A Caenorhabditis elegans life-dead-assay for the estimation of the pathogenic potential of pathogenic Escherichia coli from food samples

Shiga toxin-producing Escherichia coli (STEC) and diarrheagenic E. coli (DEC) are important food-borne pathogens. The severity of clinical symptoms can vary from diarrhea to haemolytic-uremic syndrome and death. In STEC, the Shiga toxin subtype and the occurrence of additional virulence factors have an impact on the clinical manifestation. STEC from food can vary in serotype and virulence associated genes (VAG) from those usually connected to severe clinical cases. Therefore, an assessment of the pathogenic potential of food isolates for humans would be helpful. Here, we established a Caenorhabditis (C.) elegans life-dead-assay to estimate the pathogenic potential of STEC from food samples. A collection of non-pathogenic E. coli, DEC, STEC and respective stx deletion mutants were tested concerning their impact on the lifespan of C. elegans SS104. For the life-dead-assay, worms were synchronized to the same L4 larval stadium. Fifteen worms were seeded on fresh nematode growth medium plates with either the non-pathogenic E. coli OP50 control strain or a challenge strain. Numbers of life and dead worms were counted each day. Kaplan-Meier survival curves were calculated using R and the median survival times (MSTs) of three technical and biological replicates were determined. For all tested strains, MSTs of the worm populations were reduced compared to the control strain OP50. Worms fed on OP50 had an MST of 11 days. MSTs for non-pathogenic E. coli strains were reduced to 10 to 7 days, respectively while DEC and STEC strains led to a further reduction of the MST to 6 days. Deletion of the stx gene led to an increase of the MST compared to the wild type strain. A C. elegans life-dead-assay for in vivo pathogenicity determination experiments for DEC and STEC from food samples was successfully established. Comparable reductions in the MSTs for DEC and STEC were determined. The shape of the Kaplan-Meier survival curves differed between DEC and STEC strains and the investigated deletion mutants, respectively. Furthermore, one tested STEC strain harbour the Stx2g Shiga toxin subtype, which is assumed to be less toxic, therefore STEC with other Stx-subtypes shall be tested.