AUTHOR=Milani Giovanni , Belloso Daza Mireya Viviana , Cortimiglia Claudia , Bassi Daniela , Cocconcelli Pier Sandro 

TITLE=Genome engineering of Stx1-and Stx2-converting bacteriophages unveils the virulence of the dairy isolate Escherichia coli O174:H2 strain UC4224

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1156375

DOI=10.3389/fmicb.2023.1156375

ISSN=1664-302X

ABSTRACT=<p>The past decade witnessed the emergence in Shiga toxin-producing <italic>Escherichia coli</italic> (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 (<italic>stx1</italic> and <italic>stx2</italic>) carried by Stx-converting bacteriophages, along with the intimin gene <italic>eae</italic>. 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 <italic>E. coli</italic> 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 <italic>E. coli</italic> 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 <italic>Galleria mellonella</italic> animal model, <italic>E. coli</italic> UC4224 demonstrated high pathogenicity potential with an LD<sub>50</sub> of 6 CFU/10 μL. Upon engineering <italic>E. coli</italic> UC4224 to generate single and double mutant derivatives by inactivating <italic>stx1a</italic> and/or <italic>stx2a</italic> genes, the LD<sub>50</sub> 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 <italic>stx1</italic>-<italic>stx2</italic> mutant behaviour, making it as a suitable less-virulent surrogate for conducting studies during food processing.</p>