|Debroy, Chitrita - PENN STATE|
|Bryne, Brian - ASHTOWN FOOD RESEARCH|
|Monaghan, Aine - ASHTOWN FOOD RESEARCH|
|Bolton, Declan - ASHTOWN FOOD RESEARCH|
|Fanning, Seamus -|
Submitted to: Canadian Journal of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 26, 2010
Publication Date: April 15, 2010
Citation: Fratamico, P.M., Yan, X., Debroy, C., Bryne, B., Monaghan, A., Liu, Y., Bolton, D., Fanning, S. 2010. Escherichia coli serogroup O2 and O28ac O-antigen gene cluster sequences and detection of pathogenic E. coli O2 and O28ac by PCR. Canadian Journal of Microbiology. 56:308-316. Interpretive Summary: The bacterium Escherichia coli, consists of various serogroups (types), which are usually not harmful; however many strains belonging to various serogroups cause a variety of serious diseases in humans and animals. Traditionally, a procedure called serotyping is used to distinguish among the 179 different E. coli serogroups. This procedure relies on the use of antibodies raised in rabbits against different surface components (O antigen) of the bacteria. Serotyping, however, can generally only be performed in specialized laboratories, it is a lengthy procedure and labor intensive, and one antiserum can react with multiple E. coli serogroups, rendering identification difficult. Thus, due to the lack of simple and rapid methods for detection and identification of harmful and non-harmful E. coli types, the incidence of disease caused by harmful E. coli may be underestimated, and epidemiological studies are difficult to perform. To develop a more rapid and simple method for detection and typing of the E. coli serogroups O2 and O28ac, which have caused serious diseases in humans and animals, the DNA sequence of a cluster of genes involved in the production of a specific surface polysaccharide of these two serogroups was determined. Based on the DNA sequence information, a technique based on the polymerase chain reaction (PCR), involving amplification of specific genes in the cluster that are specific for E. coli O2 and O28ac, was developed for typing of these E. coli serogroups. Additionally, multiplex PCR assays targeting genes in the respective O antigen gene clusters and genes found in E. coli serogroups associated with causing disease were developed to detect pathogenic strains belonging to E. coli serogroups O2 and O28ac. Thus, the use of the E. coli O2- and O28ac-specific PCR assays facilitates identification of these bacteria and detection of pathogenic O2 and O28ac strains potentially in food, environmental, and other types of samples.
Technical Abstract: The O antigen gene clusters of Escherichia coli serogroups O2 and O28ac were sequenced. Thirteen open reading frames (ORFs) were identified in the O antigen gene cluster of E. coli O2 strain O2:H4 U 9-41, and 7 ORFs were identified in the O antigen gene cluster of E. coli serogroup O28ac strain O28ac:H25 96-3286, all having the same transcriptional direction. The ORFs encode genes for O antigen sugar biosynthesis pathways, glycsosyl transferases, and O antigen processing. The wzx (O antigen flippase) and wzy (O antigen polymerase) genes in the E. coli O2 and O28ac O antigen gene clusters were selected as targets for PCR assays for identification of these E. coli serogroups. PCR assays targeting the O2 and O28ac wzx and wzy genes using strains belonging to E. coli O2 and O28ac, non-O2 and non-O28ac strains isolated from humans, animals, food, and the environment, representative strains belonging to 165 E. coli O serogroups, and non-E. coli bacteria showed 100 % specificity for E. coli O2 and O28ac Thus, the wzx and wzy genes of E. coli serogroups O2 and O28ac can be used as diagnostic markers in PCR assays for rapid identification of these serogroups as an alternative to serotyping. Multiplex PCR assays targeting the O2 wzx, stx1, stx2, hly, eae, and saa genes and the O28ac wzx, ial, ipaC, and ipaH genes were developed for detection of Shiga toxin-producing E. coli O2 strains and enteroinvasive E. coli O28ac strains, respectively. The multiplex PCR assays targeting the O2 and O28ac wzx and wzy genes in combination with virulence genes can be used to identify and to detect pathogenic strains of these serogroups potentially in food, fecal, and environmental samples.