Escherichia coli enterotoxigénica y enteroagregativa: prevalencia, patogénesis y modelos múridos / Enterotoxigenic and enteroaggregative Escherichia coli: prevalence, pathogenesis and murine models

Resumen Los patotipos de Escherichia coli enterotoxigénica (ETEC) y enteroagregativa (EAEC) son importantes agentes etiológicos causantes de diarrea en niños menores de cinco años de México y países en desarrollo, en quienes causan numerosas muertes. Ambos se han asociado con retraso en el crecimiento infantil y son los principales agentes causales de la "diarrea del viajero". La patogénesis de ambas bacterias se inicia cuando estas se adhieren al epitelio intestinal mediante fimbrias, denominadas factores de colonización en las cepas ETEC aisladas de humano y fimbrias de adherencia agregativa en las cepas de EAEC. Una vez que ETEC se adhiere al enterocito produce una o ambas de sus toxinas e induce la secreción de iones de cloruro, sodio y agua al lumen intestinal, produciendo su característica diarrea acusa. EAEC se une al epitelio intestinal formando una biopelícula, induce la producción de moco, libera sus toxinas y promueve inflamación. Modelos de infección de EAEC y ETEC con ratones C57BL/6 silvestres y deficientes del ligando de CD40 (con microbiotas intactas), respectivamente, revelaron que la desnutrición y la dieta baja en cinc incrementan la infección de EAEC causando retraso en el crecimiento y que ETEC coloniza, persiste e induce respuesta inmune humoral local y sistémica.

Abstract Enterotoxigenic (ETEC) and enteroaggregative Escherichia coli (EAEC) pathotypes are important etiological agents causative of diarrhea in children younger than 5 years of age in Mexico and in developing countries, where they cause numerous deaths. Both have been associated with delayed growth in children and are the main causative agents of traveler's diarrhea. The pathogenesis of both bacteria starts by adhering to the intestinal epithelium by means of fimbriae, called colonization factors in human ETEC isolates and aggregative adherence fimbriae in EAEC isolates. Once ETEC adheres to the enterocyte, it produces one or both of its toxins and induces the secretion of chloride and sodium ions and water into the intestinal lumen, producing its characteristic watery diarrhea. EAEC binds to the intestinal epithelium forming a biofilm, induces the production of mucus, releases its toxins and promotes inflammation. EAEC and ETEC infection models with wild-type C57BL/6 and CD40 ligand-deficient mice (with intact microbiota), respectively, revealed that undernutrition and low-zinc diet increases EAEC infection, causing growth retardation, and that ETEC colonizes, persists and induces local and systemic humoral immune response.

Adaptação de Escherichia coli enterotoxigênica ao eugenol / Adaptation of enterotoxigenic Escherichia coli to eugenol

O objetivo do trabalho foi verificar a capacidade de adaptação de ETEC- ATCC 35401 ao antimicrobiano eugenol. Para isso, a Concentração Mínima Bactericida (CMB) de eugenol foi determinada e em seguida,as células de ETEC foram expostas a concentrações subletais de eugenol (CMB/4, CMB/8 e CMB/16). Posteriormente testadas frente a diferentes concentrações do composto (CMB/2; CMB; 1,2CMB; 1,4CMB; 1,6CMB; 1,8CMB e 2CMB), estas foram incubadas e plaqueadas em TSA (Ágar Triptona de Soja) empregando a técnica de microgotas. As células de ETEC foram classificadas como capazes de se adaptarem quando essas cresceram em placas após cultivo em presença do componente em concentração igual ou maior que a CMB do eugenol (1,0% (v/v)). As células de ETEC apresentaram a capacidade de adaptação por crescerem na CMB, após exposta a CMB/16. Os resultados demonstram a necessidade de obtenção da concentração de uso adequado de eugenol a fim de evitar a adaptação.

Virulence profiles of enterotoxigenic Escherichia coli isolated from piglets with post-weaning diarrhea and classification according to fecal consistency / Perfis de virulência de Escherichia coli enterotoxigênica isoladas de leitões desmamados com diarreia do Sul do Brasil e classificação das amostras de acordo com a consistência fecal

