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Prevalence of sepA autotransporter-encoding gene in enteroaggregative Escherichia coli (EAEC).

Andrade FB1, Abreu AG2, Piazza RMF1, Gomes TA3, Elias WP1

1 Laboratório de Bacteriologia, Instituto Butantan, Brasil; 2 Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Maranhão, Brasil; 3 Disciplina de Microbiologia, Universidade Federal de São Paulo, Brasil

Introduction:  Enteroaggregative Escherichia coli (EAEC) are recognized worldwide as an agent of acute and persistent diarrhea in children and adults. These strains can be classified as typical or atypical, based on the presence or absence of aggR, respectively. The SPATES (Serine Proteases Autotransporters of Enterobacteriaceae) proteins, also known as autotransporters (AT), are virulence factors that EAEC strains may express. Among the AT, the SepA encoding-gene (sepA), described in Shigella flexneri 2a and EAEC, has been associated with diarrheal cases caused by EAEC. However, the role of SepA in pathogenicity is unknown.  Objectives:  The present study aimed to determine the frequency of sepA gene in a large collection of EAEC strains.  Methods:  A total of 193 strains (108 typical and 85 atypical) isolated from two studies on the etiology of acute diarrhea in children were selected. Initially, all strains were searched for the presence of sepA by PCR. Following, the sepA-positive strains were further characterized in terms of the presence of other AT genes, also by PCR. Results and Discussion:  Among the 193 EAEC, sepA was detected in 26 (13.5%) strains (10/9.2% typical and 16/18.8% atypical). The sepA-positive strains presented the following combinations of different AT-encoding genes: 5 strains harbored only sepA, 10 sepA-pic, 2 sepA-espI, 1 sepA-pet, 2 sepA-pic-sat, 2 sepA-pic-pet, 3 sepA-pic-sat-espI, and 1 sepA-pet-pic-sat. Two of the typical EAEC sepA-positive strains (BA198 e BA732) were chosen for further characterizations, based on the presence of the smaller number of additional AT genes. These strains presented the sepA-pic genetic combination. The plasmid profile and sepA location were determined in both of them by gel electrophoresis of plasmid extraction and Southern Blotting, respectively. Strain BA198 harbored three plasmid bands of 2.8, 6 and 88 MDa; while BA732 was devoid of plasmids. Hybridizations with the sepA probe demonstrated the presence of that gene in the 88 MDa plasmid of BA198 and in the chromosome of BA732. DNA sequencing analyzes, in comparison with sepA of the reference Shigella flexneri 5a (M90T), showed high identities between these sepA sequences, i.e. 96% for BA198 and 97% for BA732. We can conclude that the sepA gene occurred in low frequency in our EAEC strains, being more frequent in the atypical group. Also, in these strains sepA may be either plasmidial or chromosomal and their DNA sequences showed high similarity with the corresponding gene of Shigella flexneri 5a.

Supported by FAPESP



5 - MICROORGANISMS 17th Annual Scientific Meeting of Instituto Butantan.