Aggregative adherence fimbria I expression in enteroaggregative Escherichia coli requires two unlinked plasmid regions.

Adherence to HEp-2 cells by many enteroaggregative Escherichia coli (EAggEC) strains is associated with the expression of flexible, bundle-forming fimbriae 2 to 3 nm in diameter, designated aggregative adherence fimbriae I (AAF/I). We have previously reported the molecular cloning and TnphoA mutagenesis of AAF/I genes from the large plasmid of prototype EAggEC strain 17-2 (J. P. Nataro, Y. Deng, D. R. Maneval, A. L. German, W. C. Martin, and M. M. Levine, Infect. Immun. 60:2297-2304, 1992). Here, we report that further mapping and subcloning of AAF/I regions suggest that expression of the fimbriae requires two separate plasmid regions (designated regions 1 and 2). Approximately 9 kb of DNA unnecessary for fimbrial expression separates the two regions; this intervening segment encodes the EAggEC heat-stable enterotoxin (EAST1). Neither region was capable of conferring aggregative HEp-2 adherence (AA) when cloned individually; when the two regions were cloned as a single fragment or when each was cloned into a different vector and introduced into the same E. coli HB101 cell, AA was restored. AA-positive constructs also expressed human erythrocyte hemagglutination, autoagglutination in broth cultures, and the production of AAF/I as detected by immunogold electron microscopy.     

Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes.

Strains of enteroaggregative Escherichia coli (EAggEC) have been implicated in several studies as important agents of persistent diarrhea among infants in the developing world. We have previously shown that the aggregative adherence (AA) property of EAggEC is associated with the presence of a 60-MDa plasmid which confers AA when introduced into E. coli HB101. Here, we report the cloning of the AA determinant from EAggEC strain 17-2 into the 21.5-kb cosmid vector pCVD301. TnphoA mutagenesis of the AA cosmid clone pJPN31 implicated an AA region of approximately 12 kb. Transmission electron microscopy of HB101 (pJPN31) revealed the presence of bundle-forming fimbriae, which were absent in AA- TnphoA insertion mutants. The presence of these fimbriae, AA, and hemagglutination (HA) of human erythrocytes were all concurrently lost by single-insertion mutations. A 14-kDa protein was seen on polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of surface shear preparations from fimbriated clones. Twelve of nineteen volunteers fed EAggEC 17-2 developed rises in antibodies to the 14-kDa protein as determined by Western blot. We have termed the cloned bundle-forming fimbriae aggregative adherence fimbriae I (AAF/I); positivity with a previously described EAggEC probe and human erythrocyte HA appear to correlate with the presence of AAF/I.     

A novel cryohemagglutinin associated with adherence of enteroaggregative Escherichia coli.

Strain O42 (serotype O44:H18) of enteroaggregative Escherichia coli (EAggEC) has been shown to be pathogenic in volunteer experiments. This strain exhibited plasmid (pO42)-encoded D-mannose-resistant hemagglutinating activity (MRHA) that was detected only at low temperatures (e.g., 0 degrees C) and only with human erythrocytes. The production of this cryogenic MRHA (cryo-MRHA) was observed when the bacteria were grown in liquid media and was strictly regulated by bacterial growth temperatures. Transposon-insertion mutagenesis revealed that this MRHA is associated with (i) bacterial clump formation in liquid cultures, (ii) bacterial adherence to HEp-2 cells as well as (Formalin-fixed) human colonic mucosa, and (iii) production of a 16-kDa outer membrane protein. The PCR designed on the basis of the determined cryo-MRHA-associated DNA sequence sharply distinguished strain O42 from eight other EAggEC strains whose MRHAs were detected at both cold and room temperatures to the same (or similar) extent. Strain O42 possessed a surface layer that may enhance the pO42-mediated adherence. The data suggest that a plasmid-encoded cryo-MRHA is a candidate for a major adhesin of EAggEC strain O42.     

Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family

Enteroaggregative Escherichia coli (EAEC) is recognized as an emerging cause of diarrhea in children and adults worldwide, and recent studies have implicated EAEC in persistent diarrhea in patients infected with human immunodeficiency virus (HIV). In this study, we identified aggregative adhesion fimbria type III (AAF-III) in isolate 55989, a typical EAEC strain. Analysis of the sequence of the plasmid-borne agg-3 gene cluster encoding AAF-III showed this cluster to be closely related to the agg and aaf operons and to the afa operons carried by diffusely adherent pathogenic E. coli. We investigated the adhesion properties of a collection of 25 EAEC strains isolated from HIV-infected patients presenting with persistent diarrhea. We found that a minority of strains (36%) carried sequences similar to those of the agg and aaf operons, which encode AAF-I and AAF-II, respectively. We developed PCR assays specific for the agg-3 operon. In our collection, the frequency of AAF-III strains was similar (12%) to that of AAF-I strains (16%) but higher than that of AAF-II isolates (0%). Differences between EAEC strains in terms of the virulence factors present render detection of these strains difficult with the available DNA probes. Based on comparison of the agg, aaf, and agg-3 operons, we defined an AAF probe internal to the adhesion gene clusters and demonstrated that it was efficient for the identification of EAEC strains. We investigated 32 EAEC isolates, of which only 34.4% were detected with the classical CVD432 probe (detecting pAA virulence plasmids) whereas 65.6% were detected with the AAF probe.     

Characteristics of adherence of enteroaggregative Escherichia coli to human and animal mucosa.

An Escherichia coli strain (serotype O127a:H2) that had been isolated from a child with diarrhea in Thailand and that was negative for the virulence factors of the four categories of diarrheagenic E. coli (enterotoxigenic, enteropathogenic, enteroinvasive, and enterohemorrhagic) and that showed an aggregative pattern of adherence to HeLa cells was investigated for adherence to native or Formalin-fixed human and animal mucosa. The hemagglutinating activity and adherence ability of the bacteria were resistant to D-mannose and were strictly regulated by environmental conditions. Genetic data supported the close relation between the hemagglutinating activity and adherence ability. In accordance with the adherence pattern on tissue-cultured cells, the bacteria adhered to human and animal mucosa, as evidenced by a direct gold-labeling analysis. In human intestines, Formalin-fixed mucous coatings, epithelial cells of colonic mucosa, epithelial cells of ileal single lymphoid follicles and Peyer's patches, and the absorptive cells of jejunal or ileal villi provided adherence targets. Adherence to M cells in the Peyer's patch-associated epithelium was also confirmed. The adherence levels to native jejunal or ileal human villi were low, as was the case with the corresponding Formalin-fixed villi. In human urinary tract, the superficial epithelial cells of both native and Formalin-fixed ureter provided striking adherence targets. In animal (porcine and rabbit) small intestines, the bacteria adhered to the native villi to a lesser extent than to the Formalin-fixed villi. The adherence levels were compared with those of enterotoxigenic E. coli with colonization factor antigen (CFA)/I pili or CFA/II pili. The data suggested unique mucosa adherence characteristics of the enteroaggregative E. coli strain. The possibility of the adherence ability as a virulence factor was discussed.     

Antibodies raised against the outer membrane protein interrupt adherence of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAggEC) is a distinct category of diarrheal pathogen implicated as the cause of persistent diarrhea. The pathogen exhibits a characteristic "stacked-brick" pattern of aggregation when incubated with HEp-2 cells. The outer membrane protein (OMP) profile of a prototype EAggEC strain (F03) reflected the presence of one major 30-kDa protein. The OMP is expressed in the presence of the 60-MDa plasmid that the strain harbors. Antibodies were raised against the OMP by injecting the protein into a rabbit. The manifestation of an adherence phenotype on HEp-2 cells was observed for F03 and other strains that express OMP in the presence and absence of anti-OMP serum. Clumps of bacteria forming an aggregative pattern were observed in the HEp-2 cell assay in the absence of OMP antibodies, whereas a few bacteria attached to the cells in the presence of OMP antibodies. Mannose-resistant hemagglutination of human erythrocytes observed in the presence of EAggEC strains was inhibited in the presence of anti-OMP serum. Sequence analysis of a peptide generated by protease digestion of OMP exhibited 90% homology to a peptide of flagellin protein encoded by the hag gene of Serratia marcescens. Immunolabeling of the outer membrane by colloidal gold confirmed the protein to be an OMP. Our results suggest that the OMP of EAggEC have common antigenic properties. Antibodies raised against the protein can prevent adherence in vitro and could potentially interrupt the natural disease.     

Comparison of a modified adherence assay with existing assay methods for identification of enteroaggregative Escherichia coli.

Localized, diffuse, and aggregative adherence patterns of Escherichia coli identified with specific DNA probes were compared in cell culture adherence assays by using the Center for Vaccine Development, Baltimore, method, the University of Texas Medical School, Houston (UTH), method, and a modified UTH method. Increasing postwash incubation time from 2 to 4 h in the modified UTH method allowed identification of enteroaggregative E. coli, which was otherwise not identified by the original UTH method.     

