Killing of Bacteria by Copper Surfaces Involves Dissolved Copper

Bacteria are rapidly killed on copper surfaces. However, the mechanism of this process remains unclear. Using Enterococcus hirae, the effect of inactivation of copper homeostatic genes and of medium compositions on survival and copper dissolution was tested. The results support a role for dissolved copper ions in killing.The rapid killing of bacteria by solid copper surfaces is receiving rapidly growing attention. In laboratory experiments, it has been shown that many bacterial species, such as Escherichia coli O157, Staphylococcus aureus, Salmonella enterica, Campylobacter jejuni, Clostridium difficile, and Mycobacterium tuberculosis, are efficiently killed on copper or copper alloy surfaces (1, 3, 4, 6, 7, 12-14). In contrast, on stainless steel, living cells could be recovered even after 28 days. The antimicrobial activity of copper and copper alloys is now well established, and copper has recently been registered at the U.S. Environmental Protection Agency as the first solid antimicrobial material. A key focus is the use of copper in health care facilities, food processing plants, and other areas where clean or aseptic working procedures are required (2). In this connection, it has become important to understand the mechanism of bacterial killing, as it may bear on the possibility of the emergence of resistant organisms, cleaning procedures, and material and object engineering. We here used wild-type and mutant strains of Enterococcus hirae to investigate the influence of copper resistance genes on killing rates. We also evaluated copper dissolution by various media and its relation to killing efficiency. Our findings provide support for a prominent role for dissolved copper in the killing process.     

Copper Reduction and Contact Killing of Bacteria by Iron Surfaces

The well-established killing of bacteria by copper surfaces, also called contact killing, is currently believed to be a combined effect of bacterial contact with the copper surface and the dissolution of copper, resulting in lethal bacterial damage. Iron can similarly be released in ionic form from iron surfaces and would thus be expected to also exhibit contact killing, although essentially no contact killing is observed by iron surfaces. However, we show here that the exposure of bacteria to iron surfaces in the presence of copper ions results in efficient contact killing. The process involves reduction of Cu²⁺ to Cu⁺ by iron; Cu⁺ has been shown to be considerably more toxic to cells than Cu²⁺. The specific Cu⁺ chelator, bicinchoninic acid, suppresses contact killing by chelating the Cu⁺ ions. These findings underline the importance of Cu⁺ ions in the contact killing process and infer that iron-based alloys containing copper could provide novel antimicrobial materials.     

Mechanisms of contact-mediated killing of yeast cells on dry metallic copper surfaces

Surfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivate Candida albicans and Saccharomyces cerevisiae within minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactive S. cerevisiae Ctr1p (ScCtr1p) copper uptake transporter in Saccharomyces resulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of the C. albicans Crp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing of Candida mutant cells than of wild-type cells. Candida and Saccharomyces took up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay with Saccharomyces. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC(2)(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated.     

Advantages and challenges of increased antimicrobial copper use and copper mining

Copper is a highly utilized metal for electrical, automotive, household objects, and more recently as an effective antimicrobial surface. Copper-containing solutions applied to fruits and vegetables can prevent bacterial and fungal infections. Bacteria, such as Salmonellae and Cronobacter sakazakii, often found in food contamination, are rapidly killed on contact with copper alloys. The antimicrobial effectiveness of copper alloys in the healthcare environment against bacteria causing hospital-acquired infections such as methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli O157:H7, and Clostridium difficile has been described recently. The use of copper and copper-containing materials will continue to expand and may lead to an increase in copper mining and production. However, the copper mining and manufacturing industry and the consumer do not necessarily enjoy a favorable relationship. Open pit mining, copper mine tailings, leaching products, and deposits of toxic metals in the environment often raises concerns and sometimes public outrage. In addition, consumers may fear that copper alloys utilized as antimicrobial surfaces in food production will lead to copper toxicity in humans. Therefore, there is a need to mitigate some of the negative effects of increased copper use and copper mining. More thermo-tolerant, copper ion-resistant microorganisms could improve copper leaching and lessen copper groundwater contamination. Copper ion-resistant bacteria associated with plants might be useful in biostabilization and phytoremediation of copper-contaminated environments. In this review, recent progress in microbiological and biotechnological aspects of microorganisms in contact with copper will be presented and discussed, exploring their role in the improvement for the industries involved as well as providing better environmental outcomes.     

