Epidemiologic and Mutational Characteristics of Variable-Number Tandem Repeats in Escherichia coli O157:H7

Escherichia coli O157:H7 is an important food-borne pathogen and public health risk that infects thousands of people a year in the United States alone. While many infections may remain undetected, some develop into hemorrhagic colitis and/or hemolytic uremic syndrome especially in young children and the elderly. The development of the molecular subtyping technique pulsed-field gel electrophoresis (PFGE) greatly enhanced the detection of outbreaks caused by this organism, but its technical limitations had researchers searching for alternative techniques. The use of variable-number tandem repeats (VNTRs) for human forensics and subtyping of extremely clonal bacterial species such as Bacillus anthracis provided a potential new technique for examining E. coli O157:H7. This new technique, multi-locus VNTR analysis (MLVA), examines multiple VNTR loci, which are some of the most rapidly evolving genetic elements in the genome. We demonstrated the utility and superiority of MLVA over PFGE as a molecular subtyping technique for E. coli O157:H7. With the establishment of the MLVA protocol, the need arose to understand how often the MLVA loci mutate to help characterize which isolates are highly related. Using an experimental protocol of 10 serial subcultures, one of the 7 MLVA loci was found to be hypervariable with a tendency of single, addition TR mutation. Two other loci were found to be slightly variable while the remaining 4 loci had no mutation events during the experiments. The establishment of a protocol based on VNTRs and the initial understanding of mutational dynamics only touched upon the genotypic roles of VNTRs but not the functional roles. A preliminary examination of the functional roles of a few selected VNTRs was undertaken by performing a variety of tests. A detailed description of all this project's results is presented in the following work.