Filogenia e potencial de virulência de isolados híbridos de Escherichia coli enteropatogênica atípica/enteroagregativa (aEPEC/EAEC) do sorotipo O3:H2 obtidos durante a investigação de um surto de diarreia no Brasil
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2022-02-11
Autores
Lira, Daiany Ribeiro Paz de
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Universidade Estadual Paulista (Unesp)
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Escherichia coli enteropatogênica (EPEC) e enteroagregativa (EAEC) são dois dos principais patotipos de E. coli diarreiogênica (ECD) causadores de doenças ao redor do mundo. A EPEC atípica (aEPEC) produz a lesão attaching and effacing nas células hospedeiras infectadas, levando ao acúmulo de F-actina e à formação de estruturas semelhantes a um pedestal sob as bactérias aderentes. A EAEC adere às células epiteliais formando um arranjo semelhante a tijolos empilhados, conhecido como padrão de aderência agregativa (AA), devido à produção de várias fímbrias de aderência agregativa (AAF/I-AAF/V). O objetivo do presente estudo foi investigar as características genotípicas e fenotípicas de isolados de E. coli híbridos de aEPEC/EAEC, pertencentes ao sorotipo O3:H2, bem como de isolados de aEPEC e EAEC do mesmo sorotipo, todos obtidos durante a investigação de um surto de diarreia, com o intuito de avaliar a influência da combinação de fatores de virulência na patogenicidade e melhor compreender a evolução desses híbridos. A partir de um surto de diarreira, foram obtidos um isolado de aEPEC (eae+/bfpB-), quatro EAEC (aggR+) e três híbridos de aEPEC/EAEC (eae+/bfpB-/aggR+), sendo todos os oito isolados do sorotipo O3:H2. Esses isolados foram caracterizados quanto à classificação dos filogrupos de E. coli, presença de genes codificadores de fatores de virulência associados à patogênese de EPEC e EAEC, análise do perfil plasmidial, padrão de aderência em células HeLa, formação de biofilme e teste de FAS (fluorescent actin staining). Todos os oito isolados estudados foram atribuídos ao filogrupo A. Observou-se que as EAEC e os híbridos de aEPEC/EAEC albergam vários genes do plasmídio de aderência agregativa (pAA), como aatA, aap, orf3, aar, aggA e aggC (codificando a pilina e o usher de AAF/I, respectivamente), além de apresentarem um plasmídio de alto peso molecular. Por outro lado, os isolados de aEPEC e os híbridos aEPEC/EAEC albergam dois genes cromossômicos, nleC e nleF, que codificam efetores secretados pelo sistema de secreção tipo 3. Juntos, esses dados sugerem que o background de aEPEC deve ter servido como um receptor para o pAA, originando assim os isolados híbridos de aEPEC/EAEC. As EAEC e os híbridos aEPEC/EAEC produziram o padrão AA, enquanto a aEPEC aderiu esporadicamente às células epiteliais. Conforme observado no ensaio de FAS, a aEPEC foi incapaz de promover o acúmulo de F-actina sob as bactérias aderentes, enquanto os híbridos aEPEC/EAEC foram FAS-positivos. Diferentemente do isolado de aEPEC, os quatro isolados de EAEC e os três híbridos de aEPEC/EAEC produziram biofilme na superfície abiótica, apontando a influência do pAA para o estabelecimento deste fenótipo. Esse estudo revelou a alta plasticidade da E. coli patogênica do sorotipo O3:H2 na aquisição e manutenção de genes que codificam a virulência. No entanto, a busca por novos híbridos é continuamente necessária para melhor entender quais eventos estão envolvidos no surgimento desses híbridos e como diferentes combinações genéticas podem influenciar sua patogenicidade.
