Contamination of hospital surfaces with pathogenic microorganisms contributes to reinfection and spread of disease. Not only are enterococci hardy and able to survive on many types of “touch” surfaces (27
) for several weeks, but the ability of these organisms to easily transfer antibiotic resistance means that it is essential that any contamination of the environment from infected individuals with viable cells be effectively destroyed (41
). A combination of bactericidal surfaces providing a constant “killing surface” and regular effective disinfection could greatly reduce the spread of disease. Stainless steel is a commonly used hospital surface for many reasons, including resistance to corrosion and the ability to withstand regular disinfection. However, our research has shown that vancomycin-resistant isolates of the two main pathogenic enterococcal species are able to survive for several months on stainless steel surfaces, which could potentially contribute to the reinfection of personnel and especially of vulnerable patients.
In our studies on alloy surfaces containing at least 65% copper, enterococci at a high contamination concentration of 106 CFU/cm2 were rapidly killed over a few hours of contact, compared to survival for several months on stainless steel. Pure copper was the most effective surface at killing bacterial cells.
In general, alloys containing >90% copper were as effective as pure copper, with all of the isolates of E. faecalis and two of E. faecium achieving complete cell death by 1 h (with a few cells of the remaining E. faecium isolates remaining viable for another hour but still with a 3- to 4-log reduction in number). For both species, no viable cells were detected after 2 h of contact with alloys containing 60 to 70% copper. Occasionally, alloy C75200 (nickel-silver) outperformed alloys with a higher copper content, e.g., C26000 (cartridge brass). The other metal constituents and physical properties of each alloy, particularly the rate of release of copper, may have a role in the bactericidal activity reported.
However, at bacterial contamination levels of ≤105
, all of the copper alloys tested were virtually indistinguishable from pure copper and were very effective at killing pathogenic enterococcal cells within 1 h and in 20 min at <103
. This reinforces the potential for the use of these alloys in the clinical environment, because a recent study in the United States determined VRE contamination on surfaces to be, at most, a few hundred cells/100 cm2
at three large hospitals (M. Schmidt, personal communication; 42
Our experiments were done under worst-case scenario conditions: inoculation of surfaces with aqueous samples in a nutritious and isotonic medium. It has been reported that the presence of biological fluids or meat juices can delay the mechanism of Staphylococcus aureus
and E. coli
O157 killing by copper, respectively (38
). Recent work in our laboratories has also determined the importance of chelating substances on bacterial survival on copper surfaces (unpublished data). Experiments with a rapidly drying swabbed inoculum in PBS or water suggest that killing is even more rapid (data not shown), but these conditions may be not entirely relevant to actual contamination with organic specimens in a clinical environment.
Assessment of the number of viable cells recovered from metal surfaces by respiratory staining was not significantly different from that obtained by culture, providing more evidence that enterococci survive for long periods on stainless steel but also suggesting the absence of a viable-but-nonculturable state on copper surfaces under these conditions.
Assessment of the viability of cells recovered from stainless steel using Bac
Light SYTO 9/PI staining also demonstrated no significant difference from results obtained by culture but suggested that some damage to the cell membrane does occur on this surface. However, staining with Bac
Light does not always produce distinct “live” and “dead” populations, particularly because cells with an intact membrane are not necessarily alive (2
). The Bac
Light staining method was difficult to interpret for enterococci exposed to copper alloys because of the diminished and frequently absent staining with SYTO 9 and PI, respectively, except at the time of inoculation. This suggested that the bacterial DNA has been affected to the extent that intercalating DNA stains cannot now bind. There does not appear to be any uptake of PI by enterococci exposed to copper alloys, suggesting that damage to the cell membrane is not occurring, but these results may be misleading if the DNA is too damaged to bind PI.
Targets of copper toxicity are thought to include nucleic acid, structural and functional proteins, lipids, and inhibition of metabolic processes such as respiration and osmotic stress resulting in cell lysis. In mammalian cells, soluble copper(II) ions are known to bind to DNA bases, resulting in unwinding of the double helix, and under aerobic conditions, the Fenton reaction with bound and free ions and hydrogen peroxide results in the production of reactive oxygen species (ROS) that cause double- and single-strand breaks and intrastrand cross-linking (30
). Macomber et al. (31
). reported that exposure of E. coli
mutants lacking in copper export systems to copper solutions resulted in copper-overloaded cells and no detectable oxidative damage to the DNA using a gene-specific PCR assay. The reasons suggested were compartmentalization of hydroxyl radicals generated in the periplasm of the cell. The effect of hydroxyl radical damage is short reaching, and therefore, damage to the DNA could not occur if the sites of hydroxyl radical generation are spatially separated from the nucleic acid. They also described the existence of ligands, perhaps glutathione, complexing with copper ions and suggested that copper toxicity may primarily be due to damage of metalloenzymes by the ROS, i.e., dihydrogen peroxide, hydroxyl radicals, and superoxides.
