Rapid and sensitive detection of VBNC Escherichia coli O157: H7 in beef by PMAxx and real-time LAMP

Accurate and rapid identification of the live fraction of pathogenic bacteria in beef food samples has emerged as a pressing issue due to the serious threat posed by pathogens to human health. Escherichia coli O157: H7, one of the most common pathogens in beef, causes hemorrhagic colitis or hemolytic uremic syndrome and subsequently a viable but nonculturable (VBNC) state under low temperatures in which the bacteria do not grow in culture medium but remain potentially virulent. The objective of this study was to establish and evaluate a method that combines improved propidium monoazide (PMAxx) treatment with real-time loop-mediated isothermal amplification (qLAMP) for the detection and quantification of VBNC E. coli O157: H7 in beef. In this study, E. coli O157: H7 was induced to enter the VBNC state by simulating food storage conditions at −20 °C. The respiratory activity of VBNC E. coli O157: H7 was measured by flow cytometry. The optimal PMAxx final concentration was 10 μM with exposure to light for 10 min for photoactivation, which was found to completely inhibit amplification of DNA derived from dead cells. When there were low levels of VBNC microorganisms in beef samples, PMAxx was significantly more accurate and effective at distinguishing between viable and dead E. coli O157: H7 cells. The developed PMAxx-qLAMP assay remarkably exhibited both high sensitivity in pure culture and beef (30 colony-forming units/mL and 300 CFU/g, respectively) and rapidity (60 min). Especially for frozen beef samples, positive ratio was 4.71% (four out of eighty-five were positive) by PMAxx-qLAMP method and the mean measured value of positive samples was 2.04 ± 0.08 Log10 CFU/g. Therefore, PMAxx-qLAMP is an effective means for improving the detection and quantification accuracy of VBNC E. coli O157: H7 in beef.