Genes involved in copper resistance influence survival of Pseudomonas aeruginosa on copper surfaces

Aims:  To evaluate the killing of Pseudomonas aeruginosa PAO1 on copper cast alloys and the influence of genes on survival on copper containing medium and surfaces.
Methods and Results:  Different strains of P. aeruginosa were inoculated on copper containing medium or different copper cast alloys and the survival rate determined. The survival rates were compared with rates on copper-free medium and stainless steel as control. In addition, the effect of temperature on survival was examined.
Conclusions:  Copper cast alloys had been previously shown to be bactericidal to various bacteria, but the mechanism of copper-mediated killing is still not known. In this report, we demonstrate that P. aeruginosa PAO1 is rapidly killed on different copper cast alloys and that genes involved in conferring copper resistance in copper-containing medium also influenced survival on copper cast alloys. We also show that the rate of killing is influenced by temperature.
Significance and Impact of the Study:  To use copper surfaces more widely as bactericidal agents in various settings, it is important to understand how genes influence survival on these surfaces. Here we show that genes shown to be involved in copper resistance in P. aeruginosa PAO1 can have an impact on the length of survival time on copper cast alloys under certain conditions. This is an important first step for evaluation of future use of copper surfaces as bactericidal agents.     

Contribution of Copper Ion Resistance to Survival of Escherichia coli on Metallic Copper Surfaces

Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of Escherichia coli on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.     

Copper as an antimicrobial agent against opportunistic pathogenic and multidrug resistant Enterobacter bacteria

Infections by Enterobacter species are common and are multidrug resistant. The use of bactericidal surface materials such as copper has lately gained attention as an effective antimicrobial agent due to its deadly effects on bacteria, yeast, and viruses. The aim of the current study was to assess the antibacterial activity of copper surfaces against Enterobacter species. The antibacterial activity of copper surfaces was tested by overlying 5×10(6) CFU/ml suspensions of representative Enterobacter strains and comparing bacterial survival counts on copper surfaces at room temperature. Iron, stainless steel, and polyvinylchloride (PVC) were used as controls. The mechanisms responsible for bacterial killing on copper surfaces were investigated by a mutagenicity assay of the D-cycloserin (cyclA gene), single cell gel electrophoresis, a staining technique, and inductively coupled plasma mass spectroscopy. Copper yielded a significant decrease in the viable bacterial counts at 2 h exposure and a highly significant decrease at 4 h. Loss of cell integrity and a significantly higher influx of copper into bacterial cells exposed to copper surfaces, as compared to those exposed to the controls, were documented. There was no increase in mutation rate and DNA damage indicating that copper contributes to bacterial killing by adversely affecting cellular structure without directly targeting the genomic DNA. These findings suggest that copper's antibacterial activity against Enterobacter species could be utilized in health care facilities and in food processing plants to reduce the bioburden, which would increase protection for susceptible members of the community.     

Membrane lipid peroxidation in copper alloy-mediated contact killing of Escherichia coli

Copper alloy surfaces are passive antimicrobial sanitizing agents that kill bacteria, fungi, and some viruses. Studies of the mechanism of contact killing in Escherichia coli implicate the membrane as the target, yet the specific component and underlying biochemistry remain unknown. This study explores the hypothesis that nonenzymatic peroxidation of membrane phospholipids is responsible for copper alloy-mediated surface killing. Lipid peroxidation was monitored with the thiobarbituric acid-reactive substances (TBARS) assay. Survival, TBARS levels, and DNA degradation were followed in cells exposed to copper alloy surfaces containing 60 to 99.90% copper or in medium containing CuSO(4). In all cases, TBARS levels increased with copper exposure levels. Cells exposed to the highest copper content alloys, C11000 and C24000, exhibited novel characteristics. TBARS increased immediately at a very rapid rate but peaked at about 30 min. This peak was associated with the period of most rapid killing, loss in membrane integrity, and DNA degradation. DNA degradation is not the primary cause of copper-mediated surface killing. Cells exposed to the 60% copper alloy for 60 min had fully intact genomic DNA but no viable cells. In a fabR mutant strain with increased levels of unsaturated fatty acids, sensitivity to copper alloy surface-mediated killing increased, TBARS levels peaked earlier, and genomic DNA degradation occurred sooner than in the isogenic parental strain. Taken together, these results suggest that copper alloy surface-mediated killing of E. coli is triggered by nonenzymatic oxidative damage of membrane phospholipids that ultimately results in the loss of membrane integrity and cell death.     

