STEC O157 remains a perennial and costly food safety issue and public health concern to the beef industry at the production, processing, wholesale, distribution, retail and consumer levels. While STEC O157 strains were originally described by microbiologists as “clonal” (i.e., genetically and phenotypically invariant), an ever-growing body of emerging scientific and epidemiologic evidence suggests that this is probably not the case. Rather, there appears to be significant, frequent and informative (i.e., discriminatory) variation between and among STEC O157 field isolates in both strain phenotypic properties and in fine-resolution genomic structure. These strain differences may be manifested by the diverse ecological niches occupied, by variability in time and dose required to initiate and maintain gastro-intestinal tract infections in livestock, by capacity to survive on and to adhere (or not) to animal hides or muscle tissue (meat), and by the variable competence of strains to infect and cause disease (or not) in people exposed to STEC O157.
A growing body of recent scientific evidence suggests that human clinical STEC O157 strains represent only a genetic and phenotypic subset of the STEC O157 populations founds in animals and the ambient environment. Unfortunately, comprehensive STEC O157 characterization, especially genetic and genomic characterization, has focused almost exclusively on human clinical isolates. For example, STEC O157:H7 human clinical strains EDL 933 (from a 1982 U.S. hamburger index outbreak) and Sakai (from the 1996 Sakai City, Japan radish sprout outbreak) are the only two STEC O157 strains that have been completely DNA sequenced to date. New, relatively low-cost contract DNA sequencing systems have become available in recent years. One such system called Comparative Genome Sequencing (CGS) rapidly surveys entire microbial genomes (compared to a completely sequenced reference strain) to identify the locations of single nucleotide polymorphisms (SNPs).
The stated objectives for this work were:
E. coli O157:H7 isolates for the isolate panels were chosen from the collection at USMARC. Each isolate was genetically and phenotypically characterized as STEC O157:H7. DNA was extracted from each isolate using the Qiagen genomic tip 100/G. Genomic DNA purity and concentration were determined by 260/280 spectrophotometry and agarose gel electrophoresis. Equivalent amounts of DNA from bovine or human isolates were used to generate DNA pools of STEC O157:H7 of bovine and human origin, respectively. Three to five micrograms of DNA from the DNA pools was nebulized and used to create a library for sequencing using the 454 Genome Sequencer FLX. DNA sequence was analyzed using the GS Reference Mapper and GS De Novo Assembler software and other DNA analysis software. SNPs were scored with a system that uses primer-oligonucleotide base extension (PROBE), nano-liter dispensing of extension products onto silicon chips and fully automated mass spectrometric analysis using a MALDI-TOF MS in fully automated mode.
There have been 13,026 putative SNPs identified through 1.35X sequencing coverage of 99 bovine STEC O157:H7 isolates. STEC O157:H7 was once considered virtually clonal. Contrary to this notion, the researchers have found a DNA polymorphism for every 434 bases of the genome in STEC O157:H7. This level of genetic variation strongly works in the favor of developing a genotyping platform for distinguishing individual strains of STEC O157:H7. The researchers were able to add over ten times the original number of STEC O157:H7 strains originally proposed by switching from the CGS method to 454 GS FLX pyrosequencing. This huge boost in samples has given great power to the study. Additionally, the DNA pooling approach, which was not recommended by the 454 GS FLX specialists as being too risky, worked spectacularly well and has greatly enabled our ability to detect SNPs and determine allele frequencies.
The scientists identified thousands of differences between the DNA sequences of 190 E. coli STEC O157:H7 isolates. These genetic differences, also known as single nucleotide polymorphisms (SNPs), may be instrumental in either replacing or complementing current typing methods for E. coli STEC O157:H7.