A combination of direct viable count and fluorescent in situ hybridization for estimating Helicobacter pylori cell viability

Abstract

The viable but non-culturable (VBNC) stage of Helicobacter pylori may represent a problem of public health concern, since these cells cannot be detected by traditional culture methods. In this study, the direct viable count method (DVC) was modified and adapted to H. pylori analysis by testing different times of incubation and concentrations of DNA-gyrase inhibitors. The DVC procedure was combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of H. pylori (DVC–FISH). Incubation with 0.5 μg/ml of novobiocin for 24 h provided the optimal conditions for obtaining 3–5 times the original size of Helicobacter viable cells. Field work performed with various types of water (freshwater and seawater) using the DVC–FISH approach enabled us to confirm the presence of VBNC H. pylori cells in 16 of the 45 analyzed samples. The combination of the modified DVC procedure with FISH can provide a rapid and specific method to detect and identify viable cells of H. pylori in environmental samples.