Comparison of Escherichia coli fimbrial antigen F7 with type 1 fimbriae.

Two Escherichia coli O6:K2:H1 strains, C1212 and C1214, isolated from urinary tract infections, were compared for their capacity to adhere to various cells. After growth on solid medium, only C1212 bacteria agglutinate human erythrocytes and attach to urinary epithelial cells. Both of these reactions are mannose resistant. In contrast, C1214 bacteria cause a mannose-sensitive agglutination of guinea pig erythrocytes, show a mannose-sensitive attachment to buccal epithelial cells, and attach to urinary mucus. Immunoelectron microscopy revealed that C1214 bacteria possess type 1 fimbriae (mannose sensitive), which are not present in C1212 bacteria when this strain is grown on solid medium. The fimbriae of C1212 (mannose resistant) were also demonstrated by immunoelectron microscopy. We call these fimbriae demonstrated in C1212 the E. coli F7 antigen. Urinary mucus, and probably mucous material elsewhere, may function as a trap for Enterobacteriaceae with type 1 fimbriae by the specific adherence of such bacteria. We consider this a nonimmune resistance mechanism against disease caused by Enterobacteriaceae.     

CS31A, a new K88-related fimbrial antigen on bovine enterotoxigenic and septicemic Escherichia coli strains.

The nature of the common surface antigen of six hemagglutinating and adhesive piliated Escherichia coli strains isolated from diarrheic or septicemic calves was studied. By electron microscopy studies, the E. coli surface antigen designated CS31A was found on bacterial cells and in purified form to consist of thin (2-nm) "fibrillar" fimbriae. E. coli 31A, which was cured of a 105-megadalton plasmid, failed to express CS31A fimbriae, but retained the ability to hemagglutinate and to adhere in vitro on intestinal cells. Conversely, E. coli K-12, harboring the 105-megadalton plasmid originating from strain 31A, produced CS31A fimbriae but was not able to hemagglutinate or adhere on intestinal cells. A single fimbrial subunit of 29 kilodaltons was observed when purified fimbriae from the 105-megadalton plasmid-containing E. coli K-12 strain was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis or eluted by gel filtration after dissociation by 8.5 M guanidium hydrochloride from an S300 Sephacryl column. Western immunoblot analysis and the N-terminal sequence and amino acid composition of CS31A indicate structural and immunological relatedness between CS31A and K88 protein subunits.     

Escherichia coli O125ac:H6 encompasses atypical enteropathogenic E. coli strains that display the aggregative adherence pattern

O125 is an enteropathogenic Escherichia coli (EPEC) serogroup, which includes the O125ac:H6 serotype, defined as atypical EPEC. Strains of this serotype displayed the aggregative adherence (AA) pattern with HEp-2, Caco-2, T84, and HT-29 cells, possessed all the LEE region genes, and expressed intimin, Tir, and EspABD, although the attaching-effacing lesion was not detected in vitro. These results confirm that E. coli O125ac:H6 is atypical EPEC that displays the AA pattern and indicate the necessity of testing for EPEC genes combined with the determination of the adherence pattern for atypical EPEC identification.     

The fimbrial and non-fimbrial haemagglutinins of Escherichia coli.

Both cultures of Escherichia coli were examined for mannose-sensitive (MS) haemagglutinin in rocked-tile tests with guinea-pig red cells at ambient temperature, and agar plate cultures were examined for mannose-resistant eluting (MRE) haemagglutinins against 14 species of red cells in tests mixed at 3--5 degrees C in the presence of .05% (w/v) D-mannose. Ox, sheep, human, pig, horse, guinea-pig, and fowl red cells were required to detect the various patterns of MRE haemagglutination with the different species of cells. Of 387 strains in 155 O serogroups, 95 formed both MS and MRE haemagglutinins (MS+/MRE+), 198 formed only MS (MS+/MRE-), 21 only MRE (MS+/MRE+), and 73 neither (MS-/MRE-). Strains of more than one of these types, and MRE+ strains with different cell specificities were found in many of the serogroups. Some strains in 144 O serogroups had MS haemagglutinin and some in 50 an MRE haemagglutinin. The presence of MS haemagglutinin in a culture was invariably associated with the presence of type-1 fimbriae on the bacteria. All MS+ strains shared a common antigen in their type-1 fimbriae and three groups of these strains possessed also a group-specific fimbrial antigen. The presence of certain kinds of MRE haemagglutinin in over half the MRE+ strains was associated with that of type-MRE fimbriae, but fimbriae were not detected in the other MRE+ strains. The antigens of the MRE haemagglutinins in different strains were heterogeneous and differed from those of the type-1 fimbriae of MS+ strains. Three series of strains from normal faeces, and from patients with infantile diarrhoea and urinary-tract infections each included a minority possessing neither type of haemagglutinin, but this observation did not preclude a role of the haemagglutinins in colonization or pathogenicity.     

