Genetic diversity of intimin genes of attaching and effacing Escherichia coli strains

In this study, we determined the sequences of four intimin variant genes detected in attaching and effacing Escherichia coli isolates of human origin. Three of them were novel and were designated eae-eta (eta), eae-iota (iota), and eae-kappa (kappa). The fourth was identical to the recently described eae-zeta (zeta), isolated from a bovine E. coli O84:NM isolate. We compared these sequences with those of published intimin-alpha, intimin-beta, intimin-gamma1, intimin-gamma2, intimin- epsilon, and intimin-theta alleles. Sequence analysis of these 10 intimin alleles confirmed extensive genetic diversity within the intimin gene family in E. coli. The genetic diversity was more prominent in the 3' region (starting at bp 2,112), which encodes the binding domain of intimin. Phylogenetic analyses revealed four groups of closely related intimin genes: alpha and zeta; beta and kappa; gamma1 and gamma2/theta; and epsilon and eta. Calculation of homoplasy ratios of sequences of the 5' region of eae (positions 1 to 2,111) revealed evidence for intragenic recombination. Split decomposition analysis also indicates that recombination events have played a role in the evolutionary history of eae. In conclusion, we recommend an eae nomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing of E. coli eae.     

Biotype, serotype, and pathogenicity of attaching and effacing enteropathogenic Escherichia coli strains isolated from diarrheic commercial rabbits.

A total of 568 strains of Escherichia coli isolated from healthy and diarrheic rabbits were separated into 11 different biotypes according to the fermentation patterns of four carbohydrates. Strains belonging to biotypes 1 to 3, 6, and 8 induced lesions characteristic for attaching and effacing E. coli (AEEC). They attached to the intestinal epithelium of the terminal small intestine and the large intestine of 5-week-old rabbits after experimental infection and caused effacement of the microvillous brush border. However, pathogenicity for weaned rabbits, as judged by diarrhea score, anorexia, and reduced weight gain, varied according to the biotypes of the strains. Strains belonging to biotypes 1 and 6 produced only discrete clinical signs, strains belonging to biotypes 2 and 3+ (motile) induced diarrhea and growth depression, whereas strains belonging to biotypes 3- (immotile) and 8 caused severe clinical signs and high mortality. This confirms evidence from the field. Biotypes 3- and 8, accounting for 35.5 and 7.1% of AEEC strains in weaned diarrheic rabbits, respectively, were not detected in weaned healthy rabbits, while biotype 2 was the predominant strain in weaned healthy rabbits (62.3%). Finally, serotyping showed a close relationship between biotype and serotype of the AEEC examined. Most strains of biotypes 1+ and 2+ tested were O109:K-:H2 and O132:K-:H2, respectively, whereas all strains tested of biotype 3- were O15:K-:H- and those of biotype 8 were O103:K-:H2. These data indicate that specific clones of AEEC might be involved in juvenile rabbit enteritis. It was concluded that determination of biotypes allows the screening of highly pathogenic AEEC in weaned rabbits (biotypes 3- and 8).     

eae-negative attaching and effacing Escherichia coli from piglets with diarrhea

One hundred and ninety strains of Escherichia coli that were isolated from pigs with diarrhea in the state of S?o Paulo, Brazil, and that were negative for enterotoxins and cytotoxins were investigated. Strains which adhered to HeLa cells were examined for fluorescence actin staining (FAS), the ability to induce attaching and effacing (A/E) lesions on HEp-2 cells detectable by transmission electron microscopy and the presence of eae gene sequences detected by PCR. Intimin production was detected by western blot and serogrouping was performed. Forty-seven isolates adhered to HeLa cells in several patterns, but none adhered in a localized adherence pattern. However, seven of the 47 adherent strains were positive for the FAS reaction, although the reactions were usually weak or atypical. One FAS-negative and three FAS-positive strains, which were examined for their ability to induce A/E lesions, were all positive. Subsequently, testing of these strains for the eae gene showed that they all lacked this gene. These findings, along with earlier reports of eae-negative A/E E. coli, suggest that higher quantities of E. coli in this category might be detected if more reliance were placed on phenotypic tests rather than on gene detection tests alone.     

