The project is based on developing a test that can rapidly discriminate different subpopulations of E. coli 0157:H7. High resolution genotyping methods have previously shown that subpopulations of 0157:H7 exist that are genetically distinct and appear to have a bias as to their source. Preliminary studies indicated that Lineage I strains are more commonly isolated from humans than lineage II and that the bias was not regional, but national. This lead to the hypothesis that lineage 11 strains were less likely to be transmitted to humans from cattle or that they were less efficient at causing disease in humans. In order to test that hypothesis, high-throughput methods need to be developed to distinguish between the subpopulation. The current method of OBGS is too complex and lacking image analysis software to allow high throughput.
The specific aims of this project are to<ol>
<li> To validate the SSP (now called Lineage Specific Polymorphism-LSP) assay using sets of 0157:H7 strains that have previously been characterized
<li> To use the LSP assay to conduct preliminary studies of prevalence of the subclones from prevalence studies of E. coli 0157: H7 in cattle
<li> To use the LSP assay to examine distribution of the subclones among clinical isolates</ol>
<p>The project is part of a larger 0157:H7 comparative genomics project supported by USDA CSREES and LiCor, inc. The project outline, in brief, was to use OBGS at high density to identify lineage-specific polymorphisms (LSPs). All class I LSPs (conserved in all strains of a lineage) are being cloned and sequenced along with a large number of class II and III LSPs (lineage-specific but non-conserved). Candidate LSPs that contain short, stable mutations, are then being tested as markers by designing specific primers upstream and downstream of the mutation. The goal is to multiplex together several of these markers into a single test that can rapidly define which lineage and clade a given 0157:H7 strain belongs to. This has been accomplished and now a test has been developed and validated. The results are summarized below.<p>
Status: The genome coverage phase of the project was completed this summer. An additional 20 primer combinations were added to increase complexity and degree of genome coverage. Over 170 different OBGS primer combinations have now been run on the set of 40 representative 0157:H7 strains. Table 1 below indicates the distribution of polymorphisms that were identified. A total of 70 class I LSPs were identified, along with 897 class II and III LSPs. Thus far, 61 of them have been cloned and sequenced, of which 55 are in independent loci. Of the 55 independent loci, 8 have mutations that are suitable as discriminatory alleles (markers) by PCR assays (e.g. they are short insertions or deletions). Five of these markers have been multiplexed together into a Lineage-Specific Marker Assay (LMSA) and validated on a strain set of 174 different 0157:H7 isolates (See Figs. 1 and 2 below for an example). Lineage I strains always a single allele combination whereas lineage II strains may show one of five different combinations, thereby providing five different clades in which to group the strains. The correlation coefficient between lineage predicted by the LSP assay and by OBGS on the 174 strains is 0.96, missing only on two of 174 strains. Thus, the assay is highly robust. As with OBGS, the marker assay initially indicates a bias of human isolates to lineage 1. Currently, collaborations with clinical laboratories in the U.S. and Canada are being set up to examine the distribution of human isolates among the lineages. Nonetheless, the assay is highly robust. Five of the markers have been multiplexed together into a single PCR reaction. Additionally, collaborations with scientists in the U.S. and Canada are being developed to begin large-scale testing of strain collections using the LMSA.