Bacterial killing by dry metallic copper surfaces

Abstract

Metallic copper surfaces rapidly and efficiently kill bacteria. Cells exposed to copper surfaces accumulated large amounts of copper ions, and this copper uptake was faster from dry copper than from moist copper. Cells suffered extensive membrane damage within minutes of exposure to dry copper. Further, cells removed from copper showed loss of cell integrity. Acute contact with metallic copper surfaces did not result in increased mutation rates or DNA lesions. These findings are important first steps for revealing the molecular sensitive targets in cells lethally challenged by exposure to copper surfaces and provide a scientific explanation for the use of copper surfaces as antimicrobial agents for supporting public hygiene.

FIG. 1.

Copper uptake into cells exposed to moist or dry copper surfaces. Cells of E. coli were exposed to moist (A and B) or dry (C and D) metallic copper surfaces for the indicated times, removed, washed, and plated on solidified growth media. Survivors were counted as CFU (▪) (A and C). Parallel samples were mineralized and subjected to ICP-MS analysis for determination of cellular copper content (▴) (A and C) or were stained with the Cu(I)-specific fluorescent dye Coppersensor-1 and subjected to fluorescence microscopy (B and D). Shown are averages and standard deviations (error bars) from triplicate experiments (A and C) and representative phase-contrast (right) and fluorescence (left) microscopy images (B and D).

FIG. 2.

Prolonged contact with metallic copper results in cell disintegration. Cells of Gram-negative E. coli and Gram-positive B. cereus were exposed to pure copper for 1 min (right) or unexposed (left), removed, washed, and stained. E. coli was stained red with safranin, and B. cereus was visualized by endospore staining. This process colors endospores green and vegetative cells red after safranin counterstaining. Shown are representative light microscopy images.

FIG. 3.

Cells exposed to copper surfaces suffer membrane damage. Cells of E. coli were exposed for 1 min to copper or control surfaces or unchallenged, removed, stained (Live/Dead BacLight bacterial viability kit; Invitrogen), and visualized by fluorescence microscopy. Live bacteria with intact membranes fluoresce green, while those with damaged membranes fluoresce red.

FIG. 4.

Exposure to metallic copper surfaces does not lead to increased mutations in E. coli. A total of 10⁸ E. coli cells were exposed for 5 s to copper surfaces, stainless steel surfaces, or surfaces containing 0.25% (wt/vol) of the mutagen formaldehyde (CH₂O) plus stainless steel, removed, concentrated, and spread on solid medium containing 20 μg·ml⁻¹ of the bacteriostatic compound d-cycloserine. After 24 h of incubation at 37°C, colonies were counted as originating from mutation events leading to resistance via inactivation of CycA, a d-cycloserine uptake permease. Shown are averages from triplicate experiments, with standard deviations (error bars). The asterisk denotes significantly different values (P ≤ 0.05) for formaldehyde-challenged cells.

FIG. 5.

Exposure to metallic copper does not cause extensive DNA breakage in E. coli. E. coli cells (10⁶ cells) were challenged on copper (D and F) or stainless steel (C and E) surfaces for t₀ (C and D) or 1 min (E and F) and investigated for DNA double-strand breakage by the comet assay (32). Unchallenged (A) and ciprofloxacin-treated (B) cells indicate intact and fragmented DNA, respectively. The arrow indicates characteristic comets, highly fragmented DNA resulting from gyrase inhibition by ciprofloxacin. Images shown are representatives from three independent experiments with similar results.

FIG. 6.

Efficient DNA repair provides no protection against toxicity exerted by metallic copper. Contact killing of stationary-phase (A) and exponential-phase (B) cultures of D. radiodurans (squares) or E. coli (triangles) on stainless steel (open symbols) or copper (filled symbols) surfaces. Shown are averages and standard deviations (error bars) from three independent experiments.