Assessment of microbial safety of fresh vegetables through Caenorhabditis elegans model

Abstract

Caenorhabditis elegans is widely used as a model to predict the virulence of animal-origin human pathogens, as C. elegans and humans share similar intestinal epithelial cells. A study was undertaken to assess the feasibility of C. elegans for surveillance of foodborne pathogens in fresh vegetables. First, the virulent strains of four foodborne pathogens viz., Pseudomonas aeruginosa (PAO1), Escherichia coli (O157), Salmonella enterica (CI1), and Staphylococcus aureus (ATCC25923) were fed to the worm as sole diet and compared the survival, phenome traits, and gut integrity with standard E. coli OP50. Secondly, the vegetable surface microbiota (tomato) was enriched with gradient concentrations of PAO1, and the worm's survival and gut distension and leakage were assessed. Thirdly, the total microbiota of fresh vegetables (tomato, brinjal, and lablab) were evaluated for the worm's survival. The gut epithelial cell damage and leakage were monitored by blue dye-based smurf assay. Foodborne pathogens reduced the median lifespan (LT50) of the worm drastically, with PAO1 had 2.5 days, O157 (6 days), CI1 (8 days), ATCC25923 (6 days), while the standard diet OP50 had 16 days. The phenome traits of worms, viz., pharyngeal pumping, defecation cycle, body bends, head thrashes, touch stimulus, paralysis, gut leakage, and reproduction, were significantly negatively affected due to pathogen feeding. The gut epithelial integrity was severely affected, and blue dye leakage was the highest in O157 (73%), followed by PAO1 (55%), CI1 (40%) and ATCC25923 (38%), while the gut epithelial cells were intact in OP50 fed worms. The pathogen enrichment in tomato surface microbiota diet showed that LT50 of the worms was reduced from 8 days to 2.5 days and 10 to 8 days with increasing gradient of PAO1 in fresh tomato microbiota and 24-hours pre-enriched tomato microbiota, respectively. Smurf test also confirmed that an increase in PAO1 proportion in total microbiota distended the gut with high dye leakage. The total microbiota extracted from tomato, brinjal, and lablab did not affect the worm's health and had an extended median life span (11 – 13 days) when they were devoid of any foodborne pathogens. These findings provide a novel and straightforward approach for assessing foodborne pathogens using animal models.