Monochloramine Treatment Induces a Viable-but-Nonculturable State into Biofilm and Planktonic Legionella pneumophila Populations

Abstract

The aim of the authors' work was to study the impact of monochloramine on sessile and planktonic Legionella pneumophila populations. The authors produced Legionella biofilms on glass beads (0.5 mm diameter) in buffered-yeast extract (BYE) broth. Bacteria were grown for one week at 37°C, under static conditions. Planktonic cells were obtained in same growth conditions without glass beads. Planktonic and sessile cells were washed twice with sterile water and then treated with 0.25 to 10 mg liter-1 monochloramine solutions. Sessile bacteria were collected from glass beads by sonication and enumerated on buffered charcoal yeast extract (BCYE) agar. In order to detect metabolic activity in viable-but-nonculturable (VBNC) cells, planktonic L. pneumophila Lens cells were treated with 0.75 mg liter-1 of monochloramine in order to obtain no culturable cells. After 8 days in the resting medium, the authors were able to detect esterase activity and membrane integrity by fluorescence microscopy using the ChemChrom V6 substratum (Chemunex) in samples without culturable cells. This demonstrates that VBNC cells produced by monochloramine retained metabolic activity. Recovery of culturability was observed for sessile bacteria treated with 1 mg liter-1 of monochloramine and coincubated with Acanthamoeba castellanii.