Influence of Diagnostic Methods in Determining the Prevalence of the Top 6 Non-O157 Shiga Toxin-Producing Escherichia coli in Beef Processing

There have been increasing numbers of clinical infections and Haemolytic Uremic Syndrome cases caused by the Top 6 serotypes of non-O157 Shiga Toxin producing Escherichia coli. The following research project is a comparative study looking at three Real Time PCR diagnostic platforms to detect the Top 6 non-O157 Shiga Toxin producing Escherichia coli associated with hides, manure, contact surfaces and carcasses. Confirmation studies were performed by a combination of immunological (immunoblot), mass spectrometry (VITEK® MS system) and culture-based methods. Samples from hide (n=170), kill floor (n=48), carcass (n=50) and manure (n=60) were enriched then plated onto Chromoagar and screened using the three RT-PCR platforms (GDS, BAX and Pall GeneDisc® Cycler). The presumptive positive results of the Top 6 were 97.0%, 53.4%, 86.6% and 87.5% by Chromoagar, GDS, BAX system and Pall GeneDisc® Cycler, respectively. Serotype O103 was the most prevalent serotype detected by the Pall GeneDisc® Cycler and BAX systems and was present in 69.5% and 74.7% of samples, respectively. The least frequent serotype was O111 and was present in only around 20% of samples by the Pall GeneDisc® Cycler and BAX systems. Cultural-based methods did not recover any of the Top 6 from PCR positive cultures. The explanations for the disagreement between culture methods and the RT-PCR were suggested to be due to the presence of virulence factors in different cells, the low quantities of the targeted factors in the samples and also the transient retention of stx. The study demonstrated that strains with the full complementary of virulence factors, eae and stx1/stx2, had higher chances of recovering when cultures were stored for 14 days at 4°C. From the studies performed there was no evidence of stx prophage replication nor inhibition of growth by background microflora. Yet, it was demonstrated that the presence of virulent phage in enrichment broth at MOI’s >1 could inhibit the growth of the target STEC. The significance of the work is to highlight the challenges of using RT-PCR when screening for the Top 6 non-O157 STEC in environmental, manure and hide samples.