Accurate flow cytometric monitoring of Escherichia coli subpopulations on solid food treated with high pressure carbon dioxide
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Aims: Evaluation of flow cytometry coupled with viability markers to monitor the inactivation of Escherichia coli cells spiked on solid food following High Pressure Carbon Dioxide (HPCD), a mild processing technology. Methods and Results: Flow cytometry (FCM) coupled with SYBR-Green I and Propidium Iodide was applied to monitor the effect of HPCD treatment on E. coli cells spiked on fresh cut carrots, therefore mimicking contamination of food products by faecal coliforms. FCM allowed to distinguish E. coli cells from carrot debris and natural flora, to evaluate the reduction of total cells, and to quantify viable and dead cells based on their membrane integrity after HPCD treatment. The comparison of FCM results with conventional cultivation methods revealed that HPCD treatments performed at 120 bar, 22 or 35 degrees C for 5 min disrupted 43 and 53% of bacterial cells, respectively, and produced a large percentage of partially permeabilized (96.5 and 98%) cells. Conclusions: Although treatments at 22 degrees C for 10 min and at 35 degrees C for 7 min were sufficient to inhibit the ability of all E. coli cells to replicate with an inactivation of 8 Log, FCM analysis showed that the inactivation of intact cells was only 2-2.5 Log. A fraction of HPCD-treated cells maintained their metabolic activity and re-growth capacity, indicating that the treatment induces a transitory Viable But Not Cultivable (VNBC) state. Significance and Impact of the Study: Under stress conditions many pathogens enter in a VBNC state, thus escaping detection by traditional cultivation methods. We provide the first evaluation of HPCD-mediated bacterial inactivation on fresh food using FCM coupled with viability markers, which should assist in the prevention of food-associated health risks.