Detection and Evaluation of Viable but Non-culturable Escherichia coli O157:H7 Induced by Low Temperature with a BCAC-EMA-Rti-LAMP Assay in Chicken Without Enrichment

Abstract

In this study, a rapid and sensitive method that combines ethidium bromide monoazide (EMA) staining with real-time loop-mediated isothermal amplification (Rti-LAMP) assay was tested in detecting and evaluating viable but non-culturable state (VBNC) Escherichia coli (E. coli) O157:H7 induced by low temperature. When 1.7 × 104 CFU/ml of E. coli O157:H7 was 3 cycles of freeze-thaw, the cells were all dead. However, E. coli O157:H7 in 1.7 × 106 CFU/ml and 1.7 × 108 CFU/ml gradually transferred into VBNC state reaching 6.8 × 102 CFU/ml (0.04%) and 4.1 × 105 CFU/ml (0.24%) after 6 cycles of freeze-thaw, respectively. Keeping E. coli O157:H7 (1.7 × 108 CFU/ml) at 4 °C and − 20 °C, the culturable cells persistently decreased in plate counting. Meanwhile, the VBNC cells increased from 0 to 1.1 × 106 CFU/ml and 5.5 × 106 CFU/ml detected by both EMA-Rti-LAMP and direct epifluorescence method (DEM) up to 258-day storage at 4 °C and − 20 °C, respectively. The EMA-Rti-LAMP had similar accuracy with DEM in detecting viable including VBNC cells, the former had specificity but not the later. Furthermore, the EMA-Rti-LAMP could detect as low as 25 CFU/g of VBNC E. coli O157:H7 derived from contaminated chicken combined with bentonite-coated activated carbon (BCAC) treatment to remove DNA amplification inhibitors, and the entire assay could be completed in 5 h. In addition, one same sample for E. coli O157:H7 positive was detected by both BCAC-EMA-Rti-LAMP and plate count method from 24 retail chicken samples, but four positive samples by BCAC-Rti-LAMP assay. The results obviously suggested that the BCAC-EMA-Rti-LAMP assay might be a rapid and sensitive method for detection of viable including VBNC E. coli O157:H7 cells in food without enrichment.