Prevalence of Pathogenic Shiga Toxin-producing E. coli (pSTEC) O157 and Non-O157 in Raw Milk Samples

Introduction: Detection of pathogenic Shiga Toxin-producing E. coli (pSTEC) typically rely on two successive PCR screening steps as described in the ISO/TS 13136 (EU) and MLG 5B (US) reference methods. In order to reduce both the rate of presumptive PCR positive results and the time to result, we developed a one-step multiplex PCR based method using the GeneDisc® technology.

Purpose: With the aim to evaluate the prevalence of pSTEC in raw milk products, 4,064 raw milk samples (3,331 cow milk and 733 ewe milk samples), collected over one year in France, were analyzed according to the ISO/TS 13136 standard and the GeneDisc method. 

Methods: Twenty-five ml of raw milk sample were enriched in 225 ml BPW supplemented with 10 mg/l acriflavine for 18 h ± 2 h at 37°C. DNA extracted from the enrichment broth was analyzed with the GeneDisc Cycler, according to both PCR approaches. Confirmation of presumptive positive samples was also done following own procedures for each method. Isolated strains were characterized using qPCR on chips allowing carrying out 48 different PCR reactions (Fluidigm).

Results: One thousand nine (28.3 %) presumptive positive results for pSTEC were obtained according to the ISO/TS 13136 standard while only 456 (11.2 %) were positive with the GeneDisc multiplex PCR. The number of atypical enteropathogenic E. coli (aEPEC) and pSTEC strains isolated was, respectively, 41 and 28 with a majority of O26 strains (62%), followed by O103 (34.4 %). Molecular characterization of the isolates showed that aEPEC O26 strains might be in fact sub-divided in stx-negative pSTEC derivatives and aEPEC O26 strains.

Significance: The accuracy of the GeneDisc screening method provides a lower rate of presumptive positive for pSTEC making it suitable for routine testing condition. It would allow the dairy industry to accurately sort their milk and confidently direct it to the right processing.