Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/15/2009
Publication Date: 7/4/2010
Publication URL: http://handle.nal.usda.gov/10113/57279
Citation: Dowd, S.E., Wolcott, R., Crippen, T.L., Sun, Y., Callaway, T.R. 2010. Microarray analysis and draft genomes of two Escherichia coli 0157:H7 lineage II cattle isolates FRIK966 and FRIK2000 investigating lack of Shiga toxin expression. Foodborne Pathogens and Disease. 7:763-773. Interpretive Summary: Two genetic lines of E. coli O157:H7 have been reported in the literature. These lines differ in their pathogenicity towards humans, largely due to differential production of the Shiga toxin 2. This study was undertaken to determine some of the genetic differences that underlie this physiological difference. The structural genes of the toxin were found to be missing in E. coli that did not produce Shiga toxin 2, suggesting that genetic rearrangements may have occurred during evolutionary time, producing a lineage that is less of a human foodborne illness risk.
Technical Abstract: The existence of two separate lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that lineage I might be more pathogenic towards human hosts than lineage II. We have previously shown that lineage I expresses higher levels of Shiga toxin 2 (Stx2). To evaluate why lineage II isolates do not express appreciable levels of toxin, whole genome microarrays were performed using Agilent technology on two representative bovine lineage II isolates (FRIK966 and FRIK2000). E. coli O157:H7 EDL933 (lineage I clinical type isolate) was used as a reference. Missing or differentially expressed genes and pathways were identified, and quantitative RT-PCR was performed to validate the microarray data. Finally, draft genomes of FRIK966 and FRIK2000 were sequenced using Roche Applied Science/454 GS-FLX technology shotgun and paired-end approaches, followed by de-novo assembly. These assemblies were compared to two existing lineage I genome sequences, E. coli O157:H7 Sakai and E. coli O157:H7 EDL933. The bacteriophage 933W, which encodes the STX2 genes sequence, showed a dramatic repression in gene expression. PCR primers, based upon EDL933 genomic information, were designed against the potentially missing genes of the bacteriophage STX2. Most of the structural genes associated with the bacteriophage were found to be absent from the genome of the two bovine isolates. This analysis suggests that IS629 rearrangements may be associated with disruption of the bacteriophage encoding stx(2AB) in lineage II isolates. The results support our hypothesis that lineage II isolates may be less of a risk as human foodborne pathogens. The microarray and genome data have been made available to the scientific community to allow continuing analysis of genomes and expression data of these two E. coli O157:H7 bovine isolates.