In this study we have demonstrated a rapid killing, particularly for “dry-touch” contamination, of important pathogenic enterococci, where death occurs in minutes on copper and, perhaps more importantly, copper alloys, which could be used practically as touch surfaces in health care settings and elsewhere. An understanding of the mechanism of copper surface toxicity requires the identification of the agents responsible and the targets affected. The prolonged survival that we observed in the presence of chelators suggests that both ionic species, copper(I) and copper(II), are important, directly or indirectly, in the killing mechanism for wet and dry surface contamination under aerobic and anaerobic conditions. This was surprising, because it was expected that for the wet inoculum with longer contact times, the rate of copper ion release would have been greater. Molteni et al. (29
) previously quantified copper ion released from surfaces and observed that release was proportional to the killing rate and very dependent on the liquid matrix constitution. Perhaps, the most significant ion release is immediately upon contact. In E. faecium
, both copper oxidative states were equally important, but in E. faecalis
, copper(I) ions appeared to be more significant than copper(II) for the type strains and clinical isolates tested here. The chelation of copper ions also resulted in the protection of enterococcal genomic DNA and respiration on copper surfaces under wet and dry inoculum conditions.
Copper is an essential element in biological systems but highly toxic at elevated concentrations, which may be due to the generation of toxic radicals that damage cellular components such as superoxide (13
). In enterococci, superoxide dismutase (SOD) dismutes superoxide to hydrogen peroxide under the acidic conditions of the intestine. Our results suggest that Tiron, a membrane-permeable quencher of superoxide, significantly protected enterococci from the toxicity of copper surfaces under wet and dry conditions. However, if SOD was present, protective effects were minimal for the dry inoculum but more evident under wet conditions, particularly for E. faecalis
. However, these experiments were conducted at a neutral pH, and therefore, the dismutation rate may have been reduced. The protective effect of Tiron was initially considerable but rapidly declined after 60 min in the wet inoculum. It is unclear if this is due to a release of ROS, which occurs as part of a common lethal pathway in bacteria exposed to antimicrobials with completely different modes of action (21
). However, the addition of EDTA at the point where Tiron protection had begun to decline protected cells, suggesting that toxicity may involve a short-term generation of superoxide but prolonged copper(II) toxicity. Tiron also protected enterococci from DNA damage and respiratory failure on copper surfaces.
, enterococci exist primarily under the anoxic conditions of the gut. However, E. faecalis
is an unusual intestinal commensal, because under certain nutrient-limiting conditions, an incomplete respiratory chain results in fermentative metabolism that releases extracellular superoxide, which enhances virulence and has been linked to chromosomal instability (CIN), a possible precursor to colorectal cancer (CRC) and inflammatory bowel disease (16
). However, it may be that if small quantities of superoxide are being produced in our system, then the copper(II) released from the copper surface is reduced to copper(I).
This would explain the increased effect of the copper(I) chelator observed for E. faecalis
compared to that observed for E. faecium
. It is interesting that Baker et al. (2
) previously observed extracellular copper(I) on the surface of Staphylococcus aureus
, which is implicated in the toxicity of soluble copper. It is unclear if Tiron is quenching intracellular and extracellular superoxide, although the only partial protection afforded by SOD suggests that the majority of superoxide generation is intracellular, since the enzyme would be unlikely to penetrate the cell. The generation of superoxide, which is a virulence factor in vivo
, may be a suicidal act when cells are exposed to copper and copper alloy surfaces.
Hydrogen peroxide is a 2-electron reductant of oxygen and therefore not a true radical but has the ability to diffuse through cell membranes. It has a long half-life in the presence of superoxide and can damage biomolecules directly by the oxidation of sulfur atoms in cysteine residues (16
); in the presence of the transition metals iron and copper, it is responsible for the generation of highly toxic hydroxyl free radicals (the Fenton reaction). Catalase was used to decompose hydrogen peroxide to water and oxygen but did not have a protective effect on survival for either species in the dry inoculum and only a slight effect at 60 min of contact for the E. faecium
wet inoculum (although superoxide generated in E. faecalis
is known to inactivate catalase but not SOD, and the production of hydrogen peroxide in vivo
is a virulence factor for E. faecium
]). No protective effect on bacterial DNA or respiration was seen.
