Induction of Viable but Nonculturable (VBNC) state and the effect of multiple subculturing on the survival of Legionella pneumophila strains in the presence of monochloramine

Abstract

The aim of the study was to evaluate the response of two different Legionella pneumophila strains with their 20 times subcultured passages, regarding VBNC induction and to test the effect of multiple subculturing on cell vulnerability in the presence of monochloramine. A freshly opened of L. pneumophila ATCC 33152, a first subculture of environmental isolate of L. pneumophila and the 20(th) subcultures of both strains were tested for survival in the presence of different doses of monochloramine (24 hours). Besides culture method, live-dead cells were visualised. It was found that multiple subcultured strains were significantly more susceptible to monochloramine and not capable of entering to VBNC state in comparison with freshly opened/isolated strains. Environmental isolate was survived at 2 ppm dose of monochloramine after 24 h, whereas 20(th) subculture of this strain failed to survive. Multiple subcultured L. pneumophila strains were lost their culturability and viability significantly. This phenomenon should be considered while working with laboratory strains. After monochloramine disinfection, L. pneumophila bacteria can completely lose their cultivability but do not lose viability, which remains responsible for serious outbreaks worldwide. Even after completely loosing cultivability, it is possible to find live cells in network water in the VBNC state.