types of the eae gene tested. The types β and γ2 in healthy cattle, β, γ2 and ε in healthy sheep and goats, and β in diarrhoeic calves, lambs and goat kids were the most frequent types of the eae gene among the strains studied. Although the eaeβ gene was the most prevalent among AEEC from healthy and diarrhoeic ruminants, the percentages of AEEC strains with this type found in this study in diarrhoeic animals (66.7–100%) were higher than those found in healthy animals (33.3–40.6%). Thus, these data suggest that AEEC strains with the eaeβ gene are associated with neonatal diarrhoea in ruminants. The eaeγ1, eaeζ and eaeι genes were found in low percentages in the strains studied (4.5, 2.8 and 7.3%, respectively). All the types of the eae gene, except the type ι, showed a close correlation with the types of the espB gene: the eaeβ and eaeε genes with the espBβ gene, the eaeγ2 and eaeζ genes with the espBα gene and the eaeγ1 gene with the espBγ gene.

HEp-2 cell adherence, actin aggregation, and intimin types of attaching and effacing Escherichia coli strains isolated from healthy infants in Germany and Australia.

Fecal samples from healthy children under 2 years of age living in Berlin, Germany (205 infants), and Melbourne, Australia (184 infants), were investigated for the presence of attaching and effacing (AE) Escherichia coli (AEEC) strains by screening for eae (intimin) genes. Twenty-seven AEEC strains were isolated from 14 children (7.6%) from Melbourne and from 12 children (5.9%) from Berlin. The 27 AEEC strains were classified as enterohemorrhagic E. coli (one strain, producing Shiga toxin 1), typical enteropathogenic E. coli (EPEC) (one strain carrying an EPEC adherence factor [EAF] plasmid), and atypical EPEC (25 strains negative for Shiga toxins and EAF plasmids). The AEEC were divided into 18 different serotypes, O-nontypeable and O-rough strains. Typing of their intimin genes revealed the presence of intimin alpha in 6 strains, intimin beta in 11 strains, intimin gamma in 7 strains, intimin zeta in 2 strains, and intimin eta in one strain. Analysis of HEp-2 cell adherence showed diffuse adherence or localized adherence-like patterns in 26 AEEC strains; local adherence was found only with the EAF-positive strain. Ten AEEC strains showed an AE property with the fluorescent actin staining (FAS) test. The introduction of an EAF plasmid (pMAR7) converted 11 FAS-negative AEEC strains to FAS positive and increased the FAS reaction in six FAS-positive AEEC strains, indicating that the genes needed for the AE phenotype were functional in these strains. Our finding indicates that atypical EPEC strains could play a double role as strains that naturally immunize against intimin in humans and as reservoirs for new emerging human pathogenic EPEC strains.

Genotypic and phenotypic characterization of attaching and effacing Escherichia coli (AEEC) isolated from children with and without diarrhoea in Londrina, Brazil.

Attaching and effacing Escherichia coli (AEEC) have been implicated in diarrhoea in humans in several countries. A total of 919 E. coli strains, isolated from 125 children with diarrhoea and 98 without diarrhoea, was investigated by PCR for the presence of the EAF, bfp, eae and stx genes. Thirty-four of these isolates were found to carry the eae gene; they were isolated from 27 (79.4 %) children with diarrhoea and seven (20.6 %) controls, in the city of Londrina, Brazil. These strains were investigated for their genotypic and phenotypic characteristics. Different genetic profiles were observed; strains containing the eae gene alone were most common (47.1 %). The characteristic genetic profile of typical enteropathogenic E. coli (EPEC), eae, bfp and EAF, was only found in isolates from children with diarrhoea. The stx gene was not detected in any of the 34 strains studied. Ten (29.4 %) strains were negative in the fluorescent actin-staining test. Localized adhesion (LA) was the most common pattern of adhesion (44.1 %), followed by the aggregative adhesion (AA) (23.5 %) and localized adhesion-like (LAL) (14.7 %) patterns. The results showed a strong association between strains presenting the LA pattern and diarrhoea. Forty-seven per cent of the strains studied belonged to classical O-serogroups of EPEC. The most common serotype found was O119 : H6; these isolates all showed the LA pattern, were positive for fluorescent actin-staining and were associated with diarrhoea. Intimin beta was detected in seven strains, four of which belonged to serotype O119 : H6 and three to serotype ONT : H7; all were associated with diarrhoea. On the other hand, intimin epsilon was detected in two strains of serotype O111 : H38 and one of serotype ONT : H19, isolated from children without diarrhoea. To our knowledge, this is the first report of the occurrence of intimin epsilon in strains of E. coli isolated from humans in Brazil.