Enteropathogenic (EPEC) and enteroaggregative (EAEC) Escherichia coli are two of the major pathotypes of diarrheagenic E. coli (DEC) causing disease worldwide. Atypical EPEC (aEPEC) produces the attaching and effacing lesion in infected host cells, leading to the accumulation of F-actin and the formation of pedestal-like structures underneath adherent bacteria. EAEC adheres on epithelial cells forming an arrangement that resembles stacked bricks, known as aggregative adherence (AA) pattern, due to the production of several aggregative adherence fimbriae (AAF/I – AAF/V). The main goal of the present study was to investigate the genotypic and phenotypic features hybrid aEPEC/EAEC, belonging to the O3:H2 serotype, as well as of aEPEC and EAEC isolates of the same serotype, all of them obtained during an diarrheal outbreak investigation, in order to evaluate the influence of the virulence factors combinations on their pathogenicity, and better understand the emergence of these hybrids. Recently, we reported a diarrheal outbreak investigation, in which we obtained one aEPEC (eae+/bfpB-), four EAEC (aggR+), and three hybrid aEPEC/EAEC (eae+/bfpB-/aggR+), with all eight isolates assigned in the serotype O3:H2. Here we attempted to characterize these isolates in terms of E. coli phylogroup classification, presence of virulence factor-encoding genes, associated with the pathogenesis of EPEC and EAEC, plasmidial profile, adherence pattern on HeLa cells, FAS (fluorescent actin staining) test and biofilm formation. All eight isolates studied were assigned in the phylogroup A. We observed that the EAEC and the hybrid aEPEC/EAEC harbored several genes from the aggregative adherence plasmid (pAA), such as aatA, aap, orf3, aar, aggA and aggC (encoding the the pilin and usher of AAF/I, respectively) and share a common high molecular weight plasmid. On the other hand, the aEPEC and the hybrid aEPEC/EAEC isolates harbored two chromosomal genes, nleC and nleF, encoding for type 3 secreted effectors. Together, these data may suggest that the aEPEC background must have served as a receptor for the pAA, thus originating the hybrid aEPEC/EAEC isolates. The EAEC and the hybrid aEPEC/EAEC produced the AA pattern, while the aEPEC adhered only sporadically to the epithelial cells. As observed in the FAS assay, the aEPEC was unable to promote F-actin accumulation underneath adherent bacteria, while the hybrids aEPEC/EAEC were FASpositive. Distinct from the aEPEC isolate, the four EAEC and the three hybrid aEPEC/EAEC produced biofilm on abiotic surface, thus pointing out the influence of the pAA for the establishment of this phenotype. Our study reveals the high plasticity of pathogenic E. coli of serotype O3:H2 in acquiring and maintaining virulence-encoding genes. However, the search for new hybrids is continually needed to better understand which events are involved in the emergence of these hybrids and how different genetic combinations can influence their pathogenicity.
Enteropathogenic (EPEC) and enteroaggregative (EAEC) Escherichia coli are two of the major pathotypes of diarrheagenic E. coli (DEC) causing disease worldwide. Atypical EPEC (aEPEC) produces the attaching and effacing lesion in infected host cells, leading to the accumulation of F-actin and the formation of pedestal-like structures underneath adherent bacteria. EAEC adheres on epithelial cells forming an arrangement that resembles stacked bricks, known as aggregative adherence (AA) pattern, due to the production of several aggregative adherence fimbriae (AAF/I – AAF/V). The main goal of the present study was to investigate the genotypic and phenotypic features hybrid aEPEC/EAEC, belonging to the O3:H2 serotype, as well as of aEPEC and EAEC isolates of the same serotype, all of them obtained during an diarrheal outbreak investigation, in order to evaluate the influence of the virulence factors combinations on their pathogenicity, and better understand the emergence of these hybrids. Recently, we reported a diarrheal outbreak investigation, in which we obtained one aEPEC (eae+/bfpB-), four EAEC (aggR+), and three hybrid aEPEC/EAEC (eae+/bfpB-/aggR+), with all eight isolates assigned in the serotype O3:H2. Here we attempted to characterize these isolates in terms of E. coli phylogroup classification, presence of virulence factor-encoding genes, associated with the pathogenesis of EPEC and EAEC, plasmidial profile, adherence pattern on HeLa cells, FAS (fluorescent actin staining) test and biofilm formation. All eight isolates studied were assigned in the phylogroup A. We observed that the EAEC and the hybrid aEPEC/EAEC harbored several genes from the aggregative adherence plasmid (pAA), such as aatA, aap, orf3, aar, aggA and aggC (encoding the the pilin and usher of AAF/I, respectively) and share a common high molecular weight plasmid. On the other hand, the aEPEC and the hybrid aEPEC/EAEC isolates harbored two chromosomal genes, nleC and nleF, encoding for type 3 secreted effectors. Together, these data may suggest that the aEPEC background must have served as a receptor for the pAA, thus originating the hybrid aEPEC/EAEC isolates. The EAEC and the hybrid aEPEC/EAEC produced the AA pattern, while the aEPEC adhered only sporadically to the epithelial cells. As observed in the FAS assay, the aEPEC was unable to promote F-actin accumulation underneath adherent bacteria, while the hybrids aEPEC/EAEC were FASpositive. Distinct from the aEPEC isolate, the four EAEC and the three hybrid aEPEC/EAEC produced biofilm on abiotic surface, thus pointing out the influence of the pAA for the establishment of this phenotype. Our study reveals the high plasticity of pathogenic E. coli of serotype O3:H2 in acquiring and maintaining virulence-encoding genes. However, the search for new hybrids is continually needed to better understand which events are involved in the emergence of these hybrids and how different genetic combinations can influence their pathogenicity.
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Palavras-chave
Híbrido aEPEC/EAEC, Diarréia, Virulência, Hybrid aEPEC/EAEC, Diarrhea, Virulence