Adhesion of Diarrheagenic Escherichia coli and Inhibition by Glycocompounds Engaged in the Mucosal Innate Immunity.

Escherichia coli colonizes the human intestine shortly after birth, with most strains engaging in a commensal relationship. However, some E. coli strains have evolved toward acquiring genetic traits associated with virulence. Currently, five categories of enteroadherent E. coli strains are well-recognized, and are classified in regard to expressed adhesins and the strategy used during the colonization. The high morbidity associated with diarrhea has motivated investigations focusing on E. coli adhesins, as well on factors that inhibit bacterial adherence. Breastfeeding has proved to be the most effective strategy for preventing diarrhea in children. Aside from the immunoglobulin content, glycocompounds and oligosaccharides in breast milk play a critical role in the innate immunity against diarrheagenic E. coli strains. This review summarizes the colonization factors and virulence strategies exploited by diarrheagenic E. coli strains, addressing the inhibitory effects that oligosaccharides and glycocompounds, such as lactoferrin and free secretory components, exert on the adherence and virulence of these strains. This review thus provides an overview of experimental data indicating that human milk glycocompounds are responsible for the universal protective effect of breastfeeding against diarrheagenic E. coli pathotypes.

Diarrhea-associated biofilm formed by enteroaggregative Escherichia coli and aggregative Citrobacter freundii: a consortium mediated by putative F pili.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea. RESULTS: During an epidemiologic study focusing on infantile diarrhea, aggregative C. freundii (EACF) and EAEC strains were concomitantly recovered from a severe case of mucous diarrhea. Thereby, the occurrence of synergic events involving these strains was investigated. Coinfection of HeLa cells with EACF and EAEC strains showed an 8-fold increase in the overall bacterial adhesion compared with single infections (P < 0.001). The synergic effect was mediated by physical interactions among the bacteria and primed in the absence of chemical signaling and without the participation of host cells. Thus, significant increases (2.7-fold on average) in bacterial adhesion were also observed during the formation of mixed biofilms on abiotic surfaces. Bacterial settling assays showed that EAEC strains harboring F-pili genes (traA) were capable of forming bacterial aggregates only in the presence of EACF. Scanning electronic microscopy analyses revealed that bacterial aggregates as well as enhanced biofilms formed by EACF and traA-positive EAEC were mediated by non-bundle forming, flexible pili. Moreover, mixed biofilms formed by EACF and traA-positive EAEC strains were significantly reduced using nonlethal concentration of zinc, a specific inhibitor of F pili. In addition, EAEC strains isolated from diarrheic children frequently produced single biofilms sensitive to zinc. CONCLUSIONS: Putative F pili expressed by EAEC strains boosted mixed biofilm formation when in the presence of aggregative C. freundii.

Inhibition of enterotoxigenic Escherichia coli (ETEC) adhesion to Caco-2 cells by human milk and its immunoglobulin and non-immunoglobulin fractions

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea in children in developing countries and among travelers to ETEC endemic areas. ETEC diarrhea is caused by colonization of the small intestine mediated by colonization factor (CF) antigens, and subsequent elaboration of enterotoxins. Breast feeding has been related to protection against enteric infections. The protective effect of human milk can be ascribed to its immunoglobulin content, specially secretory immunoglobulin A (sIgA), and to non-immunoglobulin components such as free oligosaccharides, glycoproteins and glycolipids. In this study we investigated the effect of whole human milk and its fractions immunoglobulin and non-immunoglobulin on the adherence of ETEC strains possessing different CFs to Caco-2 cells, as well as the ability of sIgA and free secretory component (fSC) to bind to bacterial superficial proteins. Pooled human milk from three donors were fractionated by gel filtration and analyzed by SDS-PAGE. Our results revealed that whole human milk and its proteins fractions, containing sIgA and fSC, inhibited adhesion ETEC strains harboring different colonization factors antigens. We also verified that sIgA and fSC, using immunoblotting and immunogold labeling assays, bound to some fimbrial proteins and other material present in bacterial surface. Our findings suggest that whole human milk and its fractions may contribute to protection against ETEC infections by blocking bacterial adhesion mediated by different colonization antigens.

Escherichia coli enterotoxigênica (ETEC) é uma importante causa de diarréia infantil em países em desenvolvimento e entre os viajantes em áreas endêmicas. A diarréia por ETEC é causada pela colonização do intestino delgado, mediada pelos fatores antigênicos de colonização (CFs), e a subseqüente elaboração de enterotoxinas. A amamentação tem sido relacionada com a proteção contra infecções entéricas. O efeito protetor do leite humano pode ser atribuído ao seu conteúdo imunoglobulínico, especialmente a imunoglobulina A secretória (sIgA), e aos componentes não imunoglobulínicos tais como oligossacarídeos livres, glicoproteínas e glicolipídeos. Neste estudo nós investigamos o efeito do leite humano total e suas frações imunoglobulínica e não imunoglobulínica sobre a adesão de cepas ETEC possuidoras de diferentes fatores de colonização as células Caco-2, bem como a habilidade da sIgA e do componente secretor livre (fSC) de se ligarem as proteínas da superfície bacteriana. Uma mistura de leite de três doadoras foi fracionada por filtração em gel e analisada por SDS-PAGE. Nossos resultados revelaram que o leite humano total e suas frações protéicas, contendo sIgA e fSC, inibiu a adesão de cepas ETEC albergando diferentes fatores antigênicos de colonização. Nós também verificamos que a sIgA e o fSC, usando ensaios de imunoeletrotransferência e imunomarcação, ligaram-se a algumas proteínas fimbriais e a outro material presente na superfície bacteriana. Nossos dados sugerem que o leite humano total e suas frações podem contribuir para proteção contra as infecções por ETEC bloqueando a adesão bacteriana mediada pelos diferentes antígenos de colonização.

