Evaluation of a New Selective Enrichment Protocol to Improve the Detection of Shiga Toxin-Producing E. coli in Dairy Products

Introduction: STEC has emerged as important public health threats. Most human infections caused by STEC have been acquired by the consumption of contaminated foods especially those of bovine origin. According the ISO/TS 13136 standardized method for the detection of STEC, dairy testing requires the addition of acriflavin in the enrichment broth in order to decrease the high level of competitive flora. However, the subsequent PCR assay which should reveal the presence of STEC can be affected by the fluorescence of acriflavin, leading to non-interpretable results. In this context, a new selective enrichment protocol using Buffered Peptone Water supplemented with the STEC supplement (BPW+STEC sup.) has been developed.

Purpose: The objective of this study was to compare the ISO/TS 13136 reference method for the detection of STEC in dairy products to the new method combining the enrichment in BPW+STEC supplement and a final detection with the iQ-Check VirX PCR kit. The STEC supplement consists in a mixture of 3 selective agents inhibiting the background flora, thus enhancing the growth of STEC strains.

Methods: Twenty-five g samples of raw milk and raw milk cheese were spiked with a STEC strain at a concentration below 10 CFU/sample. They were then enriched according to the ISO/TS 13136 reference method and in BPW+STEC sup. (dilution ratio 1:9). After 16 - 20 hours of incubation, the culture obtained was analyzed either with the ISO/TS 13136 reference PCR assay or with the iQ-Check VirX PCR kit.

Results: All spiked samples were detected with the new protocol. The reference method failed to detect all spiked samples. This new method showed also lower Cqs in the final PCR tests due to higher selectivity during the bacterial growth.

Significance: Results demonstrated that the selective protocol with Buffered Peptone Water + STEC supplement is an effective and convenient method for the enrichment of STEC in dairy products.