RESUMO
Two phylogenetic methods (multilocus sequence typing [MLST] and a multiplex PCR) were investigated to determine whether phylogenetic classification of verocytotoxin-producing Escherichia coli serotypes correlates with their classification into groups (seropathotypes A to E) based on their relative incidence in human disease and on their association with outbreaks and serious complications. MLST was able to separate 96% of seropathotype D and E serotypes from those that cause serious disease (seropathotypes A to C), whereas the multiplex PCR lacked this level of seropathotype discrimination.
Assuntos
Escherichia coli/classificação , Escherichia coli/genética , Filogenia , Reação em Cadeia da Polimerase/métodos , Toxina Shiga I/metabolismo , Sequência de Bases , Análise por Conglomerados , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Genes Bacterianos/genética , Modelos Genéticos , Dados de Sequência Molecular , Saúde Pública/métodos , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , VirulênciaRESUMO
This study evaluated two generic polymerase chain reaction (PCR) protocols, and nine subtyping protocols and three PCR-restriction fragment length polymorphism (RFLP) protocols for detection of stx genes. The PCR protocols were evaluated by testing 12 reference isolates and 496 field strains of Shiga toxin-producing Escherichia coli (STEC). Both generic methods detected all stx genes. In tests with the reference isolates, all methods detected stx1 and stx2, seven subtyping methods detected stx2v(EH250), seven detected stx2e and only two detected stx2f. Four of the subtyping protocols identified stx genes in all of the field isolates. The PCR-RFLP protocols gave contradictory results for approximately 20% of the strains tested. The observed limitations of the protocols were shown to be due to nucleotide sequence variation in the region of the PCR primers. One subtyping protocol that detected the virulence-related genes, eae and ehxA, and all stx except for the stx2f gene, was modified by newly designed primers so that it identified all stx genes. This modified protocol provides comprehensive characterization of STEC in a single multiplex reaction.
Assuntos
Escherichia coli/genética , Reação em Cadeia da Polimerase/métodos , Toxina Shiga I/genética , Toxina Shiga II/genética , Animais , Chlorocebus aethiops , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/classificação , Escherichia coli/metabolismo , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Toxina Shiga I/química , Toxina Shiga I/toxicidade , Toxina Shiga II/química , Toxina Shiga II/toxicidade , Células VeroRESUMO
Verocytotoxin-producing E. coli (VTEC) of serotype O157:H7 have been shown to be important agents of foodborne disease in humans worldwide. While the majority of research effort has been targeted on this serotype it is becoming more evident that other serotypes of VTEC can also be associated with human disease. An increasing number of these non-O157:H7 VTEC have been isolated from humans suffering from HUS and diarrhea. Recently a number of foodborne outbreaks in the USA, Australia, and other countries have been attributed to non-O157:H7 VTEC serotypes. Surveys of animal populations in a variety of countries have shown that the cattle reservoir contains more than 100 serotypes of VTEC, many of which are similar to those isolated from humans. The diversity and complexity of the VTEC family requires that laboratories and public health surveillance systems have the ability to detect and monitor all serotypes of VTEC.