Enterotoxigenic Escherichia coli - an overview

Enterotoxigenic Escherichia coli is an important cause of traveler's diarrhea and diarrheal illnesses in children in the developing world. In this presentation we will focus on the main virulence attributes of this pathogenic category of E. coli, and discuss the evolution of studies conducted in our laboratory.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Immunization against the colonization factor antigen I of enterotoxigenic Escherichia coli by administration of a bivalent Salmonella typhimurium aroA strain

An expression plasmid (pCFA-1) carrying the cfaB gene that codes for the enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin colonization factor antigen I (CFA/I) subunit was constructed and used to transform a derivative of the attenuated Salmonella typhimurium aroA vaccine strain SL3261 carrying an F'lacIq. Treatment of the transformed strain with isopropyl-ß-D-thiogalactopyranoside (IPTG) resulted in elevated in vitro expression of the CFA/I subunit. Although flagellar function and lipopolysaccharide (LPS) synthesis were similar in both the parental and the recombinant strains, spleen colonization was reduced in the recombinant strain. All BALB/c mice parenterally inoculated with the recombinant strain developed significant anti-CFA/I and anti-LPS serum antibody titers (P<0.05). Moreover, 2 of 5 mice orally inoculated with the engineered Salmonella strain developed anti-CFA/I intestinal IgA (P>0.05) while 4/5 of the same mice developed anti-LPS IgA (P<0.05). The results indicate that the vaccine strain elicited an antibody response against the bacterial host both after oral and intravenous immunization while the response against the CFA/I antigen was significant only after inoculation by the intravenous route

New vaccine strategies against enterotoxigenic Escherichia coli: I: DNA vaccines against the CFA/I fimbrial adhesin

Stimulation of the mammalian immune system by administration of plasmid DNA has been shown to be an important approach for vaccine development against several pathogens. In the present study we investigated the use of DNA vaccines to induce immune responses against an enteric bacterial pathogen, enterotoxigenic Escherichia coli (ETEC). Three plasmid vectors encoding colonization factor antigen I (CFA/I), an ETEC fimbrial adhesin, were constructed. Eukaryotic cells transfected with each of these plasmids expressed the heterologous antigen in different compartments: bound to the cytoplasmic membrane (pRECFA), accumulated in the cytoplasm (pPolyCFA) or secreted to the outside medium (pBLCFA). BALB/c mice were intramuscularly (im) inoculated with purified plasmid DNA and the systemic, cellular and secreted CFA/I-specific immune responses were analyzed. The results showed that all three DNA vaccine formulations could elicit CFA/I-specific immune responses. Moreover, cellular location of the plasmid-encoded CFA/I seems to have an important role in the induced immune response. Taken together, these results indicate that DNA vaccines also represent a promising approach against enteric bacterial pathogens.

New vaccine strategies against enterotoxigenic Escherichia coli: II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine

The induction of systemic (IgG) and mucosal (IgA) antibody responses against the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC) was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

Diarrheagenic Escherichia coli categories among the traditional enteropathogenic E. coli O serogroups: a review

The socalled enteropathogenic Escherichia coli (EPEC) O serogroups include typical and atypical EPEC, enterohaemorrragic E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli. The aim of this article is to review the composition of each O serogroup and the major serotypes, clones, and additional virulence characteristics of each of these diarrheageniccategories. Their adherence patterns and genetic relationships are also presented. The review is based on the study of 805 strains of serogroups O26, O55, O86, O111, O114, O119, O125, O126, O1127, O128, and O142 most of which isolated in São Paulo from children with diarrhea between 1970 and 1990. Since some O serogroups include more than one diarrheageniccategory O serogrouping only should be abandoned as a diagnostic method. However serotyping is a reliable method for those serotypes that correspond to clones.

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.

Phylogenetic and pathotype analysis of Escherichia coli swine isolates from Southern Brazil

The current study evaluated the presence of virulence factors by a multiplex PCR technique and then phylogenetically classified the studied strains into groups A, B1, B2 and D, according to Clermont et al. (2000), in 152 intestinal and extraintestinal swine isolates of Escherichia coli. Seventy seven isolates tested were positive for virulence factors. Phylogenetic characterization placed 21 samples into group A, 65 into B1, 19 into B2 and 47 into D. Fourteen urine samples were classified as uropathogenic E. coli (UPEC), nine were both UPEC and enterotoxigenic E. coli (ETEC) and four were ETEC only. The most common phylogenetic classifications were B1 and D groups. Of the analyzed fecal samples, 25 were classified as ETEC. Phylogenetically, the group of higher occurrence was B1, followed by B2, A and D. For the small intestine samples, 20 were classified as ETEC. Phylogenetic analysis found groups B1 and A to be the most commons in these samples. Six isolated tissue samples were classified as ETEC and most of them were designated as group D by phylogenetic classification. The phylogenetic analysis could be employed in veterinary laboratories in the E. coli isolates screening, including the possibility of vaccine strain selection and epidemiological searches.

Identificação de cepas de Escherichia coli enteropatogênicas em amostras de fezes, por reação de imunofluorescência direta

Foi realizado diagnóstico etiológico de casos de diarréia aguda em 121 pacientes internados na Clínica Pediátrica do Hospital da Santa Casa de São Paulo, Brasil. Foram utilizados os métodos bacteriológico clássico e de reação de imunofluorescência direta para a identificação de cepas de Escherichia coli enteropatogênicas: para estudo da sensibilidade das cepas de Escherichia coli isoladas, a diferentes antibióticos, foi usado o método de Concentração Inibitória Minima (CIM). Dos 56 casos positivos, 89,3% correspondiam a diferentes sorotipos enteropatogênicos de Escherichia coli, quando utilizada a técnica de imunofluorescência direta. O método bacteriológico clássico revelou ainda, nos 121 casos examinados, 4 cepas de Salmonella e 2 de Shigella. No estudo da CIM verificou-se maior sensibilidade das cepas de Escherichia coli enteropatogênicas estudadas à Gentamicina e Amikacina, do que aos outros antibióticos.

