COMPARISON BETWEEN TWO METHODOLOGIES TO VERIFY BIOFILM FORMATION BY ATYPICAL ENTEROPATHOGENIC ESCHERICHIA COLI (aEPEC) STRAINS.

AC08

Hebert Fabricio Culler, Cristiane Moda Mota, Marcelo Palma Sircili, Marcia Regina Franzolin.

Laboratório de Bacteriologia, Instituto Butantan, SP, Brasil.

Atypical Enteropathogenic Escherichia coli (aEPEC) has become the most frequent bacterial pathogen in children with diarrhea in Brazil. Microorganisms can live and proliferate in the environment and can possess the capacity to adhere forming biofilms. This type of formation represents colonization mechanisms, conferring resistance to the action of antibiotics in the associated bacteria, related with bacterial persistence. The gene shf, regularly present in Enteroaggregative E. coli strains, encodes a protein possibly required for the firm biofilm formation of EAEC 042. The aim of this study was to verify the capacity of biofilm formation by atypical EPEC strains in abiotic surface and compare the method of staining with crystal violet (indirect method) with colony forming units (CFU) count (direct method). We analyzed 76 atypical EPEC strains isolated of children with acute diarrhea from Salvador, Bahia. These strains were screened for biofilm production through colorimetric assay of violet crystal using polystyrene 24-well culture dishes and was quantified using enzyme immunosorbent assay plate reader in length of wave of 595 nm. Of these, the 13 highest biofilm formation and 7 who had little biofilm formation were tested through the counting of CFU/cm² attached to the biofilm after disrupting with Triton X-100 to examine the efficiency of the violet crystal assay. The shf gene was researched through PCR. The aEPEC strains had been capable to form biofilm in polystyrene. 82.9% of the strains had presented a similar biofilm production comparing with the typical EPEC strain (E2348/69), whereas 17.1% had presented a superior potential (OD>0,100 at 595 nm), reaching maximum OD of 1,026. With regard to the method of CFU counting the largest producer strain obtained a value equals to 8,53x10⁷ CFU while the lower obtained value equals to 1,22x10⁴. The comparison of 20 samples through the direct and indirect methods showed that the majority of them did not show the same order of magnitude in both methodologies. The shf gene was found in 2 samples. The violet crystal assay does not seem to be as reproducible as the methodology by CFUs count, but the first can be employed as a presumptive test to select the samples better-producing biofilm, using as a confirmation of these potential to test of CFU adhered to the substrate. The shf gene appears not to be as important for the formation of biofilm in atypical E. coli strains, because this gene was detected only in 2 of the 20 samples analyzed, and one of them did not present a good biofilm formation. These results raise the possibility of that others structures and/or mechanisms can be involved in pathogenesis of these strains. Adesins not yet described can be involved in the multifactorial process biofilm formation.