Absolute Quantification of Viable but Nonculturable Vibrio cholerae Using Droplet Digital PCR with Oil-Enveloped Bacterial Cells

When exposed to adverse conditions, many bacterial pathogens, including Vibrio cholerae, can adapt to the environment by entering the viable but nonculturable (VBNC) state. Cells in this state cannot grow on conventional media but still survive. The VBNC state is a significant threat to aquatic safety and public health due to the enhanced ability of the bacteria to remain in the environment and escape from monitoring. Detecting and quantifying VBNC cells and distinguishing them from nonviable cells is necessary for microbiological studies and pathogen monitoring. Cell staining and microscopy are used for the observation of VBNC cells, but it is difficult to quantify VBNC cells accurately. In this study, we developed droplet digital PCR (ddPCR) with a chromosomal single-copy gene as an internal reference combined with Propidium monoazide (PMA) treatment to enumerate VBNC cells of V. cholerae. In this method, bacterial cells, but not extracted chromosomal DNA, were used directly to form oil-enveloped droplets in the ddPCR procedure. One bacterial cell possesses one copy of the chromosome. Thus, enumeration of a single-copy gene on the chromosome can be used to count VBNC cells. ddPCR showed greater accuracy and sensitivity than qPCR. This study showed that the oil-enveloped bacterial method can reduce the number of steps needed and improve the accuracy of VBNC cells quantification and has the potential to be extended to quantify bacterial VBNC cells and assess pathogen survival in the environment. IMPORTANCE The viable but nonculturable (VBNC) state of bacteria represents an important life state for their survival in adverse environments. The VBNC cells of the pathogenic bacteria in the environment and food will be a potential threat to public health because these pathogens cannot be found by the detection of culture. We developed a sensitive molecular method to detect and enumerate the VBNC cells of V. cholerae, which can distinguish the VBNC and dead cells, and count the VBNC cells in the sample without the step of DNA extraction from cells. It can be used to improve the sensitivity of pathogen detection in the surveillance, risk assessment of environment and food contamination, and outbreak warning. The accurate identification and numeration of VBNC cells will also facilitate the microbiological and genetic studies on the development, adaptation, resuscitation, and elimination of the VBNC state.