Abstract
Metallic copper to combat bacterial proliferation in drinking water systems is being investigated as an attractive alternative to existing strategies. A potential obstacle to this approach is the induction of metal resistance mechanisms in contaminating bacteria, that could severely impact inactivation efficacy. Thus far, the role of these resistance mechanisms has not been studied in conditions relevant to drinking water systems. Therefore, we evaluated the inactivation kinetics of Cupriavidus metallidurans CH34 in contact with metallic copper in drinking water. Viability and membrane permeability were examined for 9 days through viable counts and flow cytometry. After an initial drop in viable count, a significant recovery was observed starting after 48 h. This behavior could be explained by either a recovery from an injured/viable-but-non-culturable state or regrowth of surviving cells metabolizing lysed cells. Either hypothesis would necessitate an induction of copper resistance mechanisms, since no recovery was seen in a CH34 mutant strain lacking metal resistance mechanisms, while being more pronounced when copper resistance mechanisms were pre-induced. Interestingly, no biofilms were formed on the copper surface, while extensive biofilm formation was observed on the stainless steel control plates. When CH34 cells in water were supplied with CuSO4, a similar initial decrease in viable counts was observed, but cells recovered fully after 7 days. In conclusion, we have shown that long-term bacterial survival in the presence of a copper surface is possible upon the induction of metal resistance mechanisms. This observation may have important consequences in the context of the increasing use of copper as an antimicrobial surface, especially in light of potential co-selection for metal and antimicrobial resistance.
Highlights
The historical use of copper (Cu) for sterilization of drinking water and the treatment of various illnesses dates back to the third millennium B.C
We tested the survival of C. metallidurans CH34 in sterilized drinking water in contact with either a submerged Cu plate, a stainless steel plate or no plate
We evaluated the inactivation kinetics of C. metallidurans CH34 in contact with metallic Cu in drinking water
Summary
The historical use of copper (Cu) for sterilization of drinking water and the treatment of various illnesses dates back to the third millennium B.C. In addition to the toxic effects of ions present in the medium, the direct interaction of bacterial cells with the metal surface can lead to a strongly enhanced killing effect (Mathews et al, 2013), coined as contact killing and recently reviewed by Vincent et al (2018). This enhanced toxicity was demonstrated by covering the copper surface with an inert polymeric grid, which prevented direct bacterial attachment but not the release of Cu ions in the medium, resulting in a drastic decrease in toxicity. Many questions still surround the VBNC state, but it is clear that VBNC cells can still pose problems since resuscitation and subsequent toxicity of bacteria from drinking water have been reported (Lee et al, 2007; Dwidjosiswojo et al, 2011; Li et al, 2014; Dopp et al, 2017; Ye et al, 2020)
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