Detection of VBNC Vibrio cholerae by RT-Real Time PCR based on differential gene expression analysis.

The recognition of the viable but non-culturable (VBNC) state of pathogenic bacteria has brought with it many questions to answer related to the need to detect and quantify viable bacteria in the environment in an accurate way. To assess viability of Vibrio cholerae, we developed a RT-Real Time PCR technique based on differential expression analysis from mRNA deep sequencing data. We compared two induction conditions to achieve the VBNC state: a bacterial suspension induced by artificial seawater at 4°C, and the addition of 3',5'-cyclic diguanylic acid. The evaluation of the up-regulated genes in the induced bacterial samples was compared with a fresh culture in the mid-exponential phase. The data analysis was performed with the NOISeq R-package and revealed 17 up-regulated genes for induction condition I and 22 up-regulated genes for induction condition II. Only one region was found to be up-regulated for both induction conditions. The VCA0656 gene related to the aminoimidazole riboside kinase protein was detected as the most up-regulated region and used as a genetic marker to precisely detect the VBNC state in combination with the RT-Real Time PCR technique. This approach describes a novel method to differentiate the VBNC state of V. cholerae in water samples.

Effects of ozone and chlorine disinfection on VBNC Helicobacter pylori by molecular techniques and FESEM images.

Helicobacter pylori is a pathogen bacteria associated with chronic gastritis, peptic ulceration, and gastric carcinoma. H. pylori has a spiral morphology, which under certain conditions of stress becomes a coccoid form. This type of morphology has been linked to a viable but non-culturable (VBNC) state, which is thought to allow its persistence in the environment. Membrane damage in VBNC H. pylori in water as a mechanism for inactivation using ozone (O3) and chlorine disinfection has not been reported in the literature. In this paper, disinfection assays with ozone and chlorine were conducted to evaluate their effects on VBNC H. pylori cells. The use of fluorescent dyes such as propidium monoazide (PMA) coupled with quantitative real-time polymerase chain reactions produced results necessary to assess the viability of the microorganism and demonstrate the effect of each disinfectant on the bacterial count. Applying ozone showed a 5-log bacterial reduction using a disinfectant concentration and exposure time (CT) of 4 mg min/L. Chlorine disinfection for the same 5-log reduction required a higher CT value. Field emission scanning electron microscope images of ozone-treated VBNC H. pylori also showed severe cell damage. The use of PMA revealed that chlorine produced physical damage in the membrane in addition to the known inhibiting effect on cell enzymatic processes. These findings are important for the detection and control of VBNC H. pylori cells in drinking water systems.