pEO5 Plasmid of Escherichia coli of O26 serogroup: comparative analysis with other plasmids that encode alpha hemolysin in pathogenic E. coli.

O pEO5, que codifica hemolisina, foi isolado de uma amostra de EPEC do sorotipo O26. Este plasmídio mostrou ser conjugativo e compatível com o pO157, e pelos testes de hibridização observou-se que estes plasmídios não são geneticamente relacionados. Para o estudo comparativo de similaridade foi seqüenciada uma região de 9227 pb de DNA do pEO5 que compreende todo o operon hlyCABD e suas regiões a montante e a jusante. A região do operon hemolitico (7225 pb) e a região promotora do operon foram similares às mesmas regiões do pHly152, que em uma amostra de E. coli isolada de roedor, codifica uma a hemolisina. No entanto, verificou-se a presença de elementos de inserção na região a montante do gene hlyC no pHly152. O pEO5 mostrou ser semelhante a outros plasmídios que também codificam a hemolisina em cepas de EPEC O26 de origem humana e de bovinos. A presença de estruturas semelhantes a transposons em ambas as extremidades do operon a hemolítico do pEO5 indica que esse fator de virulência provavelmente foi adquirido por transferência horizontal de genes.The conjugative pEO5 encoding haemolysin in strains of EPEC O26 was investigated for its relationship with EHEC haemolysin-encoding of EHEC O26 and O157 strains. pEO5 was found to be compatible with EHEC virulence plasmids and did not hybridize in Southern blots with pO157, indicating that both plasmids were unrelated. A 9227 bp stretch pEO5 DNA encompassing the entire operon hlyCABD was sequenced and compared for similarity to plasmid and chromosomally inherited hly determinants. The a hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of pHly152, in particular, the structural a-hlyCABD and hlyR regions. pEO5 and hly of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural a-hlyCABD. The major difference found between the hly regions of pHly152 and pEO5 is caused by the IS2 upstream of the hlyC in pHly152. The presence of transposonlike structures at both ends of hly sequence indicates that pEO5 was probably acquired by horizontal gene transfer

Characterization of the alpha-haemolysin determinant from the human enteropathogenic Escherichia coli O26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding alpha-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire alpha-hlyCABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited alpha-hly determinants. The alpha-hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli alpha-hly plasmid pHly152, in particular, the structural alpha-hlyCABD genes (99.2% identity) and the regulatory hlyR regions (98.8% identity). pEO5 and alpha-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural alpha-hlyCABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS2 element upstream of the hlyC gene in pHly152. The presence of transposon-like structures at both ends of the alpha-hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.Brazil by `Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)`,[2689/06-5]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Determination of flagellar types by PCR-RFLP analysis of enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains isolated from animals in Sao Paulo, Brazil

This study evaluated the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis of fliC for typing flagella antigen (H) of Shiga toxin-producing Escherichia coil (STEC) and enteropathogenic E. coli (EPEC) strains isolated from different animals. The molecular typing of the H type was efficient in the determination of 93 (85%) strains. Two nonmotile (H-) E. coil strains showed a PCR-RFLP electrophoretic profile that did not match known H type patterns. The fliC nucleotide sequence of strains B2N and 4a revealed a nucleotide substitution at the restriction site and a nucleotide insertion that generated a stop codon, respectively. The results of this study showed that PCR-RFLP analysis of fliC is faster, less laborious and as efficient for the determination of H type E. coli isolated from animals, compared to serotyping and that it is useful in determining H type in nonmotile strains and strains expressing non-reactive H antigens. Moreover, the fliC sequence of strain B2N suggests that we could have found a new flagellin antigen type. (C) 2010 Elsevier Ltd. All rights reserved