Pathogenicity of attaching effacing enteropathogenic Escherichia coli isolated from diarrheic suckling and weanling rabbits for newborn rabbits

The pathogenicity of six strains of Escherichia coli originating from different commercial rabbitries was tested in neonatal rabbits. Two strains isolated from healthy weaned rabbits (O7:H6 and O9:H?) did not induce any clinical sign or lesion. Two strains (O109:H2) isolated from diarrheic suckling rabbits caused yellow diarrhea 36 to 60 h after inoculation and high mortality between 60 and 72 h after infection. At 12 h after infection, light and electron microscopy showed attachment to epithelial cells and effacement of microvilli from proximal small intestine to colon. Bacteria were often present in the apical cytoplasm of epithelial cells. The two strains isolated from diarrheic weanling rabbits (O109:H2 and O15:H-) did not induce any clinical sign. Attachment to epithelial cells and effacement of microvilli was observed 48 h after inoculation in distal small intestine, cecum, and colon. These data are further evidence for the existence of two groups of attaching effacing enteropathogenic E. coli in rabbits, showing different preferences for age group and intestinal compartment.     

Cytoskeletal composition of attaching and effacing lesions associated with enteropathogenic Escherichia coli adherence to HeLa cells.

The cytoskeletal lesions associated with enteropathogenic Escherichia coli adhering to cultured HeLa epithelial cells were examined by immunofluorescence microscopy. The microfilament-associated proteins actin, alpha-actinin, talin, and ezrin were localized with adherent enteropathogenic E. coli, whereas tropomyosin, keratin and vimentin (intermediate filaments), tubulin (microtubules), and vinculin were not localized. These cytoskeletal structures differed significantly from those associated with Salmonella typhimurium internalization (invasion).     

Heterogeneity of the eae genes in attaching/effacing Escherichia coli from cattle: comparison with human strains.

Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli isolated from cattle were studied by DNA colony hybridization to subtype their intimin-encoding (eae) gene with probes derived from the variable parts of the eae alpha gene of the human EPEC strain E2348/69, the eae gamma gene of the human O157:H7 EHEC strain ATCC43888, and the eae beta gene of the bovine O26:H- EHEC strain 193, whose eae gene was first cloned and sequenced during this work. The EPEC and EHEC had been isolated from diarrhoeic calves (143 EPEC and 48 EHEC) and from healthy animals at the slaughterhouse (10 EPEC and 34 EHEC). The 191 bovine EPEC and EHEC isolated from diseased calves were positive with the Eae beta probe (55 and 27% respectively) and with the Eae gamma probe (9 and 73% respectively), whereas 52 EPEC (36%) were negative with the Eae alpha, Eae beta, and Eae gamma probes. The results were different for the 44 bovine EPEC and EHEC isolated from healthy cattle at slaughterhouses: most tested positive with the Eae gamma probe (80 and 82% respectively) and the remaining (20 and 18% respectively) with the Eae beta probe. Nine O26 human EHEC tested positive with the Eae beta probe and seven O111 with the Eae gamma probe. The bovine and human EPEC and EHEC belonging to these two serogroups gave identical results: the 18 bovine and human O26 isolates tested positive with the Eae beta probe, whereas the 13 O111 isolates were positive with the Eae gamma probe. In contrast, the isolates belonging to other serogroups (O5, O15, O18, O20, and O118) gave more variable results. The eae beta and eae gamma, but not the eae alpha, variants were thus distributed amongst bovine EPEC and EHEC. The eae beta variant seemed to be more frequently associated with the presence of clinical signs in calves, but one third of EPEC from diarrhoeic calves carried an eae gene variant other than the alpha, beta, or gamma variants. In addition, the use of these gene probes did not enable differentiation between bovine and human EHEC belonging to the same O serogroup.     

HeLa cell adherence, actin aggregation, and invasion by nonenteropathogenic Escherichia coli possessing the eae gene.