Exposure to relatively low soluble copper concentrations, described by Macomber et al. (31
), is very different from continual contact with copper and copper alloy surfaces. In our system, we have reported extensive disintegration of the genomic and plasmid DNAs of Gram-positive enterococci exposed to copper and copper alloy surfaces because the DNA from cells exposed to copper appears to be (i) denatured over time in agarose gel electrophoresis, (ii) does not bind intercalating stains SYTO 9 and PI, and (iii) genomic DNA cannot be detected in the DNA fragmentation assay. These effects were not observed on stainless steel. The DNA fragmentation assay is a useful tool because it allows observation of the entire genome of individual cells without a purification step that could result in damage to DNA from stressed bacterial cells and lead to spurious results. This technology has been successfully used for the analysis of eukaryotic nucleic acid, for example, in the analysis of sperm DNA and the efficacy of specific anticancer agents on patient DNA. Fernández et al. (17
). successfully adapted the method to investigate damage to bacterial DNA following treatment with quinolone antibiotics. We have also shown how exposure to copper resulted in the inhibition of respiration with minimal damage to the integrity of the bacterial cell membrane. We suggest that the absence of an outer membrane in Gram-positive cells and lack of a periplasmic space may facilitate the access of copper(I)/(II) and generated ROS to the DNA directly and rapidly inflict damage.
The ligands, described by Macomber et al. (31
), responsible for removing copper(II) in copper-overloaded cells may still be present, but the effect is insignificant when bacteria are constantly in contact with the copper surface and binding sites are saturated with copper ions.
Espírito Santo et al. (13
) determined that ROS are generated when E. coli
is exposed to copper surfaces. They identified hydroxyl radicals generated under aerobic conditions, presumably by Haber-Weiss and Fenton reactions of reduced copper ions [supplied by redox cycling of copper(I) and copper(II)].
Preliminary experiments in our laboratory with E. coli
O157 exposed to copper surfaces using the DNA fragmentation assay have indicated that genomic DNA is also destroyed in this species but more slowly (manuscript in preparation). The DNA stability reported by Macomber et al. (31
) is probably due to exposure to soluble Cu(II) rather than the Cu(I)/Cu(II) redox cycling proposed in our studies. This DNA degradation effect on E. coli
and other species will be investigated in future studies.
Concerns have been expressed about the possibility of the development of copper resistance if alloy surfaces are constantly in use. Mutations in bacterial copper homeostasis mechanisms do affect survival times on copper alloys (11
), but because survival times on stainless steel and other commonly in-use surfaces are so much greater, the significance may not be relevant in a real-life situation. Further studies are required to elucidate the mechanism of copper killing and investigate this possibility.
There has been much concern recently that the frequency of antimicrobial resistance in bacteria has increased in concert with increasing usage of antimicrobial compounds. A recent European Commission report (16
) has summarized the scientific evidence from bacteriological, biochemical, and genetic data indicating that the use of active molecules in biocidal products may contribute to the increased occurrence of antibiotic-resistant bacteria. The selective stress exerted by biocides may favor bacteria expressing resistance mechanisms and their dissemination. Some biocides have the capacity to maintain the presence of mobile genetic elements that carry genes involved in cross-resistance between biocides and antibiotics. In enterococci, up to 25% of the genome has been found to contain mobile elements (41
). The dissemination of these mobile elements, their genetic organization, and the formation of biofilms provide conditions that could create a potential risk of development of cross-resistance between antibiotics and biocides. The case for the use of copper in antimicrobial products was considered, but there was no evidence that this might lead to antibiotic resistance in the way that the widespread use of Triclosan has been associated with the emergence of triclosan and mupirocin resistance in MRSA, although evidence for this is limited (15
). The plasmid-localized copper resistance tcrB
gene has been identified in E. faecium
and E. faecalis
thought to originate from pigs fed with copper sulfate-supplemented food (19
). The Tn1546
element and erm
genes conferring glycopeptide and macrolide resistance are located on the same plasmid, but there is no significant evidence that use of copper in animal feeds coselected for antibiotic resistance (20
) except under experimental conditions in piglets fed a high concentration of copper sulfate (21
). However, continued use of copper sulfate was not able to maintain high levels of antimicrobial resistance (21
The current study indicates that DNA is rapidly destroyed in enterococci exposed to copper surfaces, meaning that there is little chance of high-level copper or antibiotic resistance developing. Consequently, this disintegration of bacterial nucleic acid supports the use of copper alloys as contact surfaces in clinical environments to actively kill bacterial cells without the occurrence of DNA mutation and transfer of genetic material carrying antibiotic resistance genes.