Prevalence of HEp-2 cell-adherent Escherichia coli and characterisation of enteroaggregative E. coli and chain-like adherent E. coli isolated from children with and without diarrhoea, in Londrina, Brazil

A total of 919 Escherichia coli isolates from 125 children with diarrhoea (cases) and 98 controls were assayed for adherence to HEp-2 cells. Localised adherence was found only in isolates from cases. Diffuse, aggregative (AA), chain-like adherence (CLA) and variants of the AA pattern were found in both cases and controls. The AA isolates were tested for gene sequences associated with enteroaggregative E. coli (EAEC). Only 25% of the isolates hybridised with the EAEC probe, and the aafA, astA and pet gene sequences were found in 7.9%, 44.7% and 7.9% of the isolates, respectively. The aggA gene was not found, although 7.9% were positive for aggC. The CLA isolates reacted with the EAEC probe (55.6%), and the aggC, astA and pet gene sequences were found in 66.7%, 33.3% and 11.1%, respectively. The aggR (55.6%), aspU (55.6%), shf (33.3%) and she (22.2%) genes were also found in CLA isolates.     

Diversity of surface structures and virulence genetic markers among enteroaggregative Escherichia coli (EAEC) strains with and without the EAEC DNA probe sequence

The expression of surface structures and the presence of DNA sequences related to putative virulence factors were investigated in 22 enteroaggregative Escherichia coli strains (EAEC). Fimbria was the most frequent (72.7%) structure identified. Only strains hybridising with the EAEC DNA probe carried aggA, but one strain produced a similar but unrelated bundle-like structure. All probe-positive and 62.5% of the probe-negative strains carried the virulence genes tested; aspU and irp2 prevailed among the former strains. The EAEC probe-positive strains were more diverse, and some of these strains, which promoted cell detachment, also carried the hly and pap sequences, thus suggesting they might represent uropathogenic E. coli.     

Human milk fractions inhibit the adherence of diffusely adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) to HeLa cells

Binding to a specific receptor is an essential step for most enteropathogens to initiate an intestinal infection. We analyzed the inhibitory effect of human milk and its protein components on adhesion of two diarrheagenic Escherichia coli strains, diffusely adherent E. coli (DAEC) and enteroaggregative E. coli (EAEC), to HeLa cells. Defatted milk, whey proteins, immunoglobulin and non-immunoglobulin fractions, in concentrations lower than usually found in whole milk, inhibited both DAEC and EAEC adhesion, indicating that human milk components may contribute to the defense of the infants against enteropathogens.     

Cloning and nucleotide sequence of the eae gene homologue from enterohemorrhagic Escherichia coli serotype O157:H7

The eae gene has recently been shown to be necessary for the attaching and effacing (AE) activity of enteropathogenic Escherichia coli (EPEC) on intestinal epithelial cells. In this paper we report the cloning and nucleotide sequence of a similar gene from a strain of enterohemorrhagic E. coli (EHEC) serotype O157:H7. An EHEC eae sequence was identified which was 97% homologous to the EPEC eae gene for the first 2200 bp and 59% homologous over the last 800 bp. Both eae sequences show 50% homology to the central region of the Yersinia pseudotuberculosis inv gene. The receptor-binding domain of the inv gene product lies near the carboxyl terminus. This suggests that the predicted amino acid sequence divergence in the carboxyl termini of the eae gene products might result in different antigenic and receptor specificity of these putative adhesins.     

Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients

We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC.     

Magnesium ions are required for HEp-2 cell adhesion by enteroaggregative strains of Escherichia coli O126:H27 and O44:H18

Enteroaggregative strains of Escherichia coli, belonging to serotypes O44:H18 and O126:H27, were used to show that magnesium ions were essential for the adhesion of these enteroaggregative strains to HEp-2 cells. The removal of Mg²⁺ ions from culture media was correlated with the inability of strains to produce an outer membrane-associated protein of 18 kDa and a pellicle. It was concluded that magnesium ions were directly involved with the expression of an 18 kDa outer membrane-associated protein by strains of E. coli O126:H27 and O44:H18, and that the outer membrane-associated protein was involved in both HEp-2 adhesion and pellicle formation.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 522/C1 and the international type strain from Escherichia coli O 178

The structure of the O-antigenic polysaccharide (PS) from the enteroaggregative Escherichia coli strain 522/C1 has been determined. Component analysis and (1)H and (13)C NMR spectroscopy techniques were used to elucidate the structure. Inter-residue correlations were determined by (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [ structure: see text]. Analysis of NMR data reveals that on average the PS consists of four repeating units and indicates that the biological repeating unit contains an N-acetylgalactosamine residue at its reducing end. Serotyping of the E. coli strain 522/C1 showed it to be E. coli O 178:H7. Determination of the structure of the O-antigen PS of the international type strain from E. coli O 178:H7 showed that the two polysaccharides have identical repeating units. In addition, this pentasaccharide repeating unit is identical to that of the capsular polysaccharide from E. coli O9:K 38, which also contains O-acetyl groups.     

