Realizzazione di un sistema qualitativo in Real-Time PCR per l'identificazione di Escherichia Coli patogeni

Shiga toxin-producing E. coli (STEC-VTEC) are foodborne pathogens that have been associated with gastroenteric diseases in humans. Infections are often caused by ingestion of contaminated food. For this purpose, the report ‘Technical specifications for the monitoring and reporting of verotoxigenic Escherichia coli (VTEC o STEC) on animals and food (VTEC surveys on animals and food)’ was proposed by EFSA (European Food Safety Authority) in order to harmonize STEC research methods in EU Member States. Moreover, the reference method ISO 13136:2012 describes the molecular identification of Shiga toxin-producing E. coli through the detection of stx and eae virulence genes and serogroup assignment through the detection of the genes associated with O157, O111, O26, O103, O145 serogroups. The genes aggR and aaiC, distinctive for Enteroaggregative E. coli (EAEC) strains, should be also taken into account considering that the large outbreak occurred in Germany in 2011 (750 cases of HUS of which 44 deaths) was due to the consumption of sprouts contaminated by a O104 strain, which was found both STEC and EAEC (stx2, aggR and aaiC) positive. The aim of this study was to develop a multiplex real-time PCR assay for the rapid detection of pathogenic E.coli strains in food, in particular an assay for the detection of stx1, stx2, and eae genes, which identifies STEC; other three assays specifically formulated for the identification of the six main pathogenic serogroups (O157, O111, O26, O103, O145, and O104), and , lastly, another assay for the detection of aggR and aaiC genes, specific for EAEC. Furthermore, the assays were compared with official methods used in USA (USDA-FSIS, FDA/BAM) for the identification of E.coli in foodstuffs. The results obtained show that the assays for the sensitive, specific and rapid detection of STEC (stx-eae) and serogroups (O26-O103-O111-O145-O157-O104) were developed, in compliance with the reference method ISO 13136:2012. These assays were able to identify pathogenic E. coli in the main foodstuff implicated in epidemic outbreaks. Moreover, this work demonstrates that the performance of the assays is reliant on the enrichment process that remain a critical step in assay development. Since the contamination level of STEC in food is usually low, the selection of an appropriate medium is a key element of STEC detection protocol, to reduce the risk of false negative results. Therefore, alternative enrichment condition, like mBPWp + CV at 42°C, should be taken into consideration. Furthermore, colony tests carried out with EAEC E.coli detection assay showed optimal results, with 100% of exclusivity and efficiency. The comparison between molecular assays developed in this study and USDA-FSIS and FDA/BAM official methods showed equivalent results of performance and sensitivity, providing in some cases better results with our method.