Free Shiga Toxin 2-encoding Bacteriophages from Food to Feces and Beyond

Introduction: Shiga toxin-producing bacteria (STEC) are important foodborne pathogens implicated in food outbreaks and causing severe intestinal and systemic diseases. The main virulence factor is the production of Shiga toxin. The genes of Shiga toxin are encoded in the genome of temperate bacteriophages (Stx phages). Infectious Stx phages have been found free in wastewater and in food samples, suggesting they could be directly excreted through feces.

Purpose: Detection of free Stx phages in 80 fecal samples from healthy individuals to confirm if humans could excrete Stx phages independently of the presence of STEC strains.

Methods: Eighty human fecal samples were collected from humans that were not involved in a foodborne outbreak and did not show any severe gastrointestinal pathology. The DNA from the bacteriophage fraction of the samples was extracted and the stx gene was detected and quantified by real-time quantitative PCR. Positive amplicons were sequenced.

Results: A 55 % of the samples showed the presence of stx₂ in phage DNA. The densities of Stx phages were of 5.2 x 10⁴ Stx phages/g of feces (from 4.5 x 10¹ to 1.3 x 10⁶ stx/g). Bacteriological cultures for enteropathogens in these samples were negative. Sequencing suggested that the phages could carry various Stx variants (stx₂, stx_2v, stx_2c and stx_2d).

Significance: Free Stx phages circulate among the healthy population. They could be ingested through some foods and are consequently excreted by feces. Detection of stx using PCR approaches is commonly used for STEC detection in clinic and food. However, free Stx phages can also show a positive stx result in a molecular analysis, resulting in a positive STEC diagnostic, without a real presence of the pathogenic strain in the sample.