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MOTA, C. M. Influence of different culture conditions on biofilm formation by atypical enteropathogenic Escherichia coli (aEPEC) and search of related genes. 2014. 124 p. Masters tesis (Biotechnology) Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2014. Escherichia coli (EPEC) has been identified as the main agent of acute diarrhea in children in developing countries, predominantly atypical EPEC (aEPEC). The adhesion in epithelial cells forming microcolonies is a hallmark of EPEC and is an initial step in biofilm formation. Biofilms are bacterial structures which are adhered to biotic and abiotic surfaces and are surrounded by a matrix of exopolysaccharides. The aim of this study was investigate the influence of different culture conditions on biofilm formation by 23 aEPEC strainss isolated from children with diarrhea and search for the presence of genes related to biofilm formation. The sequences of the csgA, crl, fimH and bcsA genes were screened by PCR and were found in 6 (26%), 23 (100%), 22 (95.6%) and 23 (100%) of the strains respectively. The expression of type I fimbriae, curli fimbriae, cellulose and presence of motility were positive for 16 (69.6%), 11 (47.8%), 13 (56.5%) and 18 (78.3%) of the strains. MTP plates for aEPEC strains was better evidenced in LB medium without NaCl at 26 °C and in E. coli broth at 37 °C, both in number and intensity of biofilm formation. Ring (pellicle) formation in air-liquid-glass interface at 26 °C was observed in most of the analyzed strains in all media, with exception of M9 minimal, which only 9 strains (39.1%) formed ring. However, the strains were incubated at 37 ° C showed lower positivity in LB media (4.3%), LB without NaCl (13%) and M9 (4.3%), while E. coli broth, up 91.3% of the strains formed ring at both temperatures. However, when incubated at 37 °C strains showed lower positivity in LB media (4.3%), LB without NaCl (13%) and M9 minimal (4.3%), while broth E. coli, up 91.3% of the strains formed biofilm at both temperatures. These data emphasize the importance of persistence power and resistance to the environment, which suggests its role as a source of contamination. The confocal and electron microscopy provided a qualitative analysis of structures such as microcolonies and pillars, and respectively EPS. Although some strains do not express curli and type I fimbriae, as well as they do not product cellulose under the used conditions, some strains were able to form biofilms indicating that apart from them, other fimbriae and / or adhesins may influence the biofilm formation in aEPEC. The presence of several genes possibly involved in biofilm formation, as well as their expression revealed they are apparently involved in a multifactorial process. The ability of biofilm formation can be demonstrated using culture medium supplemented mainly with components found in the gastrointestinal tract of humans and animals (bile salts present in the E. coli broth). Moreover, biofilms can provide high-resistance potential of the environment and the host, enhancing its virulence. Therefore, the growing conditions should be used with caution to measure the real ability of biofilm formation by aEPEC strains.

Influência de diferentes condições de cultivo na formação de biofilme por Escherichia coli enteropatogênica atípica (EPECa) e pesquisa de genes relacionados.