Genome Engineering of Stx1-and Stx2-converting Bacteriophages Unveils the Virulence of the Dairy Isolate O174:H2 Strain UC4224

Overview

Journal Front Microbiol

Specialty Microbiology

Date 2023 Jul 10

PMID 37426006

Authors

Giovanni Milani

Mireya Viviana Belloso Daza

Claudia Cortimiglia

Daniela Bassi

Pier Sandro Cocconcelli

Affiliations

Soon will be listed here.

Abstract

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.

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