Enteroaggregative Escherichia coli virulence markers: positive association with distinct clinical characteristics and segregation into 3 enteropathogenic E. coli serogroups.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) are a heterogeneous category of emerging pathogens associated with acute and persistent diarrhea. METHODS: We performed a case-control study to evaluate the association of EAEC strains and their virulence markers with infantile diarrhea in Brasilia, Brazil. RESULTS: Of the EAEC markers that were tested, only EAEC heat-stable toxin (EAST1), protein involved in colonization (Pic), and the CVD432 sequence showed positive correlation with diarrhea; however, these virulence traits presented associations with distinct clinical characteristics. Although CVD432-positive strains were associated with persistent diarrhea, EAST1-positive strains were statistically correlated with acute diarrhea only in children >6 months of age. Typical EAEC strains were found with equal frequency in case patients and control subjects. O antigens of enteropathogenic E. coli (EPEC) were also sought, and the data showed that the EAEC markers are frequently segregated into 3 EPEC serogroups: O126, O128, and O158. CONCLUSIONS: The presence of EPEC O antigens along with EAEC markers influenced the positive association of E. coli strains with diarrhea. Concerning the detection of the AA phenotype, the CVD432 sequence presented a low positive predictive value, regardless of its high specificity as a marker.

Virulence markers of enteroaggregative Escherichia coli isolated from children and adults with diarrhea in Brasília, Brazil.

Escherichia coli strains isolated from sporadic cases of acute diarrhea in children and adults and from children without diarrhea were investigated for the presence of the pAA plasmid. Strains harboring the pAA plasmid were isolated at similar frequencies from children with (19.6%) and without (10.8%) diarrhea and from adults with diarrhea (11.8%). The genotypic and phenotypic virulence markers of these strains were further analyzed. Most of the strains were positive for EAST1 (73%), and this toxin was detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.05). Likewise, pic sequences were detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.005) and controls (P < 0.025). Furthermore, the association of pAA positivity (pAA(+)) and pic positivity (pic(+)) was more frequently found for strains from children with diarrhea than for strains from controls, indicating that pAA(+) pic(+) strains may represent a subset of pAA(+) strains associated with disease in children. Most of the strains (82.5%) adhered to cells presenting the typical aggregative pattern. The frequency of occurrence of enteropathogenic E. coli (EPEC) serogroups in the strains from children with diarrhea was very high (56%), while none of the strains from adults with diarrhea belonged to EPEC serogroups. Extraintestinal virulence markers were very commonly found in strains from adults with diarrhea. The frequencies of occurrence of the adhesins AFA and SFA were significantly higher in strains from adults with diarrhea than in strains from children with diarrhea. More than one extraintestinal virulence marker was found in 58% of the strains from adults with diarrhea but in only 7.7% of the strains from children with diarrhea. Our results show that pAA(+) strains isolated from children and adults with diarrhea present very different profiles when enteroaggregative E. coli virulence markers, serotypes, and extraintestinal virulence markers are considered.

Virulence factors of Escherichia coli strains isolated from diarrheic children: influence of storage conditions

A frequência de isolamento de Escherichia coli enterotoxigênica (ETEC), verotoxigênica (VTEC) e necrotizante (NTEC) foi determinada em 167 crianças com diarréia de uma área central do Brasil. Cepas de E. colli produtoras de verotoxina (VT), fator citotóxico necrotizante (CNF) e enterotoxina termo lábil (LT) foram encontradas, respectivamente, em 3 por cento, 3,6 por cento e 7,2 por cento dos casos. As cepas de E. coli foram também testadas quanto à capacidade de induzir hemaglutinaçäo manose resistente (MRHA) de eritrócitos humanos e bovinos. Cepas MRHA+ foram observadas em 42 por cento dos casos e os fatores de colonizaçäo I e II foram identificados em 32 por cento dos casos MRHA+. Após longo período de estocagem e temperatura ambiente, a estabilidade dos fatores de virulência produzidos por 28 cepas selecionadas foram reavaliados. A produçäo de citotoxinas foi mantida em 3 de 5 cepas VT+ e em 5 de 6 cepas CNF+. A expressäo de LT foi mantida em 9 de 12 cepas LT+. De 23 cepas CFA+ somente 7 mantiveram o padräo de hemaglutinaçäo. Expressäo de ST foi detectada em 4 cepas. Usando o método de hibridizaçäo de colônias além das 4 cepas que expressaram ST, outras 4 cepas ST+ foram detectadas. O genótipo LT foi encontrado em 11 das cepas selecionadas. Em conclusäo, através do estudo de cepas NTEC foram identificadas pela primeira vez no Brasil. Este estudo também confirma a grande importância das condiçöes de estocagem para a detecçäo de fatores de virulência de ETEC e ainda mostra que VTEC e NTEC podem perder a capacidade de produzir citotoxinas após longos períodos de estocagem