Rapid determination of viable but non-culturable Escherichia coli O157:H7 and Salmonella enterica in fresh produce by loop-mediated isothermal amplification coupling propidium monoazide treatment

Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the “viable but non-culturable” (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide (PMA)-qPCR assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting wzy gene of E. coli O157:H7 and agfA gene of S. enterica and PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce and spinach. No cross-reaction was observed in the specificity tests. The limit of detection (LOD) with PMA-LAMP was 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture, and 5.13×103-4 CFU/g for VBNC E. coli O157:H7 and 1.05×104-5 CFU/g for VBNC S. enterica in fresh produce, which was comparable to that of PMA-qPCR. Standard curves showed a correlation coefficient ranging from 0.925 to 0.996, indicating good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min vs. 1 h) and achieved comparable sensitivity and quantitative capacity to a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade the detection in the conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stresses, to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive and specific way to determine VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the pre- and post-harvest stages of fresh produce.