Quantitative proteomic profiling of the <Emphasis Type="Italic">Escherichia coli</Emphasis> response to metallic copper surfaces

Metallic copper surfaces have strong antimicrobial properties and kill bacteria, such as Escherichia coli, within minutes in a process called contact killing. These bacteria are exposed to acute copper stress under dry conditions which is different from chronic copper stress in growing liquid cultures. Currently, the physiological changes of E. coli during the acute contact killing process are largely unknown. Here, a label-free, quantitative proteomic approach was employed to identify the differential proteome profiles of E. coli cells after sub-lethal and lethal exposure to dry metallic copper. Of the 509 proteins identified, 110 proteins were differentially expressed after sub-lethal exposure, whereas 136 proteins had significant differences in their abundance levels after lethal exposure to copper compared to unexposed cells. A total of 210 proteins were identified only in copper-responsive proteomes. Copper surface stress coincided with increased abundance of proteins involved in secondary metabolite biosynthesis, transport and catabolism, including efflux proteins and multidrug resistance proteins. Proteins involved in translation, ribosomal structure and biogenesis functions were down-regulated after contact to metallic copper. The set of changes invoked by copper surface-exposure was diverse without a clear connection to copper ion stress but was different from that caused by exposure to stainless steel. Oxidative posttranslational modifications of proteins were observed in cells exposed to copper but also from stainless steel surfaces. However, proteins from copper stressed cells exhibited a higher degree of oxidative proline and threonine modifications.     

Metallic copper corrosion rates, moisture content, and growth medium influence survival of copper ion-resistant bacteria

The rapid killing of various bacteria in contact with metallic copper is thought to be influenced by the influx of copper ions into the cells, but the exact mechanism is not fully understood. This study showed that the kinetics of contact killing of copper surfaces depended greatly on the amount of moisture present, copper content of alloys, type of medium used, and type of bacteria. We examined antibiotic- and copper ion-resistant strains of Escherichia coli and Enterococcus faecium isolated from pig farms following the use of copper sulfate as feed supplement. The results showed rapid killing of both copper ion-resistant E. coli and E. faecium strains when samples in rich medium were spread in a thin, moist layer on copper alloys with 85% or greater copper content. E. coli strains were rapidly killed under dry conditions, while E. faecium strains were less affected. Electroplated copper surface corrosion rates were determined from electrochemical polarization tests using the Stern–Geary method and revealed decreased corrosion rates with benzotriazole and thermal oxide coating. Copper ion-resistant E. coli and E. faecium cells suspended in 0.8% NaCl showed prolonged survival rates on electroplated copper surfaces with benzotriazole coating and thermal oxide coating compared to surfaces without anti-corrosion treatment. Control of surface corrosion affected the level of copper ion influx into bacterial cells, which contributed directly to bacterial killing.     

Bactericidal Activity of Copper and Niobium–Alloyed Austenitic Stainless Steel

Biofouling and microbiologically influenced corrosion are processes of material deterioration that originate from the attachment of microorganisms as quickly as the material is immersed in a nonsterile environment. Stainless steels, despite their wide use in different industries and as appliances and implant materials, do not possess inherent antimicrobial properties. Changes in hygiene legislation and increased public awareness of product quality makes it necessary to devise control methods that inhibit biofilm formation or to act at an early stage of the biofouling process and provide the release of antimicrobial compounds on a sustainable basis and at effective level. These antibacterial stainless steels may find a wide range of applications in fields, such as kitchen appliances, medical equipment, home electronics, and tools and hardware. The purpose of this study was to obtain antibacterial stainless steel and thus mitigate the microbial colonization and bacterial infection. Copper is known as an antibacterial agent; in contrast, niobium has been demonstrated to improve the antimicrobial effect of copper by stimulating the formation of precipitated copper particles and its distribution in the matrix of the stainless steel. Thus, we obtained slides of 3.8% copper and 0.1% niobium alloyed stainless steel; subjected them to three different heat treatment protocols (550°C, 700°C, and 800°C for 100, 200, 300, and 400 hours); and determined their antimicrobial activities by using different initial bacterial cell densities and suspending solutions to apply the bacteria to the stainless steels. The bacterial strain used in these experiments was Escherichia coli CCM 4517. The best antimicrobial effects were observed in the slides of stainless steel treated at 700°C and 800°C using an initial cell density of approximately 10⁵ cells ml⁻¹ and phosphate-buffered saline as the solution in which the bacteria came into contact with copper and niobium–containing steel.     