Characterization of the F41 fimbrial antigen of enterotoxigenic Escherichia coli by using monoclonal antibodies.

We produced monoclonal antibodies (MAbs) from 23 different murine hybridoma cell lines against the F41 fimbrial antigen of bovine and porcine enterotoxigenic Escherichia coli. Cell lines were created by fusing myeloma cells and spleen cells of mice that were immunized with either purified F41 or with Formalin-killed whole cells. The specificity of the MAbs to the F41 antigen was proven by enzyme-linked immunosorbent assays (ELISAs) and radioimmunoprecipitation tests. Epitope analysis with a competition ELISA revealed that the 23 MAbs recognized at least five epitopes. These results were corroborated by those of immunodiffusion tests, in which all possible combinations of two MAbs were tested against ultrasonically disintegrated F41 antigen. In a double-antibody sandwich ELISA, all peroxidase-conjugated MAbs bound to the F41 antigen of all 182 bacterial strains that were tested. Apparently, the epitopes recognized by the MAbs are highly conserved. Immunoelectron microscopy revealed that the MAbs were directed to fimbrial structures 3 to 4 nm in diameter and that the epitopes were equally distributed along the fimbriae. Consequences for the replacement of polyclonal antisera by MAbs in diagnostic tests are discussed. The results of the radioimmunoprecipitation assay suggested that F41 fimbriae are composed of a single repeating 29,000-dalton protein subunit; however, we could not exclude the possibility of the existence of minor fimbrial components.     

Development of PCR for screening of enteroaggregative Escherichia coli.

In this study, we determined the sequence of the EcoRI-PstI fragment of the plasmid pCVD432, also termed the enteroaggregative Escherichia coli (EAggEC) probe. A primer pair complementary to this probe was designed for PCR amplification of a 630-bp region. Comparison of the analysis of the EAggEC probe sequence with those in database libraries revealed no significant similarity to any known bacterial gene. Pure cultures of E. coli cells, as well as mixed cultures from stool specimens, were investigated with the PCR assay, the EAggEC probe test, and the adherence test. Of 50 E. coli strains which demonstrated aggregative adherence to HEp-2 cells, 43 (86%) were positive with the EAggEC PCR. All 43 of these strains reacted with the EAggEC probe. Six EAggEC strains gave negative results by both molecular techniques. In contrast, only 4 of 418 (0.96%) strains representing other categories of diarrheagenic E. coli demonstrated a positive PCR result. The PCR was also successful in screening for the presence of EAggEC in enriched cultures grown from stool specimens. Compared with cell culture assays and colony hybridization, our findings revealed that the PCR assay was more rapid, simple, and highly sensitive and can therefore be recommended as a screening method for EAggEC in the clinical laboratory.     

T84 cells in culture as a model for enteroaggregative Escherichia coli pathogenesis.

Enteroaggregative Escherichia coli (EAEC) is an important cause of persistent diarrhea in many developing parts of the world, yet the pathogenetic mechanisms of EAEC diarrhea are unknown. Experiments with animal models suggest that EAEC strains damage the intestinal mucosa, and a putative cytotoxin has been described. To characterize the mucosal effects of EAEC, we studied strain 042, which we have shown to cause diarrhea in adult volunteers. Strain 042 was incubated in an in vitro organ culture model with biopsy-derived normal intestinal mucosa from pediatric patients. Strain 042 adhered strongly to samples of jejunal, ileal, and colonic mucosa. In addition, scanning electron microscopic examination of in vitro-infected intestinal biopsies revealed cytotoxic effects marked by exfoliation of mucosal epithelial cells. To develop an in vitro model to study these effects, we incubated 042 with polarized monolayers of the human intestinal epithelial cell lines Caco-2 and T84. Strain 042 adhered strongly to T84 cells but not to Caco-2 cells. T84 cells infected with 042 displayed marked toxic effects, most prominently in areas where bacteria were adhering. The apical membrane of damaged cells exhibited vesiculation and shedding of microvilli. The cytoplasm of affected cells displayed subnuclear vacuolization, and in some cases, nuclei of affected cells became separated from the surrounding cytoplasm. Severely affected cells ruptured, releasing their nuclei. Vacuolated remnant cells were seen throughout the monolayer. Strain 042 was not internalized by T84 cells. We concluded that EAEC strain 042 alters intestinal cell morphology, ultimately leading to cell death. Although the factor(s) required for this effect remains to be elucidated, T84 cells may serve as a valuable model in EAEC pathogenesis studies.     