Enteropathogenic Escherichia coli (EPEC) produce diarrhea in humans by a mechanism that involves close adherence to epithelial cells in the intestine and colon. Close adherence is associated with effacement of microvilli and condensation of actin beneath the bacteria, a process termed attaching/effacing adherence. Attaching/effacing adherence of EPEC occurs in vitro in tissue culture, simplifying the study of the molecular genetics of this process. An EPEC gene (eae) necessary for attaching/effacing adherence was recently characterized. Enterohemorrhagic E. coli and the rabbit-specific RDEC-1 strain adhere in a like fashion in vivo and hybridize with eae. However, these strains adhere poorly to tissue culture cells, complicating the in vitro study of attaching/effacing adherence. In order to develop an in vitro model for the study of attaching/effacing activity of non-EPEC bacteria, a plasmid encoding the F1845 adhesin of an E. coli strain (C1845) isolated from a patient with diarrhea was transformed into RDEC-1 and enterohemorrhagic E. coli. The transformed strains adhered in a diffuse pattern to HeLa cells, and they aggregated HeLa cell actin at points of adherence in the fluorescein-isothiocyanate-labeled phalloidin assay. They also invaded HeLa cells in a gentamicin invasion assay, although not to the extent seen with EPEC. The construction of adherent non-EPEC strains facilitates the molecular study of the attaching/effacing properties and invasiveness of these strains in tissue culture models.     

Experimental infection of young chicks with attaching and effacing Escherichia coli.

Young chicks were inoculated with six different strains of attaching and effacing Escherichia coli isolated from the feces of calves, pigs, chicks, and humans. Colibacilli of some serotypes had colonized the cecum of chicks by 7 days after inoculation. The characteristic lesions associated with bacterial attachment were also seen on the mucosal surface of the cecum. Electron microscopy revealed numerous colibacilli closely attached to the surface membrane of enterocytes. Cell membranes formed cups and pedestals at the base of the attached bacilli. The results of this study support the conclusion that young chicks can be used as a model for the study of the lesions caused by attaching and effacing E. coli strains.     

Decreased adherence of enterohemorrhagic Escherichia coli to HEp-2 cells in the presence of antibodies that recognize the C-terminal region of intimin

Antiserum raised against intimin from enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 has been shown previously by our laboratory to inhibit adherence of this strain to HEp-2 cells. In the present study, we sought to identify the region(s) of intimin important for the effect of anti-intimin antisera on EHEC adherence and to determine whether antisera raised against intimin from an O157:H7 strain could reduce adherence of other strains. Compared to preimmune serum controls, polyclonal sera raised against the histidine-tagged intimin protein RIHisEae (intimin(O157)) or against His-tagged C-terminal fragments of intimin from strain 86-24 reduced adherence of this strain. Furthermore, an antibody fraction purified from the anti-RIHisEae serum that contained antibodies to the C-terminal third of intimin, the putative receptor-binding domain, also reduced adherence of strain 86-24, but a purified fraction containing antibodies to the N-terminal two-thirds of intimin did not inhibit adherence. The polyclonal anti-intimin(O157) serum raised against RIHisEae inhibited, to different degrees, the adherence of another O157:H7 strain, an EHEC O55:H7 strain, one of two independent EHEC O111:NM isolates tested, and one of two EHEC O26:H11 strains tested. Adherence of the other O26:H11 and O111:NM strains and an EPEC O127:H6 strain was not reduced. Finally, immunoblot analysis indicated a correlation between the antigenic divergence in the C-terminal third of intimins from different strains and the capacity of anti-intimin(O157) antiserum to reduce adherence of heterologous strains. Taken together, these data suggest that intimin(O157) could be used as an immunogen to elicit adherence-blocking antibodies against O157:H7 strains and closely-related EHEC.     

Evaluation of a simplified HEp-2 cell adherence assay for Escherichia coli isolated from south Indian children with acute diarrhea and controls.

A simplified HEp-2 cell adhesion assay was performed with stored Escherichia coli isolates from 761 children with acute diarrhea and 531 matched controls, and the results were evaluated by means of fluorescent actin staining and hybridization with DNA probes. The prevalence rates of localized adherence and aggregative adherence were significantly higher for patients (9.9 and 7.6%, respectively) than for controls (3.7 and 3.9%, respectively).     

Genotypic and phenotypic characterization of Escherichia coli isolates from dogs manifesting attaching and effacing lesions.

