Detection Of O Antigens In Escherichia Coli Using Molecular Methods

Open Access
Author:
Porter, Morgan Leigh
Area of Honors:
Veterinary and Biomedical Sciences
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Chitrita Debroy, Thesis Supervisor
  • Dr. Lester C Griel Jr., Honors Advisor
Keywords:
  • Escherichia coli
  • Real-Time Polymerase Chain Reaction
Abstract:
Escherichia coli, a food-borne pathogen, causes gastroenteric diseases in both humans and animals. Cattle are the reservoirs for the pathogenic strains, therefore, a common mode of infection in humans is through the ingestion of contaminated beef or other food and water laced with E. coli.3 Pathogenic strains of E. coli belonging to serogroups O157, O26, O45, O103, O111, O121, and O145 that produce Shiga toxins, cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. While E. coli O157:H7 was considered an adulterant in meat in 1996, the Food Safety and Inspection Services of the United States Department of Agriculture recently declared the other six O groups as adulterants in meat and mandated that these seven serogroups be routinely screened for in ground beef. Therefore, a rapid method for detecting O groups are urgently needed. E. coli are classified based on O antigens that are part of the lipopolysaccharide present on the surface of the bacteria. There are 187 different O antigens currently known. The traditional method of detection of the serogroups is by serotyping, utilizing antisera raised in rabbits against the 187 O antigens that are allowed to react with antigens for agglutination. This method is laborious and can exhibit equivocal results. Real-Time Polymercase Chain Reaction (RT-PCR) assays were developed for the detection the Escherichia coli serogroups O116, O133, and O134 targeting the unique wzx (O-antigen flippase) gene, found in the O-antigen gene cluster for each serogroup. The RT-PCR assays developed were found to be specific for detecting the respective serogroups. The assays were validated using cultures belonging to the respective serogroups as well as those of other serogroups and other non E. coli bacteria. These assays may be used for determining E. coli O serogroups rapidly and accurately as an alternative method to serotyping commonly used for detecting O antigen of E. coli.