Principal

DETECTION OF VIRULENCE GENES IN ATYPICAL EPEC STRAINS



AC10


Santos, TO, Porangaba, TM, Franzolin, MR, Sircili, MP.


Laboratório de Bacteriologia, Instituto Butantan.


thiagosantos@butantan.gov.br

Introduction: Diarrhea continues to be one of the most common causes of morbidity and mortality among infants and children, especially in developing countries. Among the bacterial pathogens, diarrheagenic Escherichia coli (DEC) is an important agent of endemic and epidemic diarrhea worldwide. The diarrheagenic E. coli strains can be classified in six main pathotypes, based upon specific virulence properties, clinical features, association with serotypes O:H, epidemiological aspects, and patterns of interaction with host cells. Enteropathogenic Escherichia coli (EPEC) cause a histopathological lesion known as “attaching and effacing” (A/E). Typical EPEC differs from atypical EPEC by the presence of a plasmid called EPEC adherence factor (EAF) that encodes the bundle-forming pilus (BFP). Atypical EPEC comprises a very heterogeneous group. We developed multiplex PCR reactions in order to identify virulence genes present in other DEC pathotypes in a collection of 120 Atypical EPEC strains isolated from children with diarrhea in Salvador , Bahia, Brazil. Objective: Search virulence genes of DEC in atypical EPEC. Methods: DNA templates for PCR were obtained from overnight E. coli cultures that were pelleted, ressuspended in 500µl of sterile deionized water and boiled for 10 min. The PCR was developed by combining specific primer pairs. The genes analyzed in this study were efa, pic, sat, ldaH, toxB and saa. Each multiplex reaction was performed in a 50 µl final volume containing 1µl of template DNA, 0.2mM DNTPs, 10mM Tris-HCl (pH8.8), 1.5 mM MgCl2, 50mM KCl, 2 U Taq DNA polymerase (Invitrogen) and 10 pmol of each primer (BiosynthesisAmplified). Amplified samples were detected by 1% agarose gel electrophoresis in Tris-borate-EDTA buffer and ethidium bromide staining. Results: Of the 120 samples studied, 7 tested positive for the gene efa1/lifA; 3 for the gene toxB; 13 for gene sat; 8, for the gene ldaH and 0 for the gene saa. The results obtained by the reaction of PCR were confirmed by sequencing.   Discussion: All samples submitted to the sequencing showed at least 90 % similarity with  the genes described in the literature. Conclusion: As we expected, atypical EPEC strains carries virulences genes common to other DEC.

Gene virulence, PCR, aEPEC

FAPESP; CNPQ

Categorias: Parasitologia, Microbiologia, Imunologia e Hematologia