Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold‐starvation using propidium monoazide real‐time polymerase chain reaction

Abstract

Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation‐based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real‐time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80 days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15–16·69. During cold‐starvation, PMA qPCR selectively excluded DNAs from heat‐killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150 days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). Significance and Impact of the Study Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold‐starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real‐time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold‐starvation conditions.

Study on the Viable but Non-culturable (VBNC) State Formation of Staphylococcus aureus and Its Control in Food System

“…The detection limit of PMA-PCR for detection of the VBNC state S. aureus in rice products was 10 4 CFU/mL. Compared with the conventional culturing-based method, which uses the LIVE/DEAD ® BacLight TM fluorescence staining method to detect VBNC cells, the PMA-PCR assay can detect specific concentrations of VBNC cells with high rapidity and sensitivity ( Yoon et al, 2019 ).…”

Effect of sunlight and salinity on the survival of pathogenic and non‐pathogenic strains of Vibrio parahaemolyticus in water microcosms

“…Sunlight exposure was accompanied by the transformation of Vibrio cells into VBNC, which is considered a natural defense mechanism for bacteria to withstand under stressful conditions (Salive et al, 2020;Yoon et al, 2019) and recovers once environmental conditions become more favorable (Fern andez-Delgado et al, 2015;Wei & Zhao, 2018).…”

Quantitative Detection of Viable but Nonculturable Vibrio parahaemolyticus in Frozen Bivalve Molluscs

“…During the breeding, processing, and storage of seafood, V. parahaemolyticus can enter a viable but nonculturable (VBNC) state due to the low temperatures or excessive salinity commonly used for food preservation [ 37 , 38 ]. In the VBNC state, microorganisms become undetectable by routinary control tests since they do not form colonies on standard culture media [ 39 , 40 ].…”