A new integrated approach to analyze bulk tank milk and raw milk filters for the presence of the E. coli serogroups frequently associated with VTEC status

We optimized a combination of microbiological and molecular methods to quickly identify the presence of the O157 and the six non-O157 serogroups (O26, O45, O103, O111, O121 and O145) most frequently associated with VTEC status, at herd level. The lower detection limit of this methodology is 101 CFU/ml for each of the serogroups tested. We tested 67 bulk tank milk (BTM) and raw milk filters (RMF) derived from dairy herds located in Lombardy and Trentino Alto Adige. We identified 3 positive samples and 20 positive samples out of 67 respectively in the BTM and RMF. Interestingly, several samples showed positivity for more than one serogroups at the same time. We also identified the presence of E. coli O45 and O121 for the first time in raw milk and raw milk filters. Once screened the seven serogroups of interest in our samples, we evaluated the real pathogenicity of our positive, non-O157 samples through two parallel molecular biology methods: virulence gene research by PCR, and HRMA and sequencing. The most frequently isolated serogroups in milk were O157 (2.64%), O103 (2.11%), and O145 (1.06%), while in RMF the frequencies were, respectively 14.92%, 4.48%, and 2.98%. Moreover, this is the first published report in Italy of positive recovery of O45 and O121 serogroups in milk and milk filters. The new diagnostic approach proposed investigate the presence of the O157 and big six non-O157 serogroups at farm level and not to identify VTEC hazard only once the product is processed and/or is ready to be consumed.