Detection of viable but non-culturable Escherichia coli O157:H7 from vegetable samples using quantitative PCR with propidium monoazide and immunological assays

Quantitative differentiation of the live fraction of pathogens in raw food samples is highly critical from a public health risk perspective, as many studies have shown that under stress conditions major foodborne pathogens enter a viable but non-culturable (VBNC) state in which bacteria can remain for long periods of time and maintain the potential for virulence. The objective of this study was to evaluate the applicability of propidium monoazide (PMA) quantitative PCR (qPCR) and immunological methods for detection of Escherichia coli O157:H7 VBNC populations induced by low temperature on the surface of lettuce and spinach plants. The primer/probe set selected influenced the qPCR signal in mixtures with a defined ratio of viable and non-viable cells. The PMA qPCR used in a background of added dead pathogens and epiphytic bacteria gave a detection limit of 103 CFU/g leaf and a linear quantitative detection range of 5 log. During quantification of VBNC cells from lettuce and spinach samples there was a good correlation between the PMA qPCR results and viable counts detected by microscopy, showing that PMA qPCR gives an accurate indication of the VBNC population. However, the commercially available immunoassay methods used to detect Shiga-like toxin production and the O157 antibody in vegetable samples with no detectable culturable pathogen underestimated the number of samples contaminated with E. coli O157:H7 VBNC cells. Results indicate that PMA qPCR is a suitable technique for the detection and quantification of VBNC cells of foodborne pathogens in contaminated raw lettuce and spinach.