Structural and immunochemical relationship between the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 396/C-1 and Escherichia coli O126

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 396/C-1 has been determined. Sugar and methylation analyses together with 1H and 13C NMR spectroscopy were the main methods used. Inter-residue correlations were determined by 1H,1H-NOESY, 1H,13C-heteronuclear multiple-bond correlation and dipole-dipole cross-correlated relaxation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [structure: see text]. Analysis of NMR data reveals that on average the PS consists of approximately 13 repeating units and indicates that the biological repeating unit contains an N-acetylglucosamine residue at its reducing end. This structure is different to that reported for the O-antigen polysaccharide from E. coli O126. Monospecific anti-E. coli O126 rabbit serum from The International Escherichia and Klebsiella Centre did not distinguish between the E. coli strain 396/C-1 and the E. coli O126 reference strain, neither in slide agglutination nor in an indirect enzyme immunoassay. Subsequent successful serotyping of the E. coli strain 396/C-1 showed it to be E. coli O126:K+:H27.     

Expression of antigenically distinct fimbriae with hemagglutination and HeLa cell adherence properties by an enteroaggregative Escherichia coli strain belonging to the enteropathogenic serogroup

Abstract An enteroaggregative Escherichia coli (EAggEC) strain (DS92), isolated from a case of infantile diarrhea, was shown to express mannose-resistant hemagglutination and HeLa cell adhering properties when grown at 37°C but not at 28°C. Cellular adherence properties of DS92, which belonged to enteropathogeci serogroup 0125, were shown to correlate well with the expression of fimbriae that were encoded by a 112 kb plasmid. The fimbriae of the EAggEC strain DS92 were composed of 20 kDa subunit proteins and were serologically distinct from fimbrial or non-fimbrial cell surface antigen(s) of other diarrheagenic E. coli strains including the reference EAggEC strain 17-2. Interestingly, the 20-kDa fimbrial protein was found to be antigenically related to 18- and 14.5-kDa cell surface proteins of two other locally isolated EAggEC strains belonging to the enteropathogenic serogroup 086.     

Structural elucidation of the O-antigenic polysaccharide from the enteroaggregative Escherichia coli strain 180/C3 and its immunochemical relationship with E. coli O5 and O65

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 180/C3 has been determined. Sugar and methylation analysis together with (1)H and (13)C NMR spectroscopy were the main methods used. The PS is composed of tetrasaccharide repeating units with the following structure: -->2)beta-D-Quip3NAc-(1-->3)beta-D-RIBf-(1-->4)beta-D-Galp-(1-->3)alpha-D-GalpNAc-(1-->. Analysis of NMR data indicates that the presented sequence of sugar residues also represents the biological repeating unit of the O-chain. The structure is closely related to that of O-antigen polysaccharide from E. coli O5 and partially to that of E. coli O65. The difference between the O-antigen from the 180/C3 strain and that of E. coli O5 is the linkage to the D-Quip3NAc residue, which in the latter strain is 4-O-substituted. The E. coli O65 O-antigen contains as part of its linear pentasaccharide repeating unit a similar structural element, namely -->4)-beta-d-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->2)-beta-D-Quip3NAc-(1-->, thereby indicating that a common epitope could be present for the two polysaccharides. Monospecific anti-E. coli O5 rabbit serum did not distinguish between the two positional isomeric structures neither in slide agglutination nor in an indirect enzyme immunoassay. The anti-O65 serum did react with both the 180/C3 and O5 LPS showing a partial cross-reactivity.     

Production of an autoinducer of growth by norepinephrine cultured Escherichia coli O157:H7

Abstract Escherichia coli O157:H7 were cultured in the presence or absence of norepinephrine to generate conditioned media. The presence of a growth-inducing factoKs) in the conditioned media was examined by measurement of the ability of conditioned media to support the growth of fresh cultures of E. coli O157:H7. Supplementation of fresh cultures with as little as 0.024% (v/v) norepinephrine conditioned medium resulted in increased growth as compared to controls, thereby indicating the presence of an autoinducer of growth. Analysis of the production kinetics for the autoinducer during the generation of conditioned media indicates that it differs from other more well characterized autoinducers. It is proposed that the neurohumoral environment of the host may contribute to the production of bacterial growth factors.     

