Sequence Variations in the Flagellar Antigen Genes <i>fliC</i>_H25 and <i>fliC</i>_H28 of <i>Escherichia coli</i> and Their Use in Identification and Characterization of Enterohemorrhagic <i>E. coli</i> (EHEC) O145:H25 and O145:H28

<div><p>Enterohemorrhagic <i>E</i>. <i>coli</i> (EHEC) serogroup O145 is regarded as one of the major EHEC serogroups involved in severe infections in humans. EHEC O145 encompasses motile and non-motile strains of serotypes O145:H25 and O145:H28. Sequencing the <i>fliC</i>-genes associated with the flagellar antigens H25 and H28 revealed the genetic diversity of the <i>fliC</i>_H25 and <i>fliC</i>_H28 gene sequences in <i>E</i>. <i>coli</i>. Based on allele discrimination of these <i>fliC</i>-genes real-time PCR tests were designed for identification of EHEC O145:H25 and O145:H28. The <i>fliC</i>_H25 genes present in O145:H25 were found to be very similar to those present in <i>E</i>. <i>coli</i> serogroups O2, O100, O165, O172 and O177 pointing to their common evolution but were different from <i>fliC</i>_H25 genes of a multiple number of other <i>E</i>. <i>coli</i> serotypes. In a similar way, EHEC O145:H28 harbor a characteristic <i>fliC</i>_H28 allele which, apart from EHEC O145:H28, was only found in enteropathogenic (EPEC) O28:H28 strains that shared some common traits with EHEC O145:H28. The real time PCR-assays targeting these <i>fliC</i>_H25[O145] and <i>fliC</i>_H28[O145] alleles allow better characterization of EHEC O145:H25 and EHEC O145:H28. Evaluation of these PCR assays in spiked ready-to eat salad samples resulted in specific detection of both types of EHEC O145 strains even when low spiking levels of 1–10 cfu/g were used. Furthermore these PCR assays allowed identification of non-motile <i>E</i>. <i>coli</i> strains which are serologically not typable for their H-antigens. The combined use of O-antigen genotyping (O145<i>wzy</i>) and detection of the respective <i>fliC</i>_H25[O145] and <i>fliC</i>_H28[O145] allele types contributes to improve identification and molecular serotyping of <i>E</i>. <i>coli</i> O145 isolates.</p>