Thirteen Escherichia coli isolates from dogs manifesting attaching and effacing lesions were characterized genetically with respect to the presence of the following virulence determinants associated with human enteropathogenic E. coli (EPEC): eaeA, encoding the outer membrane protein intimin; eaeB, which is necessary for inducing signal transduction; bfpA, encoding the bundle-forming pilus; and the EAF (stands for EPEC adherence factor) plasmid. These isolates were also analyzed phenotypically with respect to adherence to mammalian cells in vivo and in vitro. Nine of these 13 isolates were found to be eaeA positive by PCR: four of these nine were eaeB positive. The 5' end, but not the 3' end, of the eaeA gene was amplified by PCR when primers derived from the eaeA gene of EPEC were used. Six and eight of these 13 isolates were found to be bfpA positive and EAF positive, respectively. The bfpA gene and EAF locus were found on high-molecular-weight plasmids, whereas the eaeA and eaeB genes were chromosomally located when present. Only one canine E. coli isolate, 4221, which was positive for eaeA, eaeB, bfpA, and EAF, adhered to HEp-2 cells in a localized manner and was positive in the fluorescence actin staining test. The nine eaeA-positive isolates adhered to the mucosal surface of piglet ileal explants and induced some microvillus effacement. However, when tested in experimentally inoculated gnotobiotic piglets, isolate 4221 did not induce attaching and effacing lesions at any level of the intestinal tract. Our results indicate that canine E. coli isolates associated with attaching and effacing lesions share some properties with human EPEC but form a heterogeneous group.     

paa, originally identified in attaching and effacing Escherichia coli, is also associated with enterotoxigenic E. coli

Previous studies on virotypes and antimicrobial resistance in a collection of porcine enterotoxigenic Escherichia coli (ETEC) O149 strains from Quebec revealed an increase in the number of multiresistant strains (in particular to tetracycline) and the appearance of new virulence factors with time. Among these factors is paa (for porcine attaching- and effacing-associated), originally identified in a porcine enteropathogenic strain, but also present in enterohemorrhagic E. coli O157:H7. In the present study, the association of paa with other ETEC virulence genes, its conservation and expression were investigated in the O149 ETEC collection. All 37 paa-positive strains possessed estB, elt, astA and faeG, and more than half also carried the estA gene, defining two main virotypes, estA(+) and estA(-). Most strains were tetA- or tetB-positive, or both. paa is carried on high molecular weight plasmids. On tetA plasmids, paa is mostly found with enterotoxin gene estA and autotransporter gene sepA. Paa, a 30 kDa protein, is highly conserved and expressed in these strains. Moreover, paaETEC and porcine EPEC/EHEC contain IS signatures, suggesting that paa could be derived from a common ancestor. All these observations suggest a broader role than previously assessed in virulence for paa.     

Truncated enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimin (EaeA) fusion proteins promote adherence of EHEC strains to HEp-2 cells.

Intimin, the product of the eaeA gene in enterohemorrhagic Escherichia coli O157:H7 (EHEC), is required for intimate adherence of these organisms to tissue culture cells and formation of the attaching and effacing lesion in the gnotobiotic pig. Because of the importance of intimin in the pathogenesis of EHEC O157:H7 infection in this animal model, we began a structure-function analysis of EaeA. For this purpose, we constructed amino-terminal fusions of the intimin protein with six histidine residues to form two independent fusions. The longer fusion, RIHisEae, contained 900 of the 935 predicted amino acids and included all but the extreme amino terminus. The second fusion, RVHdHisEae, consisted of the carboxyl two-thirds of the protein. Purified extracts of either construct enhanced binding of wild-type 86-24 to HEp-2 cells and conferred HEp-2 cell adherence on 86-24eaeDelta10, an eaeA deletion mutant, and B2F1, an EHEC O91:1-121 eaeA mutant strain. When 86-24eaeDelta10 was transformed with either of the plasmids encoding the intimin fusion proteins, the transformant behaved like the wild-type parent strain and displayed localized adherence to HEp-2 cells, with positive fluorescent-actin staining. In addition, polyclonal antisera raised against RIHisEae reacted with both fusion constructs and recognized an outer membrane protein of the same mass as intimin (97 kDa) in EHEC and enteropathogenic E. coli but not E. coli K-12. The intimin-specific antisera also blocked adherence of EHEC to HEp-2 cells. Thus, intimin (i) is a 97-kDa outer membrane protein in EHEC that serves as a requisite adhesin for attachment of the bacteria to epithelial cells, even when the protein is truncated by one-third at its amino terminus and (ii) can be added exogenously to specifically facilitate HEp-2 cell adherence of EHEC but not E. coli K-12.     

