Submitted to: Journal of Food Analytical Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/4/2008
Publication Date: 8/1/2009
Citation: Fratamico, P.M., Liu, Y., Debroy, C. 2009. Sequencing of the Escherichia coli O22 O Antigen Gene Cluster and Detection of E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and the wzy Gene in the Respective O-Antigen Gene Clusters. Journal of Food Analytical Methods. 2:169-179. Interpretive Summary: Escherichia coli is a bacterium, which causes various diseases in humans and animals, and non-harmful E. coli types or serogroups, also exist. A procedure called serotyping has traditionally been used to distinguish among the ca. 170 different E. coli serogroups. This procedure relies on the use of antibodies produced in rabbits against different E. coli surface components. Serotyping, however, can generally only be performed in specialized laboratories, is labor intensive and may require several days to complete, and one antiserum can react with multiple E. coli serogroups, rendering identification difficult. Thus, due to the lack of simple and rapid methods for identification of harmful and non-harmful E. coli serogroups, the incidence of disease caused by harmful E. coli may be underestimated, and epidemiological studies are difficult to perform. To develop rapid, specific, and simple methods for identification of E. coli serogroups O22 and O91, which have caused serious human illnesses, the DNA sequence of a cluster of genes involved in the synthesis of a specific surface polysaccharide of E. coli O22 was determined. Based on the DNA sequence information for E. coli O22 and similar information already available for E. coli O91, assays based on the polymerase chain reaction (PCR), involving amplification of specific genes in the cluster, were developed for identification of these two E. coli serogroups. The PCR assays targeting the O22 and O91 genes involved in synthesis of the E. coli polysaccharides were specific for the respective serogroups and can be used as diagnostic markers for rapid and accurate identification of these serogroups as an alternative to serotyping. Furthermore, multiplex PCR assays targeting genes in the cluster of each of the two serogroups, as well as genes harbored by pathogenic E. coli O22 and O91 (virulence genes) were developed to detect these pathogens. The multiplex PCR assays targeting the O22- and O91-specific genes and virulence genes can be used to provide information on the disease-causing capability of these bacteria and to identify and to potentially detect them in food, environmental, and clinical samples.
Technical Abstract: The O antigen gene cluster of Escherichia coli O22, an extraintestinal pathogenic E. coli (ExPEC) serogroup was sequenced, and eight open reading frames encoding O antigen sugar biosynthesis, transfer, and processing were identified. Multiplex PCR assays were developed targeting the wzx and wzy genes in the O antigen gene clusters of E. coli O22 and E. coli O91, a serogroup associated with hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS), to identify these serogroups. The assays were serogroup specific when tested against 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, humans, animals, and environmental sources, representative strains belonging to 168 Escherichia coli O serogroups, and non-E. coli bacteria. Furthermore, 72 E. coli O22 strains were tested by the PCR for the presence of six virulence genes associated with ExPEC strains, and 57 E. coli O91 strains were tested for the presence of 19 virulence genes associated with diarrheagenic E. coli. Based on the PCR screening results, multiplex PCR assays targeting the O22 wzy gene and the cnf-1 and sfa genes in E coli O22 and the O91 wzy gene and conserved sequences of stx1 and stx2 and the astA and cdt-III genes in E. coli O91 were developed to detect and identify ExPEC O22 strains and diarrheagenic E. coli O91 strains. The results demonstrate that the E. coli O22 and O91 wzx and wzy gene sequences were specific for the respective serogroups and can be used as diagnostic markers for rapid identification of these serogroups as an alternative to serotyping. Furthermore, the multiplex PCR assays targeting the O22 and O91 wzy genes and virulence genes can be used to identify and to detect pathogenic strains of these serogroups potentially in food, fecal, and environmental samples.