Bacterial killing by dry metallic copper surfaces

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.     

Contact killing and antimicrobial properties of copper

With the emergence of antibiotic resistance, the interest for antimicrobial agents has recently increased again in public health. Copper was recognized in 2008 by the United States Environmental Protection Agency (EPA) as the first metallic antimicrobial agent. This led to many investigations of the various properties of copper as an antibacterial, antifungal and antiviral agent. This review summarizes the latest findings about ‘contact killing’, the mechanism of action of copper nanoparticles and the different ways micro‐organisms develop resistance to copper.     

Lack of Involvement of Fenton Chemistry in Death of Methicillin-Resistant and Methicillin-Sensitive Strains of Staphylococcus aureus and Destruction of Their Genomes on Wet or Dry Copper Alloy Surfaces

The pandemic of hospital-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has declined, but the evolution of strains with enhanced virulence and toxins and the increase of community-associated infections are still a threat. In previous studies, 10⁷ MRSA bacteria applied as simulated droplet contamination were killed on copper and brass surfaces within 90 min. However, contamination of surfaces is often via finger tips and dries rapidly, and it may be overlooked by cleaning regimes (unlike visible droplets). In this new study, a 5-log reduction of a hardy epidemic strain of MRSA (epidemic methicillin-resistant S. aureus 16 [EMRSA-16]) was observed following 10 min of contact with copper, and a 4-log reduction was observed on copper nickel and cartridge brass alloys in 15 min. A methicillin-sensitive S. aureus (MSSA) strain from an osteomyelitis patient was killed on copper surfaces in 15 min, and 4-log and 3-log reductions occurred within 20 min of contact with copper nickel and cartridge brass, respectively. Bacterial respiration was compromised on copper surfaces, and superoxide was generated as part of the killing mechanism. In addition, destruction of genomic DNA occurs on copper and brass surfaces, allaying concerns about horizontal gene transfer and copper resistance. Incorporation of copper alloy biocidal surfaces may help to reduce the spread of this dangerous pathogen.     

Survival of bacteria on metallic copper surfaces in a hospital trial

Basic chemistry of copper is responsible for its Janus-faced feature: on one hand, copper is an essential trace element required to interact efficiently with molecular oxygen. On the other hand, interaction with reactive oxygen species in undesired Fenton-like reactions leads to the production of hydroxyl radicals, which rapidly damage cellular macromolecules. Moreover, copper cations strongly bind to thiol compounds disturbing redox-homeostasis and may also remove cations of other transition metals from their native binding sites in enzymes. Nature has learned during evolution to deal with the dangerous yet important copper cations. Bacterial cells use different efflux systems to detoxify the metal from the cytoplasm or periplasm. Despite this ability, bacteria are rapidly killed on dry metallic copper surfaces. The mode of killing likely involves copper cations being released from the metallic copper and reactive oxygen species. With all this knowledge about the interaction of copper and its cations with cellular macromolecules in mind, experiments were moved to the next level, and the antimicrobial properties of copper-containing alloys in an “everyday” hospital setting were investigated. The alloys tested decreased the number of colony-forming units on metallic copper-containing surfaces by one third compared to control aluminum or plastic surfaces. Moreover, after disinfection, repopulation of the surfaces was delayed on copper alloys. This study bridges a gap between basic research concerning cellular copper homeostasis and application of this knowledge. It demonstrates that the use of copper-containing alloys may limit the spread of multiple drug-resistant bacteria in hospitals.     

Preparation of LL-37-grafted titanium surfaces with bactericidal activity

Modification of material surfaces aimed at bestowing them with antimicrobial properties is a promising approach in the development of new biomaterials. Antimicrobial peptides (AMPs) are an attractive alternative to conventional antibiotics because of lack of toxicity, inherently high selectivity, and absence of immune response. As the antimicrobial mode of action of the AMP cathelin LL37 is formation of pores and disruption of microbial membrane, the purpose of the present study was to develop and test a method of covalent immobilization of LL37 on titanium surface. The application of a flexible hydrophilic poly(ethylene glycol) spacer and selective N-terminal conjugation of LL37 resulted in a surface peptide layer which was capable of killing bacteria on contact.     

