Characterization of Escherichia coli strains of serotype O119:H6 that co-express the patterns of localized and aggregative adherence and produce biofilm

Abstract

Escherichia coli (EPEC) was an important agent of diarrhea in children in the first year of life in our country for decades. EPEC causes the so called Attaching and Effacing lesion (A/E) in intestinal cells and carries the EAF plasmid (pEAF), which encodes the Bundle-Forming Pilus (BFP). BFP mediates the formation of compact microcolonies that characterize the pattern of Localized Adherence (LA), in HeLa and HEp-2 cells. Enteroaggregative E. coli (EAEC), another diarrheagenic E. coli pathotype that is common in our environment, is characterized by the formation of the of Aggregative Adherence (AA) pattern in HeLa and HEp-2 cells, and biofilm production. The identification of the expression of LA and AA contributes to the classification of E. coli strains as EPEC or EAEC, respectively. In Brazil, for several decades, EPEC strains of serotype O119:H6 were isolated with high frequency, always producing the typical LA pattern. Interestingly, in recent epidemiological studies, our group has isolated O119:H6 E. coli strains that co-express LA and AA (LA/AA) and produce biofilm. In our previous studies, we sequenced the bfpA gene (which encodes the structural subunit of BFP) of one of these strains (EC404/03), which showed 99.9% identity with the bfpA sequence of a prototype O119:H6 strain producing LA. We than constructed an isogenic mutant in bfpA that lost the ability to produce LA. Next, we transferred by conjugation, a high molecular weight plasmid (devoid of bfpA) of EC404/03 to a non-adherent E. coli strain. A preliminary characterization of the resulting transconjugants revealed the production of AA and the occurrence of a plasmid distinct from pEAF. To understand potential changes suffered by EPEC O119:H6, with respect to the adherence pattern, we intend to extend our studies, characterizing and comparing different phenotypic and genotypic properties of the other two O119:H6 LA/AA strains as well as comparing them with the characteristics of strains of the same serotype, but which express only LA. We also intend to obtain new transconjugants with the other two LA/AA strains in an attempt to identify whether there is a common plasmid associated with the AA pattern and biofilm formation in O119:H6 LA/AA strains. Using confocal microscopy, we also intend to compare the thickness and distribution of bacterial cells in biofilm formed in biotic and abiotic surfaces, by strains EC404/03, its bfpA mutant (obtained in our previous studies), and transconjugants. Results will be analyzed in combination in an attempt to assess the possible occurrence of evolutionary relationship among the O119:H6 LA and LA/AA strains, by horizontal acquisition of plasmid DNA containing genes that confer AA. (AU)