Detection and Survival of Viable But Non-culturable Escherichia coli O157 in Soil

Introduction: Contaminated fresh produce is responsible for more disease cases than any other food commodity, yet current detection methods for foodborne pathogens overlook cells in the viable but non-culturable (VBNC) state. It has been shown that the VBNC state can be induced in the foodborne pathogen Escherichia coli O157, while on the lettuce phylloplane. Additionally, the expression of the pathogen’s Shiga-like toxin genes has been observed in VBNC E. coli O157, suggesting that it can remain pathogenic despite being non-culturable.

Purpose: This study aimed to develop a direct method of detection of foodborne pathogens in soil including VBNC cells, overcoming the shortcomings associated with molecular methods when analyzing complex environmental matrices.

Methods: Bacterial adherents were released from soil particles into suspension using a Pulsifier and concentrated by two rounds of vacuum filtration, reducing 25 g of soil to a 1 ml suspension. Escherichia coli O157 was quantified in eleven soil samples by qPCR, targeting the tir (translocated intimin receptor) gene. Cells were elongated via incubation with pipemidic acid and visualized through PNA-FISH under epifluorescence microscopy.

Results: The assay was capable of detecting E. coli O157 at 10 CFU per gram of peat-based compost. The tir gene was detected in seven of eleven soil samples tested, despite disparate compositions and geographical origins. Peat-based compost contained the highest concentration, at 200 gene copies per gram. The presence of VBNC E. coli O157 was confirmed in the compost using PNA-FISH.

Significance: The assay described here provided a rapid and quantitative pre-screen of foodborne pathogens in soil, with applications in determining the extent of their presence throughout the environment. VBNC populations of E. coli O157 have been observed within compost, indicating its presence in agricultural soils across the UK.