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The pathogen Escherichia coli O157:H7 is responsible for a large number of infections yearly in the United States and other countries, which sometimes result in life-threatening diseases such as hemolytic uremic syndrome. The reservoir of E. coli O157:H7 is the gastrointestinal tract of cattle, and while many routes of transmissions from animal to humans are reported, the majority of human infections are foodborne, and ground beef represents the major food vehicle. <P>
Molecular subtyping methods are DNA-based approaches for tracking the spread of foodborne pathogens from farm to fork. Only once the route(s) of transmission are defined can effective intervention strategies be implemented to stop this spread. For E. coli O157:H7, this is currently done using pulsed field gel electrophoresis (PFGE). However this method suffers from many limitations including the length of the protocol, technical expertise needed, and the ambiguous data that sometimes results. <P>Multilocus sequence typing (MLST) is a more rapid, cost effective method that has been applied to other pathogens, but has not been effectively developed for O157:H7 because of the high sequence identity found among genomes of different strains. We hypothesize that high-throughput sequencing of a large number of E. coli O157:H7 strains will identify genomic targets for discriminating between different strains, and lead to the development of a tracking method that is superior to PFGE. <P>This research project has three objectives: <OL> <LI> Sequence the genomes of 24 Escherichia coli O157:H7 strains using low cost, next-generation technology. <LI> Identify sequence variations within these genomes that effectively distinguish between all current and previously sequenced strains, and develop an MLST subtyping approach for strain discrimination. <LI> Test the effectiveness of the MLST method to track and control the spread of E. coli O157:H7 strains within meat-processing facilities. </ol> The goal of this research is the development of an MLST scheme for E. coli O157:H7 that would be useful for tracking the source of contamination during epidemiologic investigations. Additionally, the data generated will provide a great deal of information concerning single nucleotide polymorphisms (SNPs) that exist between strains of E. coli O157:H7. Identifying SNPs and/or other genomic regions that can be used as markers of highly pathogenic strains would provide risk assessment data concerning the potential pathogenicity of E. coli O157:H7 isolate(s) found within the food system.
NON-TECHNICAL SUMMARY: Escherichia coli O157:H7 is foodborne pathogen that is responsible for an estimated 73,000 illnesses each year within the United States. This organism causes diarrhea that may become bloody, and approximately 5-15% of cases progress into a life-threatening condition called haemolytic uremic syndrome. E. coli O157:H7 can be isolated from the intestinal tract of cattle, and is transmitted to humans when contaminated fecal material or cattle hides come into contact with food materials. Most foodborne infections are the result of contaminated ground beef, however a growing number of outbreaks are linked to fresh produce such as lettuce and spinach. As part of an epidemiologic investigation, DNA-based methods are used to track the spread of specific strains of E. coli O157:H7 from farm-to-fork. Only once the route(s) of transmission are defined can effective intervention strategies be implemented to stop this spread. For E. coli O157:H7, this is currently done using a technique called pulsed field gel electrophoresis (PFGE). While PFGE has proven effective this method suffers from many limitations including the length of the protocol, technical expertise needed, and the ambiguous data that sometimes results. Techniques that differentiate between strains of E. coli O157:H7 based upon differences in DNA sequence, such as multilocus sequence typing (MLST), have potential to be more rapid and cost effective. The development of such methods has suffered due to the large similarity found within the DNA of different E. coli O157:H7 strains. We hypothesize that sequencing the DNA of a large number of E. coli O157:H7 strains will identify genomic targets for discriminating between different strains and lead to the development of a tracking method that can complement or even replace PFGE. Once we have developed an MLST approach, we will test its effectiveness in microbial source tracking within beef processing facilities. This research therefore has the potential to improve the safety of our food supply by improving our ability to rapidly trace the source of contamination and to apply control points to break this chain of transmission.
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APPROACH: Twenty-four strains of E. coli O157:H7 that were previously characterized as coming from distinct phylogenetic groups, as well as strains that cannot be distinguished using current DNA sequence-based typing methods, will be shotgun sequenced using SOLiD sequencing. The sequences will be uploaded into the Galaxy system, which will be used for data storage and for quality analysis of sequence reads. The Mapping and Assembly with Quality tool (MAQ), which is integrated into the Galaxy system, will be used to align shotgun sequences from each strain to a reference genome (E. coli O157:H7 strain Sakai), and to identify SNPs between the two genomes. Additionally, the program LASTZ will be used to identify SNPs between E. coli strain Sakai and other previously sequenced strains deposited within GenBank. This data will be imported into Microsoft Excel, and we will identify approximately 50 genomic regions that are conserved among all strains, and show the greatest sequence variation between strains. PCR primers will be designed that amplify these targeted regions, and these genomic loci will be amplified and sequenced from a collection of 48 E. coli O157:H7 strains obtained from the E. coli Reference Center at Penn State. We will identify no more than 15 loci that will permit the greatest discrimination between strains. This newly developed MLST method will next be used to determine whether it is able to effectively type strains of O157:H7 obtained from beef processing plants in Pennsylvania. Strains obtained from plant surveys will be typed using MLST, as well as PFGE. Discriminatory power will be calculated to determine if this MLST method is as effective as PFGE at E. coli O157:H7 source tracking within processing facilities.