The aim of this study was to assess the frequency and association of virulence factors of Escherichia (E.) coli isolated from weaned piglets with diarrhea and to correlate it with fecal consistency. A total of 152 rectal swabs were collected from 25-40 day-old piglets with diarrhea, in farms of Southern Brazil. Phenotypical and molecular techniques were used for bacterial isolation, characterization and classification of enterotoxigenic E. coli (ETEC) pathotypes. Statistical analysis was carried out to determine the frequency of virulence factors and virotypes, of fimbriae F4, F5, F6, F18, F41 and toxins LT, STa, STb and STx2e. Out of 456 E. coli isolates, 287 (62.9%) samples showed significant growth of E. coli. Among them, 194 (67.6%) samples showed at least one virulence factor, indicating that ETEC is an important etiological agent of diarrhea in weaned piglets. Higher frequencies were found of fimbria F4 and F18 and enterotoxins LT, STa and STb. Significant association was found to F4, LT, STa and STb; between F18 and STa and STx2e; between F5 and LT, STa and STb. The most frequent virotypes were F18-STa, F4-LT-STa-STb, F4-STa, F4-LT-STb and F18-STa-STx2e. Beta-hemolysis was observed in 47.4% of samples and there was significant association between hemolytic samples and virulence factors F4, F18, STa and STx2e. Regarding fecal consistency, there was significant association of liquid feces and F4 fimbria, STa toxin and virotypes F4-STa and F4-F5-LT-STa-STb. Since there was significant association of ETEC and liquid feces in nursery piglets, it is important to prioritize the sampling of liquid feces for the diagnosis etiologic cause of diarrhea.

O objetivo deste estudo foi avaliar a frequência e associação de fatores de virulência de Escherichia (E.) coli isoladas de leitões desmamados com diarreia e correlacioná-la com consistência fecal. Suabes retais foram coletados em leitões com 25-40 dias de idade com sinal clínico de diarreia, em granjas do Sul do Brasil, totalizando 456 amostras. Foram utilizadas técnicas fenotípicas e moleculares para isolamento bacteriano, caracterização e classificação de patotipos de E. coli enterotoxigênica (ETEC). A análise estatística foi realizada para determinar a frequência de fatores de virulência e virotipos, de fímbrias F4, F5, F6, F18, F41 e toxinas LT, STa, STB e STx2e. Duzentas e oitenta e sete (62,9%) amostras apresentaram crescimento significativo de E. coli. Entre os quais, 194 (67,6%) amostras apresentaram pelo menos um fator de virulência, indicando que ETEC é um importante agente etiológico de diarreia em leitões desmamados. As frequências mais elevadas foram encontradas para as fímbrias F4 e F18 e enterotoxinas LT, STa e STb. Associação significativa foi encontrada para F4, LT, STa e STb; entre F18 e STa e STx2e; entre F5 e LT, STa e STb. Os virotipos mais frequentes foram F18-STa, F4-LT-STa-STb, F4-STa, F4-LT-STb e F18-STa-STx2e. Beta-hemólise foi observada em 47,4% das amostras e houve associação significativa entre amostras hemolíticas e fatores de virulência F4, F18, STa e STx2e. Quanto consistência fecal, houve associação significativa de fezes líquidas e fímbria F4, toxina STa e virotipos F4-STa e F4-F5-LT-STa-STb. A associação significativa da ETEC e fezes líquidas em leitões de creche, é importante para priorizar a amostragem de fezes com essa consistência para no diagnóstico etiológico da diarreia.

Biofilm formation and binding specificities of CFA/I, CFA/II and CS2 adhesions of Enterotoxigenic Escherichia coli and CfaE-R181A mutant

Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. Adhesion is the first step in pathogenesis of ETEC infections and ETEC pili designated colonization factor antigens (CFAs) are believed to be important in the biofim formation, colonization and host cell adhesions. As a first step, we have determined the biofilm capability of ETEC expressing various types of pili (CFA/I, CfaE-R181A mutant/ CfaE tip mutant, CFA/II and CS2). Further, enzyme-linked immunosorbent assay (ELISA) assay were developed to compare the binding specificity of CFA/I, CFA/II (CS1 - CS3) and CS2 of ETEC, using extracted pili and piliated bacteria. CFA/II strain (E24377a) as well as extracted pili exhibited significantly higher binding both in biofilm and ELISA assays compared to non piliated wild type E24377a, CFA/I and CS2 strains. This indicates that co-expression of two or more CS2 in same strain is more efficient in increasing adherence. Significant decrease in binding specificity of DH5αF'lacIq/∆cotD (CS2) strain and MC4100/pEU2124 (CfaE-R181A) mutant strain indicated the important contribution of tip proteins in adherence assays. However, CS2 tip mutant strain (DH5αF'lacIq/pEU5881) showed that this specific residue may not be important as adhesions in these strains. In summary, our data suggest that pili, their minor subunits are important for biofilm formation and adherence mechanisms. Overall, the functional reactivity of strains co expressing various antigens, particularly minor subunit antigen observed in this study suggest that fewer antibodies may be required to elicit immunity to ETEC expressing a wider array of related pili.