Potential action of copper surfaces on meticillin-resistant Staphylococcus aureus

Aims: Studies to date have shown rapid killing of bacterial cells when exposed to copper surfaces. The mechanistic action of copper on bacterial cells is so far unknown. Methods and Results: To investigate potential mechanisms involved, meticillin‐resistant Staphylococcus aureus (MRSA) cells (10⁷ CFU) were inoculated onto coupons of copper or stainless steel and stained with either the viability fluorophore 5‐cyano‐2,3‐ditolyl tetrazolium (CTC), to detect respiration, or BacLight? (SYTO9/propidium iodide), to determine cell wall integrity. Coupons were then observed in‐situ using epifluorescence microscopy. In addition, DNA from cells inoculated onto either copper or stainless steel surfaces was isolated and analysed by agarose gel electrophoresis. An effect on cellular respiration with CTC reduction was evident but no effect on cell membrane integrity (BacLight?) was observed. Results from the DNA isolation indicated a copper‐induced detrimental effect on MRSA genomic material as no bands were observed after exposure to copper surface. Conclusions: The results indicate that exposure to copper surfaces rapidly kills MRSA by compromising cellular respiration and damaging DNA, with little effect on cell membrane integrity. Significance and Impact of the study: This research provides a mechanistic explanation in support of previous suggestions that although copper surfaces do not affect membrane integrity of cells, there is still a rapid antimicrobial effect.     

Inactivation of bacterial and viral biothreat agents on metallic copper surfaces

In recent years several studies in laboratory settings and in hospital environments have demonstrated that surfaces of massive metallic copper have intrinsic antibacterial and antiviral properties. Microbes are rapidly inactivated by a quick, sharp shock known as contact killing. The underlying mechanism is not yet fully understood; however, in this process the cytoplasmic membrane is severely damaged. Pathogenic bacterial and viral high-consequence species able to evade the host immune system are among the most serious lethal microbial challenges to human health. Here, we investigated contact-killing mediated by copper surfaces of Gram-negative bacteria (Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis tularensis and Yersinia pestis) and of Gram-positive endospore-forming Bacillus anthracis. Additionally, we also tested inactivation of monkeypox virus and vaccinia virus on copper. This group of pathogens comprises biothreat species (or their close relatives) classified by the Center for Disease and Control and Prevention (CDC) as microbial select agents posing severe threats to public health and having the potential to be deliberately released. All agents were rapidly inactivated on copper between 30 s and 5 min with the exception of B. anthracis endospores. For vegetative bacterial cells prolonged contact to metallic copper resulted in the destruction of cell structure.     

