Enumeration of Vibrio parahaemolyticus in VBNC state by PMA-combined real-time quantitative PCR coupled with confirmation of respiratory activity

Abstract

Vibrio parahaemolyticus, a worldwide pathogen, has been proved to be capable of entering into viable but nonculturable (VBNC) state and survive under stressful conditions. In this study, a method of real-time quantitative PCR coupled with propidium monoazide (PMA-qPCR) was developed and evaluated for the reliable quantification of the VBNC cells of V. parahaemolyticus. The samples of different ratios of viable and dead cells were detected by PMA-qPCR and qPCR to assess this method. Then the method was used to monitor the VBNC induction process of V. parahaemolyticus and quantify the viable cells along with confirmation of cell integrity and respiratory activity by fluorescence microscopy and confocal laser scanning microscope (CLSM) observation, flow cytometry (FCM) analysis. In addition, the applicability of the method was verified to detect artificially contaminated seafood samples with different states of V. parahaemolyticus. The results showed that the optimal PMA treatment condition was 5 min-PMA incubation in dark followed by 15 min-light exposure. This method can significantly distinguish viable from dead cells and specifically enumerate viable cells. By PMA-qPCR method, the number change of cells during VBNC induction was monitored successfully. The cells incubated at 4 °C in sterile 3% NaCl entered into VBNC state, and 106 cell/mL of VBNC cells (10% of the original viable cells) were detected on the fifth day of induction, then the density of VBNC cells was 5.8 × 104 cell/mL on the 40th day when no culturable cells were observed. FCM analysis showed the viability of the VBNC cells which were also observed red revealing respiratory activity by CTC/DAPI staining under CLSM. For the detection of the spiked seafood samples (shrimp, pomfret and scallop) with different states of V. parahaemolyticus, it could detect as low as 1.2 × 102 cell/g of the viable or VBNC state of V. parahaemolyticus without any interference from food matrices and dead cells. In summary, the PMA-qPCR method developed in this study is rapid and reliable to quantify VBNC cells of V. parahaemolyticus.