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VASCONCELLOS, F. M. Study of the aggreggative adherence pattern of Escherichia coli from serotype O142:H34. 2014. 112 p. Ph. D. thesis (Biotechnology) – Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2014. Enteropathogenic Escherichia coli is one of the diarrheagenic E. coli (DEC) pathotypes, an important agent of acute diarrhea in children, especially in developing countries. Strains of this pathotype induce histopathological changes in the intestinal epithelium known as attaching and effacing (A/E) lesion. The classification of EPEC in the subgroups typical and atypical is based on the presence or absence of the plasmid E. coli adherence factor (pEAF), respectively. Somatic antigen (O) typing of EPEC strains associated with diarrhea led to the definition of 12 classical EPEC serogroups, among which is the serogroup O142. A previous study characterizing EPEC O142 described a strain of serotype O142:H34 (Ec48/66) presenting some important characteristics: the presence of the eae gene (which encodes the adhesin intimin of EPEC), the absence of the pEAF (determinant of typical EPEC), reactivity with the probe fragment CVD432 (indicative of the virulence plasmid of enteroaggregative E. coli, or EAEC) and the expression of the aggregative adherence pattern (characteristic of EAEC). This strain has an unusual combination of EPEC and EAEC virulence genes, making difficult its classification. Furthermore, the adhesin mediating the aggregative adherence othis strain was not characterized. Thus, the present study aimed to evaluate the role of fimbrial structures in the establishment of the aggregative adherence pattern of E. coli strain serotype O142:H34 and evaluate its classification. The strain Ec46/88 was able to adhere to HeLa, HEp-2, HT-29 and T84 cell in the aggregative adherence pattern, as well as to cause the A/E lesion in HeLa and HT-29 cells. A survey of several virulence factors of EAEC and other E. coli pathotypes detected the presence of aggDBCA, which comprise the operon encoding the EAEC AAF/I fimbriae, and ecpRABCDE, which encode the E. coli common pilus (ECP). The agg operon was sequenced exhibiting high identity (96%) of aggDBC with the same genes present in EAEC 17-2, and aggA (99%) with a gene encoding the variant AggA457. The genes of the ecp operon showed high identity (99%) with the same operon of EPEC E2348/69. Mutants in the genes encoding AAF/I and ECP ushers (aggC and ecpC, respectively) were obtained. The analysis of the interaction of both mutants with HeLa cells showed the maintenance of the aggregative adherence and the ability to cause the A/E lesion. However, the aggC mutant presented significant decrease in the number of adherent bacteria. Phylogenetic analysis in relation to other DEC pathotypes situated this strain in the group consisting predominantly of EPEC. The data obtained indicated that the Ec48/66 strain is an atypical EPEC able to cause the A/E lesion in vitro and to express the aggregative adherence pattern, which is partially dependent on a variant of AAF/I fimbriae, but not on ECP fimbriae.

Estudo do padrão de adesão agregativa de Escherichia coli do sorotipo O142:H34.