Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/26/2009
Publication Date: 6/1/2009
Citation: Fratamico, P.M., Debroy, C., Miyamoto, T., Liu, Y. 2009. PCR Detection of Enterohemorrhagic Escherichia coli O145 in Food by Targeting Genes in the E. coli O145 O-Antigen Gene Cluster and the Shiga Toxin 1 and Shiga Toxin 2 Genes. Foodborne Pathogens and Disease. 6(5):605-611. Interpretive Summary: The bacterium, Escherichia coli, causes a variety of diseases in humans and animals, and non-harmful types also exist. Traditionally, a procedure called serotyping is used to distinguish among all of the different E. coli types. This procedure relies on use of antisera raised in rabbits that react against surface components of E. coli. Serotyping, however, can generally only be performed in specialized laboratories, and it is labor intensive and may require several days to complete. Moreover, one antiserum can react with multiple E. coli serogroups, and E. coli often loses the ability to react with the antisera, thus rendering identification difficult. Due to the lack of simple, rapid, and accurate methods for detection and identification of harmful E. coli types, the incidence of disease caused by these organisms may be underestimated, and epidemiological studies are difficult to perform. To develop a more rapid and simple method for detection and identification of the E. coli serogroup O145, a pathogenic serogroup, the DNA sequence of a cluster of genes involved in production of a specific surface component of E. coli O145 was determined. Based on the sequence information, an assay called a multiplex polymerase chain reaction (PCR) was developed. This assay involves amplification of the DNA sequences of two genes in the cluster, as well as two genes involved in causing disease, called the Shiga toxin 1 and Shiga toxin 2 genes. The assay was used to detect this bacterium in ground beef seeded with less than or equal to 2 E. coli O145 bacteria per 25 grams. In addition, this procedure can easily be used for detection and identification of the other known E. coli serogroups. Thus, use of a specific PCR assay enhances the ability to identify and type E. coli O145 strains and to detect the pathogen at low levels in food and potentially also in animals and in environmental samples.
Technical Abstract: Enterohemorrhagic Escherichia coli serogroup O145 strains are an important cause of hemorrhagic colitis and hemolytic uremic syndrome worldwide. Cattle and other animals are potential reservoirs for this E. coli serotype. To develop PCR assays for detection and identification of E. coli O145, the DNA sequence of the O antigen gene cluster was determined, and 15 open reading frames were identified. The wzx (O antigen flippase) and wzy (O antigen polymerase) genes in the serogroup O145 O antigen gene cluster were selected as targets for development of PCR assays specific for E. coli O145. Results of PCR assays using DNA from strains of E. coli O145, non-O145 E. coli, and non-E. coli bacteria showed 100% specificity for E coli O145. In addition, a multiplex PCR assay targeting the E. coli O145 wzx and wzy genes and the Shiga toxin 1 (stx1) and Shiga toxin 2 (stx2) genes was developed and used to detect Shiga toxin-producing E. coli O145 in ground beef seeded with less than or equal to 2 CFU/25g of meat. The PCR assays can be employed to identify E. coli serogroup O145 and to detect low levels of the organism in ground beef and potentially in other foods, in animals, and in environmental samples.