Patterns of adherence to HEp-2 cells and actin polymerisation by toxigenic Corynebacterium diphtheriae strains

Corynebacterium diphtheriae strains displayed different degrees of attachment to HEp-2 cell monolayers with two distinct adherence patterns, termed localised (LA) and diffuse (DA). The LA phenotype predominated over the DA phenotype. The non-sucrose fermenting strains expressing DA pattern adhered mostly with high index values (> or =10bact/cell). Low adhesion index (<10bact/cell) was mainly observed among sucrose fermenting strains. The fluorescein isothiocyanate (FITC)-labelled phalloidin assay (fluorescent-actin staining test) showed positive results for microorganisms of both LA and DA phenotypes. The FITC-labelled C. diphtheriae non-fimbrial surface proteins 67-72p interacted directly with HEp-2 cell membranes. Therefore, toxigenic C. diphtheriae exhibited LA and DA adherence patterns and ability to induce actin polymerisation. The experimental evidences also pointed to 67-72p as putative adhesins of C. diphtheriae to HEp-2 cells.     

Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli.

Attaching and effacing (A/E) intestinal lesions are produced by enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and RDEC-1, a pathogen of weanling rabbits. We recently identified a chromosomal locus (eae[E. coli A/E]) which is required for A/E activity in a wild-type EPEC strain. Sequences homologous to those of an eae gene probe were detected in EPEC, RDEC-1, and EHEC isolates. We report here that the eae gene is chromosomally encoded in all EPEC and EHEC strains tested and in RDEC-1. In addition, the eae probe was found to be 100% sensitive and 98% specific in detecting E. coli of EPEC serogroups that demonstrate A/E activity. Ten percent of E. coli of EPEC serogroups that hybridized with the eae probe and produced A/E activity did not hybridize with the EAF (EPEC adherence factor) probe, a plasmid-associated diagnostic probe which is currently used to identify EPEC. In addition to A/E factors, plasmid-associated adhesins also contribute to the pathogenesis of EPEC and RDEC-1. To further investigate the role of plasmid-associated adherence, a hybrid RDEC-1-EPEC strain containing the adherence plasmid of an EPEC strain in the A/E background of RDEC-1 was constructed. This hybrid strain, unlike the parent RDEC-1 strain, produced A/E lesions on human tissue culture cells, which suggests that the EPEC adherence plasmid provides tissue specificity to the hybrid strain and that the A/E factors of RDEC-1 are not host restricted.     

HeLa cell adherence and cytotoxin production by enteropathogenic Escherichia coli isolated from infants with diarrhea in Thailand.

Enteropathogenic Escherichia coli (EPEC) strains isolated from hospitalized infants with diarrhea in Thailand were examined for HeLa cell adherence and cytotoxin production. Of 101 strains examined, 56 adhered to HeLa cells in a localized pattern (LA), 27 adhered in a diffuse pattern (DA), and 18 did not adhere. All 56 LA EPEC strains were O:K serotype O119:K69. A total of 20 (83%) of 24 EPEC O86:K61 strains and 7 (38%) of 19 EPEC strains belonging to six other O:K serotypes exhibited DA. All LA EPEC strains hybridized with a DNA probe for genes encoding EPEC adherence factor, whereas none of the 27 DA or 18 nonadherent EPEC strains hybridized with EPEC adherence factor probe. Sonic extracts of 57 (58%) of 98 EPEC strains tested at a dilution of 1:100 caused greater than 25% mortality of HeLa cell monolayers. A total of 50 (88%) of 57 cytotoxic sonic extracts were inhibited to various degrees by a 1:500 dilution of polyclonal rabbit antisera to purified Shiga toxin. The mean percent inhibition of cytotoxic sonic extracts by anti-Shiga toxin was 67% (range, 29 to 89%). Fifty percent (38 of 56) of LA EPEC strains, fifty-two percent (14 of 27) of DA EPEC strains, and fifty-three percent (8 of 15) of nonadherent EPEC strains produced Shiga-like toxins. Both adherence and low levels of cell-associated cytotoxins were identified in EPEC strains from Thailand, but there did not appear to be an association between these two factors.     

