Influence of copper ions on the viability and cytotoxicity of Pseudomonas aeruginosa under conditions relevant to drinking water environments

Copper plumbing materials can be the source of copper ions in drinking water supplies. The aim of the current study was to investigate the influence of copper ions on the viability and cytotoxicity of the potential pathogen Pseudomonas aeruginosa that presents a health hazard when occurring in building plumbing systems. In batch experiments, exposure of P. aeruginosa (106 cells/mL) for 24 h at 20 °C to copper-containing drinking water from domestic plumbing systems resulted in a loss of culturability, while total cell numbers determined microscopically did not decrease. Addition of the chelator diethyldithiocarbamate (DDTC) to copper-containing water prevented the loss of culturability. When suspended in deionized water with added copper sulfate (10 μM), the culturability of P. aeruginosa decreased by more than 6 log units, while total cell counts, the concentration of cells with intact cytoplasmic membranes, determined with the LIVE/DEAD BacLight kit, and the number of cells with intact 16S ribosomal RNA, determined by fluorescent in situ hybridization, remained unchanged. When the chelator DDTC was added to copper-stressed bacteria, complete restoration of culturability was observed to occur within 14 d. Copper-stressed bacteria were not cytotoxic towards Chinese hamster ovary (CHO-9) cells, while untreated and resuscitated bacteria caused an almost complete decrease of the concentration of viable CHO-9 cells within 24 h. Thus, copper ions in concentrations relevant to drinking water in plumbing systems seem to induce a viable but non-culturable (VBNC) state in P. aeruginosa accompanied by a loss of culturability and cytotoxicity, and VBNC cells can regain both culturability and cytotoxicity, when copper stress is abolished.