Caracterización genotipica de aislamientos de Escherichia coli obtenidos de cerdos con diarrea posdestete y enfermedad de los edemas / Genotypic characterization of toxigenic Escherichia coli isolated from pigs with postweaning diarrhea (PWD) and edema disease (ED)

El objetivo del trabajo fue caracterizar mediante PCR 47 aislamientos de Escheríchia coli recuperados de 32 cerdos con diagnóstico clínico de diarrea posdestete (DPD) y de 3 cerdos con enfermedad de los edemas (ED). Sobre 44 aislamientos provenientes de cerdos con DPD, 42 (95,5 %) fueron caracterizados como E. coli enterotoxigénicos (ETEC) y 2 (4,5 %) como E. coli productores de toxina Shiga (STEC). Catorce aislamientos de ETEC (33,3 %) fueron positivos para los genes estl/estlI/fedA. El genotipo más complejo fue eltA/estll/east1/faeG/aidA. Los aislamientos provenientes de cerdos con ED se clasificaron como STEC porcinos y fueron portadores de stxJaidA. Once aislamientos (25 %) fueron portadores del gen que codifica la expresión de la adhesina AIDA-I. Sin embargo, en ningún aislamiento se detectaron los genes que codifican la expresión de las adhesinas F5, F6, F41, de intimina y de "Paa". La prevención de la DPD y de la ED podría realizarse mediante el desarrollo de vacunas que generen anticuerpos contra las adhesinas de las cepas de E. coli prevalentes en la Argentina.

The purpose of this work was to characterize 47 Escherichia coli strains isolated from 32 pigs diagnosed with postweaning diarrhea and tree pigs with edema disease by PCR. Forty two (95.5 %) of the strains isolated from diarrheic pigs were characterized as enterotoxigenic E. coli (ETEC) and 2 (4.5 %) as Shiga toxin-producing E. coli (STEC). Fourteen (33.3 %) ETEC strains were positive for est/estll/fedA genes. The most complex genotype was eltA/estl/faeG/aidA. Strains isolated from pigs with ED were classified as porcine STEC and were stxjaidA carriers. Eleven (25 %) strains carried the gene encoding adhesln protein AIDA-I. However, genes coding for F5, F6, F41, intimin and Paa were not detected. The development of vaccines generating antibodies against prevalent E. coli adhesins in Argentina could be useful for the prevention of PWD and ED.

QRDR mutations, efflux system & antimicrobial resistance genes in enterotoxigenic Escherichia coli isolated from an outbreak of diarrhoea in Ahmedabad, India.

Background & objectives: Diverse mechanisms have been identified in enteric bacteria for their adaptation and survival against multiple classes of antimicrobial agents. Resistance of bacteria to the most effective fluoroquinolones have increasingly been reported in many countries. We have identified that most of the enterotoxigenic Escherichia coli (ETEC) were resistant to several antimicrobials in a diarrhoea outbreak at Ahmedabad during 2000. The present study was done to identify several genes responsible for antimicrobial resistance and mobile genetic elements in the ETEC strains. Methods: Seventeen ETEC strains isolated from diarrhoeal patients were included in this study. The antimicrobial resistance was confirmed by conventional disc diffusion method. PCR and DNA sequencing were performed for the identification of mutation in the quinolone resistance-determining regions (QRDRs). Efflux pump was tested by inhibiting the proton-motive force. DNA hybridization assay was made for the detection of integrase genes and the resistance gene cassettes were identified by direct sequencing of the PCR amplicons. Results: Majority of the ETEC had GyrA mutations at codons 83 and 87 and in ParC at codon 80. Six strains had an additional mutation in ParC at codon 108 and two had at position 84. Plasmid-borne qnr gene alleles that encode quinolone resistance were not detected but the newly described aac(6’)-Ib-cr gene encoding a fluoroquinolne-modifying enzyme was detected in 64.7 per cent of the ETEC. Class 1 (intI1) and class 2 (intI2) integrons were detected in six (35.3%) and three (17.6%) strains, respectively. Four strains (23.5%) had both the classes of integrons. Sequence analysis revealed presence of dfrA17, aadA1, aadA5 in class 1, and dfrA1, sat1, aadA1 in class 2 integrons. In addition, the other resistance genes such as tet gene alleles (94.1%), catAI (70.6%), strA (58.8%), blaTEM-1(35.2%), and aphA1-Ia (29.4%) were detected in most of the strains. Interpretation & conclusions: Innate gene mutations and acquisition of multidrug resistance genes through mobile genetic elements might have contributed to the emergence of multidrug resistance (MDR) in ETEC. This study reinforces the necessity of utilizing molecular techniques in the epidemiological studies to understand the nature of resistance responsible for antimicrobial resistance in different species of pathogenic bacteria.

The prevalence of enterotoxigenic E. coli isolated from the stools of children aged 0-10 years with diarrhea in mid-anatolia region, Turkey

The stool samples from 245 patients with diarrhea were tested for heat labile toxin (LT) and heat stable toxins (ST) by passive latex agglutination and enzyme immunoassay methods respectively. Twelve (4.9 percent) enterotoxigenic E. Coli ETEC strains were isolated. Five strains (2 percent) expressed ST, and 7 (2.8 percent) expressed LT.