Isolation and serological identification of enteropathogenic Escherichia coli in pasteurized milk in Brazil

OBJECTIVE: To evaluate the microbiological quality of pasteurized milk commercialized in Rio de Janeiro, Brazil, and determine serologically enteropathogenic Escherichia coli (EPEC) strains in E. coli isolates obtained from milk samples. METHODS: Ninety samples of pasteurized milk -- types B and C -- of three different commercial brands, purchased in supermarkets and bakeries in Rio de Janeiro, were examined. The amount of total and fecal coliform bacteria was estimated using the Most Probable Number technique. Mesophilic, psychrotrophic, and thermoduric microorganism counts were determined by the Standard Plate Count technique. Isolation and identification of E. coli were carried out using conventional physiological tests. Commercial antisera were used for serological characterization of EPEC. RESULTS: The three milk brands analyzed revealed bacterial counts above the regulated values of the Brazilian government. It was found that among 208 strains of E. coli isolated, 46 (22.1%) were serologically classified as EPEC. The most common EPEC serogroup was O55 (15.2%). CONCLUSIONS: Though recent studies on virulence factors indicate that not all strains serologically classified as EPEC are able to attaching/effacing lesion, it is believed that the isolation of EPEC serogroups from pasteurized milk represent a potential risk for children, as well as an indicative of the presence of other enteropathogens.

Incidência de escherichia coli enterotoxigênica (ETEC), rotavirus e Clostridium perfringens de casos de diarréia em crianças, na região de Campinas, SP, Brasil

Foi realizada uma pesquisa na região de Campinas, SP, Brasil, sobre a presença de Escherichia coli enterotoxigênica (ETEC), rotavírus e Clostridium perfringens enterotoxigênico em fezes diarréicas de crianças com até 2 anos de idade. Dos 132 espécimens fecais examinados quanto à presença de ETEC 27 (20,45%) foram positivos. Destes foram isoladas 41 amostras de ETEC, das quais 40 produziram apenas a enterotoxina termolábil (LT) detectada pelo teste de imuno hemólise radial modifi cado. Entre as 183 amostras de fezes examinadas para rotavírus, 29 (15,84%) foram positivas pelas técnicas de eletroforese em gel de poliacrilamida (PAGE) e ensaio imunoenzimático (EIE), sendo que destas, 15 (51,7% ) foram provenientes de materiais coletados nos meses de inverno. Todas as amostras pertenciam ao grupo A e, através da técnica de PAGE, pode-se observar que o tipo eletroforético mais freqüente (9 amostras) foi designado Ib, IIc, Illb, IVa, de acordo com a classificação por nós adotada. Apenas 113 amostras de fezes foram examinadas para a presença de C. perfringens enterotoxigênico. Para a detecção da enterotoxina nos sobrenadantes das culturas foram utilizadas as técnicas de hemaglutinação passiva reversa e inoculação intravenosa em camundongos, sendo encontradas 12 (10,61%) amostras entero-toxigênicas. Diante destes resultados é chamada a atenção sobre o valor apenas relativo de uma coprocultura convencional para fins de diagnóstico, ressaltando-se a importância da criação de métodos simplificados que favoreçam a detecção e identificação dos grupos de agentes enteropatogênicos estudados na presente pesquisa.

Purification and parcial characterization of a hemagglutinating factor (HAF): a possible adhesive factor of the diffuse adherent of Escherichia coli (DAEC)

The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains

Asymptomatic infections by diarrheagenic Escherichia coli in children from Misiones, Argentina, during the first twenty months of their lives

Diarrheagenics Escherichia coli are the major agents involved in diarrheal disease in developing countries. The aim of this study was to evaluate the time of appearance of the first asymptomatic infection by the different categories of diarrheagenic E. coli in 44 children since their birth and during the first 20 months of their lives. In all of the children studied, we detected at least one category of diarrheagenic E. coli through the 20 months of the study. 510 diarrheagenic E. coli (33.5%) were obtained from the 1,524 samples collected from the 44 children during the time of the study (31.4% EAggEC, 28.8% EPEC, 27.1% DAEC, and 12.7% ETEC). Neither EHEC nor EIEC were identified. The median age for diarrheagenic E. coli colonization was 7.5 months. The mean weaning period was 12.8 months and the mean age for introduction of mixed feeding (breast fed supplemented) was 3.8 months. A significantly lower incidence of diarrheal disease and asymptomatic infections was recorded among the exclusively breast-fed rather than in the supplemented and non breast-fed infants. For ETEC, EPEC and EAggEC the introduction of weaning foods and complete termination of breast-feeding were associated with an increase of asymptomatic infections.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil

The aim of the study was to determine the occurrence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin among a hundred Escherichia coli isolates obtained from in-and outpatients of a tertiary-care teaching hospital, between July and August 2000, showing clinical and laboratory signs of urinary tract infection (UTI). The presence of genes (pap, afa, sfa) for fimbriae expression was assayed using specific primers in a polymerase chain reaction. Among the isolates studied, the prevalence of the virulence factors was 96.0%, 76.0%, 24.0%, for hemolysin, aerobactin and colicin, respectively; the prevalence of genes coding for fimbrial adhesive systems was 32.0%, 19.0% and 11.0% for pap, sfa and afa respectively. The strains isolated from the outpatients displayed a greater number of virulence factors compared to those from hospitalized subjects, emphasizing the difference between these two kinds of patients.