Isolation and Characterization of Bacteria Resistant to Metallic Copper Surfaces

Metallic copper alloys have recently attracted attention as a new antimicrobial weapon for areas where surface hygiene is paramount. Currently it is not understood on a molecular level how metallic copper kills microbes, but previous studies have demonstrated that a wide variety of bacteria, including Escherichia coli, Staphylococcus aureus, and Clostridium difficile, are inactivated within minutes or a few hours of exposure. In this study, we show that bacteria isolated from copper alloy coins comprise strains that are especially resistant against the toxic properties exerted by dry metallic copper surfaces. The most resistant of 294 isolates were Gram-positive staphylococci and micrococci, Kocuria palustris, and Brachybacterium conglomeratum but also included the proteobacterial species Sphingomonas panni and Pseudomonas oleovorans. Cells of some of these bacterial strains survived on copper surfaces for 48 h or more. Remarkably, when these dry-surface-resistant strains were exposed to moist copper surfaces, resistance levels were close to those of control strains and MICs for copper ions were at or below control strain levels. This suggests that mechanisms conferring resistance against dry metallic copper surfaces in these newly isolated bacterial strains are different from well-characterized copper ion detoxification systems. Furthermore, staphylococci on coins did not exhibit increased levels of resistance to antibiotics, arguing against coselection with copper surface resistance traits.Copper in its ionic form is a required trace element for most pro- and eukaryotic organisms, including humans. While needed in small amounts, copper can easily become toxic when in surplus. This toxicity is caused mainly by the intrinsic properties of copper, as free copper ions undergo redox cycling reactions alternating between Cu(I) and Cu(II). This also results in the transfer of electrons to hydrogen peroxide and the concomitant generation of hydroxyl radicals that readily attack and damage cellular biomolecules. Recently, it was found that the majority of copper stress in Escherichia coli, as indicated by hydroxyl radical formation, occurs within the periplasm, away from the cytoplasmic DNA, and is thus copper-mediated oxidative stress (25). The cytoplasm might thus be better protected from copper-mediated oxidative stress, and indeed cells usually prevent accumulation of significant intracellular concentrations of free copper ions either by producing copper-binding chaperones (26, 36) or unspecific chelators such as glutathione (20, 30) or by efflux (14, 35). Nevertheless, copper ions within the cytoplasm also cause damage. Surprisingly, this damage is not related to oxidative stress but is exerted directly by the metal ions. It seems that copper ions attack and displace iron atoms from enzymes with solvent-exposed iron sulfur clusters such as those of hydratases (24). Thus, the presence of oxygen is not needed for this reaction, and there is no copper-mediated oxidative stress involved in this damage (24).While we are now gaining a more detailed picture of why copper ions are toxic to cells, we do not understand why metallic copper surfaces kill single-celled organisms such as bacteria and yeasts. Earlier studies have demonstrated that metallic copper surfaces efficiently inactivate microbes upon contact (9, 11, 32), especially when exposed to dry surfaces (10). These beneficial properties led to the official registration of copper alloys as antimicrobials through the U.S. Environmental Protection Agency in 2008. There is now great hope that metallic copper surfaces will be able to help control hospital-acquired (nosocomial) infections. Indeed, there are ongoing trials in which dry touch surfaces in hospitals around the world are replaced by copper alloys. Results from a German hospital trial indicate that copper surfaces such as door knobs, light switches, and push plates diminished the bacterial load by up to 30% compared to stainless steel control surfaces (A. Mikolay et al., unpublished data). Similar studies in Great Britain and South Africa found that the numbers of bacteria on the surfaces of copper-containing items such as trolleys, desks, toilet seats, tap handles, or push plates were 71% (28) or 90% to 100% (5) lower than those on their stainless steel, wood, or tile control equivalents.A potential challenge when applying metallic copper might be the probable emergence and spread of resistant bacteria, similar to what was observed after the introduction of antibiotics. The goal of this study was to investigate if bacteria that can withstand dry metallic copper surfaces can be isolated and if there is a link to multiple drug resistance. Where can potentially pathogenic bacteria that are in contact with both humans and metallic copper surfaces be found? Actually, people handle copper surfaces every day. Most coins around the world are made from copper or copper alloys. This includes the U.S. penny, which is composed of copper plated over a zinc core, and the nickel, dime, and quarter, which are cupronickel alloys (www.usmint.gov/). Coins of the European Union, such as the 50-cent coin, are made from an 89% copper alloy, as are the bicolored one- and two-Euro coins, which consist of different copper alloys (http://www.copperinfo.co.uk/coins/).In the present study we isolated and initiated characterization of aerobic heterotrophic bacteria from copper alloy coins as an example of heavily used copper surfaces and person-to-person vectors. We believe that knowledge of the physiology and resistance mechanisms of these microbes will help us to adapt our strategies for using metallic copper surfaces in hygiene-sensitive areas. This might not only diminish total bacterial numbers but also prevent the emergence and spread of multiple-drug-resistant strains in hospitals equipped with copper surfaces.     

Copper toxicity and the origin of bacterial resistance--new insights and applications

The bioavailability of different metals has likely changed over the course of Earth's history. Based on geochemical models, copper became much more bioavailable with the advent of an oxidizing atmosphere. This posed both a challenge and an opportunity for the organisms at that time. Specifically, copper resistance mechanisms were required first and to do this Bacteria appear to have modified already existing protein structures. Later, Cu-utilizing proteins evolved and continue to be used sparingly, at least relative to later evolving Eukarya, by Bacteria but with significant biogeochemical consequences. Copper is a strong soft metal that can attack intracellular iron-sulfur centers of various proteins under primarily anoxic conditions. In oxic conditions, copper can catalyze a Fenton-like reaction that may cause lipid peroxidation and protein damage. The inherent ability of copper to inflict damage upon multiple cellular functions has been harnessed by macrophages and perhaps amoeba to kill and later digest bacteria and other microorganisms. Notably, these organisms, unlike Bacteria, most likely evolved after increases in copper availability, implying that Eukarya utilized their own trafficking and resistance mechanisms, in addition to the natural toxicity of copper, as leverage in interactions with Bacteria. In an "arms race," some pathogenic bacteria have evolved new mechanisms for copper resistance, which is relevant given renewed interest in the use of copper surfaces due to their antimicrobial properties.