Genome engineering of Stx1-and Stx2-converting bacteriophages unveils the virulence of the dairy isolate O174:H2 strain UC4224.

The past decade witnessed the emergence in Shiga toxin-producing (STEC) infections linked to the consumption of unpasteurized milk and raw milk cheese. The virulence of STEC is primarily attributed to the presence of Shiga toxin genes ( and ) carried by Stx-converting bacteriophages, along with the intimin gene . Most of the available information pertains to the "Top 7" serotypes associated with STEC infections. The objectives of this study were to characterize and investigate the pathogenicity potential of UC4224, a STEC O174:H2 strain isolated from semi-hard raw milk cheese and to develop surrogate strains with reduced virulence for use in food-related studies. Complete genome sequence analysis of UC4224 unveiled the presence of a Stx1a bacteriophage, a Stx2a bacteriophage, the Locus of Adhesion and Autoaggregation (LAA) pathogenicity island, plasmid-encoded virulence genes, and other colonization facilitators. In the animal model, UC4224 demonstrated high pathogenicity potential with an LD of 6 CFU/10 μL. Upon engineering UC4224 to generate single and double mutant derivatives by inactivating and/or genes, the LD increased by approximately 1 Log-dose in the single mutants and 2 Log-doses in the double mutants. However, infectivity was not completely abolished, suggesting the involvement of other virulence factors contributing to the pathogenicity of STEC O174:H2. Considering the possibility of raw milk cheese serving as a reservoir for STEC, cheesemaking model was developed to evaluate the survival of UC4224 and the adequacy of the respective mutants as reduced-virulence surrogates. All tested strains exhibited the ability to survive the curd cooking step at 48°C and multiplied (3.4 Log CFU) in cheese within the subsequent 24 h. These findings indicate that genomic engineering did not exert any unintended effect on the double - mutant behaviour, making it as a suitable less-virulent surrogate for conducting studies during food processing.