Prevalence and characteristics of attaching and effacing strains of Escherichia coli isolated from diarrheic and healthy sheep and goats.

To determine the prevalence and characteristics of attaching and effacing Escherichia coli (AEEC) in diarrheic and healthy small ruminants. 502 lambs and kids with diarrhea and 511 healthy sheep and goats. Fecal samples from diarrheic and healthy sheep and goats were screened for the eae gene. In addition, E coli isolates with positive results for the eae gene (E coli eae+) were analyzed for the espB gene, production of verotoxins (VT), and serogroup. A significantly higher prevalence of healthy lambs and kids were infected with AEEC, compared with diarrheic lambs and kids and healthy adult sheep and goats. Some differences in the characteristics of E coli eae strains isolated from diarrheic and healthy animals were detected. Thus, the espB gene was detected more frequently among E coli eae+ strains isolated from healthy animals than in those isolated from diarrheic animals, and VT production was only detected in E coli eae+ strains isolated from healthy lambs and kids. The E coli eae+ isolates belonged to several O serogroups. However, 17 of 40 (42.5%) isolates from diarrheic lambs and only 4 of 168 (2.4%) isolates from healthy sheep belonged to serogroup 026. Our results suggest that E coli eae+ 026 strains may play a role in diarrheal disease in lambs, whereas E coli eae+ strains that also had VT production and eae+ strains that had positive results for the espB gene did not appear to be associated with diarrhea in small ruminants.

Association between intimin (eae) and EspB gene subtypes in attaching and effacing Escherichia coli strains isolated from diarrhoeic lambs and goat kids.

Attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic lambs and goat kids were characterized for intimin (eae) and EspB (espB) gene subtypes by PCR and sequencing, and for genetic relatedness by PFGE. Fifty (23 ovine and 27 caprine) AEEC strains of 398 (246 ovine and 152 caprine) analysed were detected by colony blot hybridization. These strains were epidemiologically unrelated since they were isolated from different outbreaks of neonatal diarrhoea over a long period. Ovine AEEC strains belonged to serogroups O2, O4, O26, O80, O91 or were untypable, and caprine strains belonged to serogroups O3, O153 and O163. Two intimin subtypes were detected among the ovine and caprine strains studied. Most of the strains (43/50) had the beta type intimin gene, but seven ovine strains possessed a variant gamma type intimin gene (gamma(V)). Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of beta intimin of ovine and caprine strains were virtually identical to those of beta intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the gamma(V) intimin present in seven ovine strains had 75-76% identity with gamma intimin of human EHEC clone 1 strains, and 96% of identity with intimin of the human EHEC strain 95NR1 of serotype O111:H-. A PCR test was developed to identify the three different espB gene subtypes, espB of human EPEC clone 1 (espBalpha), espB of human EHEC clone 1 (espBgamma) and espB of rabbit EPEC and human EPEC clone 2 (espBbeta). There was close correlation between the intimin beta type and the espBbeta gene subtype in the ovine and caprine AEEC strains. The seven ovine strains possessing the gamma(V) intimin gene possessed the espBalpha gene subtype. None of the strains studied possessed the espBgamma gene found in human O157:H7 EHEC strains. PFGE analysis of genomic DNA of selected strains showed a great diversity among strains. Cluster analysis of PFGE patterns showed greater divergence between strains with the gamma(V) intimin gene than between strains with the beta intimin gene. This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed beta intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.