Enhanced diagnostic protocol to identify E.coli VTEC from milk filters

The EHEC (enterohemorrhagic E. coli) are a subgroup of VTEC with strong pathogenicity. The most well-known EHEC serotype is E. coli O157:H7, which has been implicated in many large outbreaks of deadly human diseases. However, EHEC strains of other serotypes have increasingly been implicated in sporadic cases and outbreaks of serious illness in humans, e.g., serotypes O26, O45, O103, O111, O121 and O145. Epidemics studied from 1982 to date have shown that ruminants, and in particular bovine, appear almost always involved in the transmission of these bacteria to humans through direct or indirect fecal contamination of foods. Unpasteurized milk and milk products are considered minor, but important sources of infection. The possible ways to the entrance of VTEC in milk are fecal contamination and mammary excretion during E.coli mastitis. Between the two ways, the first is considered much more frequent in practice, but it cannot be excluded that a small part of EHEC found in milk resulting from mammary gland, as reported. Previous investigation suggested that milk filters used in milking machines could be a useful control point to identify the presence of EHEC in dairy herds. However, conventional methods to identify the presence of EHEC have a poor sensitivity due the high content of fecal bacteria of these filters. In order to set up a monitoring scheme to identify herds at risk, we developed and tested a diagnostic protocol involving VIDAS® UP E.coli serogroups (ESPT) which is a method using phage recombinant proteins for the immuno-concentration (IC) of E.coli serogroups O157, O26, O103, O111, O145, O45 and O121 from food, multiplex PCR and high resolution melting analysis (HRMA). Practically, bulk tank milk or washing solution obtained from milk filters after stomacher mixing were analyzed by Vidas ESPT. After incubation, the solution obtained was analyzed by multiplex PCR based on serotype-specific primers coding for O-antigen regions of the seven major VTEC serogroups available in literature. If PCR was positive for any of the seven serogroups, a HRMA-based protocol to detect virulence-predictive SNPs, as discovered by Norman et al., 2012, was applied to confirm the presence of a EHEC strain. The protocol was preliminary validated by inoculation of milk and milk filters with a known concentration of the seven EHEC serotypes (O26, O45, O103, O111, O121, O145, O157). The results confirmed that this protocol was able to identify as low as 101 UFC in both milk and milk filters. The protocol applied to milk and milk filters obtained from 70 dairy herds allow to identify 2 EHEC from milk and 17 from milk filters for one or more of the EHEC serogroups considered. The proposed protocol confirmed to be useful in detecting the presence of EHEC and that milk filters are an important critical control point to identify herd at risk.