Enumeration of Viable Non-Culturable Vibrio cholerae Using Droplet Digital PCR Combined With Propidium Monoazide Treatment.

Shuo Zhao,Yu Han,Jingyun Zhang,Zhe Li,Biao Kan

doi:10.3389/fcimb.2021.753078

Abstract

Many bacterial species, including Vibrio cholerae (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, thyA, and recA). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of V. cholerae. The method can be extended to other bacterial species.

Highlights

Toxigenic Vibrio cholerae, a part of autochthonous aquatic ecosystems, is a reservoir for human infections and a threat to public health globally (Islam et al, 1994; Kaper et al, 1995; Harris et al., 2012)
We developed quantitative PCR-based methods, including real-time quantitative PCR and droplet digital PCR, to enumerate viable but non-culturable (VBNC) V. cholerae cells by counting the numbers of single-copy genes in samples during VBNC-state development
Our results show that droplet digital PCR (ddPCR) of single-copy genes combined with Propidium monoazide (PMA) treatment is a practical and effective approach for counting VBNC V. cholerae cells

Summary

Introduction

Toxigenic Vibrio cholerae, a part of autochthonous aquatic ecosystems, is a reservoir for human infections and a threat to public health globally (Islam et al, 1994; Kaper et al, 1995; Harris et al., 2012). Previous research has shown that V. cholerae can enter into a dormant state, called the viable but non-culturable (VBNC) state (Xu et al, 1982; Oliver, 2010). The first systematic study of ddPCR for VBNC the VBNC state in V. cholerae serogroup O1 was reported by Xu et al in 1982 (Xu et al, 1982). This state was defined as metabolically active but lacking the ability to reproduce on routine culture media (Oliver, 2005). The VBNC state of V. cholerae is usually induced by incubation in artificial sea water (ASW) at 4°C (Asakura et al, 2007)

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Results

Conclusion