The hydroxyl radical is a highly reactive oxidant with a half-life in aqueous solution of less than 1 nanosecond. This moiety has the ability to damage biomolecules directly, for example, by inducing strand breaks and base modifications in DNA at diffusion-limited rates (17
). In this study, the quenching of hydroxyl radical generation with d
-mannitol did not significantly prolong survival on copper surfaces or protect DNA or bacterial respiration even at a range of concentrations (data not shown). Tkeshelashvili et al. (44
) observed previously that d
-mannitol did not completely abolish lethal damage by soluble copper ions on purified E. coli
DNA and suggested that other ROS may be involved in DNA damage, including copper peroxides. Savoye et al. (40
) previously investigated the binding of soluble copper ions to purified irradiated DNA and found that conformational changes restricted the access of mannitol to the hydroxyl ions generated. It is uncertain if hydroxyl radical formation is occurring in enterococci, and we have not detected it either, because the site of generation is shielded from the quencher or too short-lived and escaped detection in our system. The enterococcal DNA destruction observed in vitro
(cells exposed to copper surfaces and removed for analysis) and in situ
on copper and copper alloy surfaces does appear to be part of the killing process, because significant breakdown begins to occur immediately upon contact but does not appear to be the result of hydroxyl radical toxicity. The DNA breakdown of dead cells exposed to copper surfaces that could be protected with EDTA suggests direct copper(II) involvement. In eukaryotic DNA, metal ions are known to bind at separate sites on DNA and can unwind the helix, affect DNA-associated proteins, and induce lesions (24
). Copper bound to peptides is also known to result in damage to the DNA (42
). Perhaps, the constant influx of ions into the bacteria on copper surfaces produces intracellular copper complexes with unknown proteins that induce damage to DNA and possibly free-ion-induced lesions. However, in viable cells the DNA breakdown is much faster than that in dead cells, suggesting that there is still a role for unknown radicals possibly generated by superoxide and cellular metabolism. Other radicals, including peroxynitrite, which are generated from superoxide and nitric oxide and are known to produce further radicals that cause DNA strand breakage and base damage, may possibly have a role but have not been addressed here. Moore et al. (30
) observed previously that in vivo
, E. faecalis
produces thiyl radicals from the oxidation of cysteine residues, which, like superoxide, are virulence factors that damage epithelial cell DNA and affect the fluidity of the membrane. Tiron did not protect dead cell DNA on copper, allaying fears that Tiron may also be chelating ions as well as quenching superoxide, as reported previously by Ghosh et al. (12
). A recent report (9
) suggested that DNA damage is not occurring in E. coli
but that the mutagenicity and comet assays used detect limited damage and not the extensive overall effect on the entire genome that we have observed for enterococci. Those authors also determined that the superior DNA repair mechanisms of the polyextremophile Deinococcus radiodurans
did not protect the organism from death on copper surfaces, but it is unclear if the DNA was already extensively degraded.
Our results suggest that for Gram-positive enterococci, the Fenton reaction-generated hydroxyl radicals may not be as important on copper surfaces, in contrast to recent reports for Gram-negative organisms (7
). Fenton chemistry has been observed in vitro
with purified DNA, but concern about its relevance in vivo
has been expressed (39
). Macomber et al. (25
) found previously that soluble copper decreased the rate of hydrogen peroxide-induced DNA damage in E. coli
and suggested that oxidative stress was not the only mechanism responsible for copper killing. The survival of enterococci on copper surfaces was prolonged in the presence of 10% sucrose, which was also observed previously for E. coli
by Espirito Santo et al. (7
). This may be due to protection from osmotic stress, the reduction of water activity (1
), or antioxidant-scavenging properties that have been attributed to some sugars (38
). Membrane damage in E. coli
on copper has been reported (9
); however, our in situ
staining with the lipophilic cationic dye rhodamine 123 indicates that extensive membrane depolarization does not occur upon the prolonged contact of enterococci on copper and alloy surfaces when DNA destruction and cell death have already occurred. Consequently, membrane damage cannot be assumed to be a universal mechanism of copper toxicity.
To summarize, in the two main pathogenic species of enterococci, copper surface toxicity is implemented by copper(I) and copper(II) ions and superoxide under both fomite and dry-touch conditions. Killing is 80 to 90% faster under dry conditions, and rapid DNA degradation is followed by a reduction in bacterial respiration. Finally, the membrane is slowly depolarized. The same killing mechanism also exists with cartridge brass, a commonly used copper alloy, but takes slightly longer, presumably due to the reduced copper content.
The bacterial DNA is denatured, primarily by copper(II) but also by superoxide, whose toxicity is thought to arise indirectly from the generation of hydroxyl radicals (Fenton and Haber-Weiss reactions) (19
). We have not found any evidence to support hydroxyl radical generation in enterococci, with superoxide being the principal ROS generated: superoxide was reported previously to have some direct effects on enzymes and small molecules (11
). The DNA of dead cells also denatures on copper surfaces but much more slowly, suggesting that metabolic activity is required for the initial superoxide-mediated killing, while direct copper effects take longer.
Respiration is also affected by copper ions and superoxide on copper surfaces. E. faecalis
possesses cytochrome bd
in the membrane (56
), and copper(II) ions are known to bind and inhibit certain cytochromes by altering the conformation and electron transfer of associated reductases (22
). This may explain why respiration is affected so quickly in the dry inoculum. Moreover, a respiratory block may result in the buildup of toxic intermediates. Although the ATPase activity in Enterococcus hirae
cell membranes was reported previously to be affected by soluble copper (46
), our results suggest that little damage to the enterococcal membrane occurs on copper surfaces until after cells are dying. It is unlikely that the thick cell wall of Gram-positive organisms helps to maintain the integrity of the cell, shielding the membrane from direct contact with copper, since copper ion uptake is rapid and contributes quickly to DNA destruction and respiratory inhibition, but bacterial morphology does appear to affect the mechanism of copper toxicity.
Our results also highlight that if any contaminating substance can inhibit the access of copper ions to pathogens contained within it, this can affect the efficiency of the copper surface as a killing surface. Molteni et al. (29
) observed previously that ion release in Tris HCl buffer was 100 times greater than that in water or phosphate buffer. These experiments were performed by using phosphate buffer, and a rapid killing of enterococci was achieved unless chelating substances were present. This was observed by previous studies of contaminated copper coins, where blood, pus, and natural soiling delayed the copper killing mechanism, presumably due to a chelation effect (8
Regardless of differences in mechanisms, copper alloy surfaces may prove invaluable for the reduction of the spread of viable organisms in health care facilities and food preparation areas, but because copper ion release is the limiting factor in surface efficacy, constant surface care to ensure that soiling or cleaning agents do not interfere with copper(I) and copper(II) ion release is essential.