Serotypes,virulence genes,and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from healthy sheep in Spain

Fecal swabs obtained from 1,300 healthy lambs in 93 flocks in Spain in 1997 were examined for Shiga toxin-producing Escherichia coli (STEC). STEC O157:H7 strains were isolated from 5 (0.4%) animals in 4 flocks, and non-O157 STEC strains were isolated from 462 (36%) lambs in 63 flocks. A total of 384 ovine STEC strains were characterized in this study. PCR showed that 213 (55%) strains carried the stx(1) gene, 10 (3%) possessed the stx(2) gene, and 161 (42%) carried both the stx(1) and the stx(2) genes. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 106 (28%) and 23 (6%) of the STEC strains, respectively. The STEC strains belonged to 35 O serogroups and 64 O:H serotypes (including 18 new serotypes). However, 72% were of 1 of the following 12 serotypes: O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O136:H20, O146:H8, O146:H21, O156:H-, O166:H28, and ONT:H21 (where NT is nontypeable). Although the 384 STEC strains belonged to 95 different seropathotypes (associations between serotypes and virulence genes), 49% of strains belonged to only 11. O91:H- stx(1) stx(2) (54 strains) was the most common seropathotype, followed by O128:H- stx(1) stx(2) (33 strains) and O6:H10 stx(1) (25 strains). Three strains of serotypes O26:H11, O156:H11, and OX177:H11 had intimin type beta1; 5 strains of serotype O157:H7 possessed intimin type gamma1; and 15 strains of serotypes O49:H-, O52:H12, O156:H- (12 strains), and O156:H25 had the new intimin, intimin type zeta. The majority (82%) of ovine STEC strains belonged to serotypes previously found to be associated with human STEC strains, and 51% belonged to serotypes associated with STEC strains isolated from patients with hemolytic-uremic syndrome. Thus, this study confirms that healthy sheep are a major reservoir of STEC strains pathogenic for humans.     

HEp-2 cell adherence patterns, serotyping, and DNA analysis of Escherichia coli isolates from eight patients with AIDS and chronic diarrhea.

Three morphologic patterns of interaction between bacteria and enterocytes have been observed in colonic biopsy specimens from AIDS patients with chronic diarrhea in the United States. The DNA encoding virulence factors and the HEp-2 cell adherence patterns of Escherichia coli strains isolated from the stools of eight symptomatic AIDS patients were compared with those of five control strains with known adherence patterns. One clinical isolate from a patient with attaching-and-effacing enteropathy displayed the localized adherence attaching-and-effacing pattern typical of enteropathogenic E. coli on HEp-2 cells, five isolates displayed the "stacked-brick" aggregative adherence pattern typical of enteroaggregative E. coli strains, and one isolate showed the pattern characteristic of diffusely adherent E. coli. One patient's isolate displayed features of all three patterns. No clinical isolate hybridized with standard probes for enteropathogenic, enteroaggregative, diffusely adherent, enterotoxigenic, and enteroinvasive E. coli strains. Thus, isolates from symptomatic AIDS patients in the United States can display the same interactive patterns with HEp-2 cells as the agents of pediatric or traveler's diarrhea, but lack their typical virulence factors.     

Localization of a determinant for HEp-2 adherence by enteropathogenic Escherichia coli.

pMAR2, a 60-megadalton plasmid encoding localized HEp-2 adherence in enteropathogenic Escherichia coli, was mapped with BamHI, HindIII, and SalI. Deletion and insertion mutants were constructed and used to define a potential DNA probe. Preliminary results indicate that this probe is sensitive and specific for the genes encoding the enteropathogenic E. coli adherence factor.