The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens

The probiotic Escherichia coli strain Nissle 1917 (Mutaflor) of serotype O6:K5:H1 was reported to protect gnotobiotic piglets from infection with Salmonella enterica serovar Typhimurium. An important virulence property of Salmonella is invasion of host epithelial cells. Therefore, we tested for interference of E. coli strain Nissle 1917 with Salmonella invasion of INT407 cells. Simultaneous administration of E. coli strain Nissle 1917 and Salmonella resulted in up to 70% reduction of Salmonella invasion efficiency. Furthermore, invasion of Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and even of Listeria monocytogenes were inhibited by the probiotic E. coli strain Nissle 1917 without affecting the viability of the invasive bacteria. The observed inhibition of invasion was not due to the production of microcins by the Nissle 1917 strain because its isogenic microcin-negative mutant SK22D was as effective as the parent strain. Reduced invasion rates were also achieved if strain Nissle 1917 was separated from the invasive bacteria as well as from the INT407 monolayer by a membrane non-permeable for bacteria. We conclude E. coli Nissle 1917 to interfere with bacterial invasion of INT407 cells via a secreted component and not relying on direct physical contact with either the invasive bacteria or the epithelial cells.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 87/D2 and international type strains from E. coli O128

The O-antigen of the lipopolysaccharide (LPS) from the enteroaggregative Escherichia coli strain 87/D2 has been determined by component analysis together with NMR spectroscopy. The polysaccharide has pentasaccharide repeating units in which all the residues have the galacto-configuration. The repeating unit of the O-antigen, elucidated using the O-deacylated LPS, is branched with the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately 0.7 per repeating unit) distributed over two positions. Subsequent analysis showed that the galactose residue carries acetyl groups at either O-3 or O-4 in a ratio of approximately 2:1. The international reference strain from E. coli O128ab was investigated and the repeating unit of the O-antigens has the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately one per repeating unit) distributed over two positions. The integrals of the resonances for the O-acetyl groups indicated similarities between the O-antigen from E. coli O128ab and that of E. coli strain 87/D2, whereas the O-acetyl substitution pattern in the E. coli O128ac O-antigen differed slightly. Enzyme immunoassay using specific anti-E. coli O128ab and anti-E. coli O128ac rabbit sera confirmed the results.     

The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5

A DNA fragment that can functionally substitute for cfaD, the positive regulatory gene involved in expression of CFA/I fimbriae, has recently been cloned from an Escherichia coli strain of serotype O167:H5 that produces CS5 fimbriae. Nucleotide sequence determination showed that the fragment contained a gene, csvR (Coli Surface Virulence factor Regulator) homologous to the cfaD gene, which encoded for a protein of 301 amino acid residues. The csvR gene was found to be located between two different insertion sequences. Comparison of the amino acid sequence of the CsvR and CfaD proteins showed that CsvR is 34 amino acid residues longer at the C-terminus and, in the sequence, it also contains an insertion of two amino acid residues. The similarity between CfaD and Rns, the positive regulator of CS1 and CS2 expression, is much higher (97%) than between CsvR and CfaD (87%). This is reflected by the fact that the level of expression of CFA/I fimbriae induced by CsvR is not as high as when expression is induced by CfaD or Rns.     

Serologically distinct fimbriae on enterotoxigenic Escherichia coli of serotype O6:K15:H16 or H-

Abstract Serologically distinct, surface-associated, mannose-resistant haemagglutinins have been reported on enterotoxigenic Escherichia coli of serotype O6:K15:H16 or H- of human origin. Using immune electron microscopy, these haemagglutinins, termed the CS1 and CS2 antigens, have been identified as serologically distinct fimbriae, the presence of which correlates with the rhamnose fermentation phenotype of strains. The CS1 and CS2 fimbriae are morphologically indistinguishable from common type fimbriae. In contrast, using the same technique, no labelling of fimbriae was obtained with specific antibodies to another protein surface antigen termed the CS3 antigen, which is common to most strains of this serotype and also found on certain enterotoxigenic E. coli of serotype O8:H9. The morphological nature of the CS3 antigen was not disclosed.     

