Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment

Abstract

<i>Escherichia coli</i> O157:H7 and <i>Salmonella enterica</i> are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC <i>E. coli</i> O157:H7 and <i>S. enterica</i> in fresh produce. The performance of PMA-LAMP assays targeting the <i>wzy</i> gene of <i>E. coli</i> O157:H7 and the <i>agfA</i> gene of <i>S. enterica</i> and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for <i>E. coli</i> O157:H7 and 4.6 CFU/reaction for <i>S. enterica</i> in pure culture and were 5.13 × 10³ or 5.13 × 10⁴ CFU/g for VBNC <i>E. coli</i> O157:H7 and 1.05 × 10⁴ or 1.05 × 10⁵ CFU/g for VBNC <i>S. enterica</i> in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a 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 versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC <i>E. coli</i> O157:H7 and <i>S. enterica</i> in fresh produce.<b>IMPORTANCE</b> VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both <i>E. coli</i> O157:H7 and <i>S. enterica</i> have been reported to enter the VBNC state under a range of environmental stress conditions and 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 levels of VBNC <i>E. coli</i> O157:H7 and <i>S. enterica</i> 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 preharvest and postharvest stages of fresh produce.