Isolation and characterization of F12 adhesive fimbrial antigen from uropathogenic Escherichia coli strains

The adhesive fimbrial antigen F12 from a strain of uropathogenic Escherichia coli has been isolated and characterized. The antigen was purified by ammonium sulfate precipitation and gel chromatography. The protein subunit of the F12 fimbria has a molecular weight of 18,200; the N-terminal amino acid sequence of the subunit shows close resemblance to that of the subunits of other F fimbriae and the type 1 fimbriae. We identified in these proteins a pattern of alternating conserved and variable amino acid residues which could indicate a special structural and functional feature.     

Integron-associated antibiotic resistance in enteroaggregative and enteroinvasive Escherichia coli

Ten enteroinvasive (EIEC) and 25 enteroaggregative (EaggEC) E. coli strains isolated from Senegalese patients were analyzed for their integron content. All strains were resistant to at least two antibiotics. Four EIEC and 15 EaggEC were found to carry a class 1 integron. An identical integron carrying a single dfrA5 cassette, conferring resistance to trimethoprim, was identified in all four EIEC strains. Five EaggEC strains harbored an integron with a single cassette, dfrA7, while the remaining 10 strains carried two integrons, one with a single cassette, aadA1a conferring resistance to streptomycin and spectinomycin, and the second one bearing two cassettes, dfrA13 and oxa5, the later being a beta-lactam resistance cassette. The presence of these integrons is worrying, because trimethoprim is largely used for diarrheal disease therapy in Africa. Thus, the presence of integrons in diarrheagenic strains is of public health importance because a limited number of antibiotics are available in developing countries.     

Colonization by enteroaggregative Escherichia coli in travelers with and without diarrhea.

Enteroaggregative Escherichia coli (EAggEC) has been found to be associated with pediatric diarrhea in developing countries. In order to determine the role of EAggEC as an agent of traveler's diarrhea, we used a sensitive and specific DNA probe for EAggEC to screen bacterial colony blots from 278 volunteers before and after travel. Colonization with EAggEC was infrequent (2.5%) prior to travel but rose to 27 to 33% after travel in volunteers who took either placebo or trimethoprim-sulfamethoxazole. Travelers who took trimethoprimsulfamethoxazole were colonized with organisms that were uniformly resistant to that antimicrobial agent; when volunteers received ciprofloxacin, colonization with EAggEC was prevented (2.0%). Although colonization rates were high in the placebo and trimethoprim-sulfamethoxazole groups, only a minority of travelers who were colonized with EAggEC experienced diarrhea. On the basis of our data, we suggest that colonization with EAggEC alone is not sufficient to cause traveler's diarrhea.     

Monoclonal antibodies against the nonhemagglutinating fimbrial antigen 1C (pseudotype 1) of Escherichia coli.

Hybridoma-derived monoclonal antibodies were produced with fimbrial preparations from Escherichia coli 20025 (04:K12:H-) with fimbrial (F) antigens 1C, 13, one related to 12, and one preliminarily termed y and from E. coli 2980 (018ac:K5:H-) with F antigens 1C and 8. Two clones of subclonal hybrid cells were studied which produced monoclonal antibodies (mc-20025-F2b, immunoglobulin G2b [IgG2b]; mc-2980-F2, IgG1) that were reactive with E. coli 20025, 2980, and a number of additional strains which exhibited the F1C antigen. Results of enzyme-linked immunosorbent assay and Western blot analysis indicated that the antibodies had F1C specificity, and competitive enzyme-linked immunosorbent assay with 125I-labeled antibodies showed that they recognized different epitopes on the fimbrial subunit. Neither of the antibodies agglutinated F1C-fimbriated E. coli but bound to the bacteria. There was no binding to E. coli without F1C fimbriae.     

Studies with enteroaggregative Escherichia coli in the gnotobiotic piglet gastroenteritis model.

Two strains of enteroaggregative Escherichia coli of human origin fed to gnotobiotic piglets caused diarrhea or death in the majority of them. Histological examination revealed moderate hyperemia of the distal small intestine and cecum, swelling of small intestinal villi, and layers of aggregated bacteria stacked together in a mucus gel-like matrix overlying intact epithelium. These findings confirm that enteroaggregative E. coli strains produce distinctive intestinal lesions different from those caused by other major categories of diarrheagenic E. coli.