The coexistence of Escherichia coli serotype O157:H7 and its specific bacteriophage in continuous culture

For the development of phage therapy, systematic understanding mechanisms of bacteriophage resistance will be required. We describe a new strain of Escherichia coli O157:H7, named Mu(L), which stably co-exists with the O157:H7-specific lytic bacteriophage PP01. Chemostat cultures of E. coli O157:H7 infected with PP01 showed unchanging cell concentration, but phage concentrations which increased by approximately 10(8) PFU mL(-1). However, the latent period, burst size, and growth rate of Mu(L) were the same as in a PP01-susceptible strain. The binding rate of PP01 to the cell surface was diminished 8.5-fold in Mu(L). By observation of the binding of fluorescently labeled O157:H7-specific phage to individual Mu(L) cells, we found that clonal Mu(L) cultures were heterogeneous in their ability to bind bacteriophage. 15% of the Mu(L) population was completely resistant to PP01 infection. Mu(L) also co-existed with bacteriophages unrelated to PP01. Broad-range phage resistance by clonal heterogeneity represents a new class of bacteria-phage interactions.     

Establishment of three categories of P-fimbriated Escherichia coli strains that show different toxic phenotypes and belong to particular O serogroups

Eight hundred and nineteen strains of Escherichia coli isolated in Spain between 1986 and 1991 from extraintestinal infections and feces of healthy controls were investigated for expression of P-fimbriae using a particle agglutination test. Among strains causing urinary tract infections, sepsis and other extraintestinal infections, P-fimbriae were found in 31% (130/420) (P < 0.001), 25% (30/118) (P < 0.001) and 12% (11/92) (P < 0.5) respectively. In contrast, only 7% (14/189) of faecal isolates from healthy individuals carried P-fimbriae. According to two more common toxic markers detected in this study (alpha-haemolysin and cytotoxic necrotizing factor type 1), P-fimbriated E. coli strains were grouped into three categories: haemolysin+cytotoxic necrosing factor+ (Hly+CNF1+) (68/185; 37%), haemolysin+cytotoxic necrosing factor- (Hly+CNF1-) (61/185; 33%) and Hly-CNF1- (56/185; 30%). The 185 P-fimbriated strains belonged to 17 different O serogroups. However, 148 (80%) were of one of six serogroups (O1, O2, O4, O6, O7 and O18). The most frequent serogroups determined in the Hly+CNF1+ strains were the O4 and O6 (53/68; 78%), in the Hly+CNF1- strains it was the O18 (27/61; 44%) and in the Hly-CNF1- strains the O1, O2 and O7 (41/56; 73%). The majority (160/185; 86%) of P-fimbriated E. coli expressed the mannose-resistant haemagglutinin type IVa.     

Type 1, P and S fimbriae, and afimbrial adhesin I are not essential for uropathogenic Escherichia coli to adhere to and invade bladder epithelial cells

Fimbrial (type 1, P, and S) and afimbrial adhesins, the unique virulence traits of uropathogenic Escherichia coli (UPEC), are well recognized for their role in the initial step of uropathogenesis. In this study, we investigated whether these adhesins are dispensable for UPEC in adherence and invasion of uroepithelial cells by using E. coli isolates (n=40) from cystitis patients and T-24 cells, the bladder carcinoma cell line. We found all isolates adherent to T-24 cells within 15 min of infection. In invasion assay, all isolates could invade T-24 cells to a variable degree; 22.5% of them were found highly invasive. About 33% of isolates that do not have any recognized adhesins were as invasive as other isolates. The amplitude of invasiveness was also independent of the adhesins. In conclusion, this study demonstrates that type 1 fimbriae, P fimbriae, S fimbriae, and afimbrial adhesin I are not required for UPEC to adhere to and invade uroepithelial cells.     

Characterization of an outer membrane protein associated with haemagglutination and adhesive properties of enteroaggregative Escherichia coli O111:H12

Diarrhoeagenic Escherichia coli strains of serotype O111:H12 are characterized by their aggregative pattern of adherence on cultured epithelial cells and thus are considered enteroaggregative E. coli (EAEC). We have previously shown that these EAEC strains lack the genes encoding the aggregative fimbriae I and II described in other heterologous EAEC strains. In this paper, we show compelling data suggesting that a plasmid-encoded outer membrane 58 kDa protein termed aggregative protein 58 (Ap58) produced by EAEC O111:H12 strains, is associated with the adherence capabilities and haemagglutination of animal red blood cells. This conclusion is supported by several lines of evidence: (i) adherent O111:H12 strains are able to produce Ap58; (ii) non-adherent O111:H12 strains are unable to produce Ap58; (iii) antibodies raised against Ap58 inhibited adherence and haemagglutination of epithelial and bovine red blood cells, respectively; (iv) a non-adherent E. coli K-12 host strain containing the ap58 gene determinant on plasmid pVM15 displayed abundant adherence to cultured HEp-2 cells; and (v) the purified Ap58 bound specifically to HEp-2 and bovine red blood cells. Our findings indicate that the aggregative adherence in the O111:H12 strains may be also mediated by non-fimbrial adhesins. We believe our data contribute to the understanding of the adherence mechanisms of these organisms.