Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Escherichia coli enteroagregativa (EAEC) à um patÃtipo de E. coli que tem sido cada vez mais identificado como agente etiolÃgico das doenÃas diarrÃicas. Esse trabalho teve como objetivos determinar a prevalÃncia de EAEC e investigar a importÃncia de alguns genes associados à virulÃncia no grau de severidade das doenÃas diarrÃicas causadas pelo microorganismo, alÃm de avaliar o impacto dessas infecÃÃes na inflamaÃÃo intestinal e no estado nutricional de crianÃas carentes de Fortaleza, CearÃ. CrianÃas na faixa etÃria entre 2 e 36 meses, com histÃrico ou nÃo de diarrÃia nos Ãltimos 14 dias, tiveram suas medidas antropomÃtricas avaliadas e suas amostras fecais coletadas. O diagnÃstico de EAEC foi realizado por reaÃÃo de polimerase em cadeia (PCR) dos genes aaiC (cromossomal) e aatA (plasmidial). Amostras positivas foram pesquisadas quanto à presenÃa dos genes de virulÃncia aggR (regulador transcricional), aap (dispersina), pic (enterotoxina), pet (enterotoxina) e astA (enterotoxina). A sequÃncia nucleotÃdica do gene aggR foi analisada atravÃs de seqÃenciamento. AlÃquotas fecais foram submetidas à quantificaÃÃo de lactoferrina (LFF) e citocinas (IL-4, IL-10, TNF-α e IFN-γ) atravÃs de reaÃÃo imunoenzimÃtica (ELISA). Esse estudo analisou 83 crianÃas com diarrÃia (casos) e 83 crianÃas sem diarrÃia (controles). EAEC foi encontrada na mesma proporÃÃo entre ambos os grupos (41,0%). CrianÃas com diarrÃia apresentaram reduÃÃo significativa na espessura da prega cutÃnea, Ãndice de massa corporal e escores-z peso-por-idade (WAZ) e peso-por-altura (WHZ), mas a presenÃa da bactÃria nÃo foi associada com alteraÃÃes nos Ãndices antropomÃtricos analisados. Entre as amostras positivas para EAEC, nÃo houve diferenÃa quanto à presenÃa isolada dos fatores de virulÃncia pesquisados nas crianÃas que desenvolveram ou nÃo diarrÃia. Avaliando as freqÃÃncias desses genes em combinaÃÃo, cepas de EAEC expressando os genes aggR, aap, pic, pet e astA foram isoladas em freqÃÃncia superior significativa de crianÃas doentes quando comparadas com cepas de EAEC expressando somente aggR, aap, pic e astA (excluindo o gene pet). A anÃlise da seqÃÃncia codificadora do gene aggR apresentou 27 polimorfismos em um Ãnico nucleotÃdeo (SNPs), distribuÃdos entre cinco amostras caso e trÃs controles. Mais de 80,0% das crianÃas estudadas apresentaram inflamaÃÃo intestinal caracterizada por elevados nÃveis de LFF, independente da presenÃa de diarrÃia e EAEC. Todas as crianÃas com diarrÃia associada com EAEC apresentaram altas concentraÃÃes de LFF. NÃveis basais de citocinas fecais foram observados entre crianÃas de ambos os grupos. A variabilidade na presenÃa dos fatores de virulÃncia pesquisados ratifica a heterogeneidade das cepas de EAEC. A combinaÃÃo de genes codificadores de fatores de virulÃncia mostrou que a presenÃa do pet està associada com a doenÃa causada por EAEC. A infecÃÃo pela bactÃria nÃo causou impacto significativo nos Ãndices antropomÃtricos analisados. As elevadas concentraÃÃes observadas de LFF sugerem a existÃncia de fatores adicionais desencadeadores do processo inflamatÃrio. / Enteroaggregative Escherichia coli (EAEC) is a pathotype of diarrheagenic E. coli, which has increasingly been identified as an etiological agent of diarrheal disease. This purpose of this study is to determine the prevalence of EAEC and examine the importance of some virulence-related genes in the severity of the diarrheal disease caused by this microorganism, and further evaluate the impact of these infections on intestinal inflammation and the nutritional status of poor children from Fortaleza, Ceara. Children aged 2 to 36 months, with and without an occurrence of diarrhea in the previous 14 days, had their anthropometric data evaluated and their stools collected. Diagnosis of EAEC was done by polymerase chain reaction (PCR) of the aaiC (chromosomal) and aatA (plasmidial) genes. Positive samples were further analyzed for the presence of the virulence genes aggR (transcription regulator), aap (dispersin), pic (enterotoxin), pet (enterotoxin) and astA (enterotoxin). The nucleotide sequence of aggR gene was also analyzed by sequencing. Aliquots of stool samples were quantified for lactoferrin (LFF) and cytokines (IL-4, IL-10, TNF-α e IFN-γ) by enzyme-linked immunosorbent assay (ELISA). This study analyzed 83 children with diarrhea (cases) and 83 children without diarrhea (controls). EAEC was found in the same proportion in both groups (41.0%). Children with diarrhea presented with significantly reduced skin thickness, body mass index and weight-for-age (WAZ) and weight-for-height (WHZ) z-scores. However, the presence of the bacteria was not associated with changes in the analyzed anthropometric measures. Among the positive samples for EAEC, there was no difference in the presence of the isolated virulence genes in the children with or without diarrhea. Observing the frequencies of these genes in combination, EAEC strains carrying the aggR, aap, pic, pet and astA genes together were isolated in significantly higher frequencies from sick children when compared to EAEC strains expressing only aggR, aap, pic, and astA (excluding pet). Nucleotide sequence analysis of the aggR gene presented 27 polymorphisms of a single nucleotide (SNPs), distributed among five case samples and three control samples. More than 80.0% of studied children had intestinal inflammation characterized by elevated levels of LFF, regardless of the presence of illness and EAEC. All children with diarrhea associated with EAEC presented with high concentrations of LFF. Basal levels of fecal cytokines were observed among children from both groups. Variability in the presence of the evaluated virulence factors confirms the heterogeneity of EAEC strains. The combination of the virulence related genes expressed showed that pet has an association with illness caused by EAEC. The infection by this bacterium did not cause significant impact on the anthropometric index analyzed. The high concentrations of LFF observed suggest that there may be additional factors triggering the inflammatory process.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:2142 |
| Date | 12 November 2008 |
| Creators | Ila Fernanda Nunes Lima |
| Contributors | Aldo Ãngelo Moreira Lima, Helena Serra Azul Monteiro, Cristiane Cunha Frota, Marcos FÃbio Gadelha Rocha, Alexandre Havt Bindà |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em Farmacologia, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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