Current Medicinal Chemistry, 2005, 12, 2163-2175 2163

Copper as a Biocidal Tool

Gadi Borkow* and Jeffrey Gabbay

Cupron Inc.

Abstract: Copper ions, either alone or in copper complexes, have been used to disinfect liquids, solids and human tissue
for centuries. Today copper is used as a water purifier, algaecide, fungicide, nematocide, molluscicide as well as an anti-
bacterial and anti-fouling agent. Copper also displays potent anti-viral activity. This article reviews (i) the biocidal
properties of copper; (ii) the possible mechanisms by which copper is toxic to microorganisms; and (iii) the systems by
which many microorganisms resist high concentrations of heavy metals, with an emphasis on copper.

1. COPPER AS BIOCIDE 1.1. Copper as a Bactericide

Metal ions, either alone or in complexes, have been used The bacteriostatic effect of copper was noted by Dr.
to disinfect fluids, solids and tissues for centuries [1,2]. The Phyllis J. Kuhn [7], who was involved in the training of
ancient Greeks in the time of Hypocrates (400 BC) were the housekeeping and maintenance personnel at the Hamot
first to discover the sanitizing power of copper. They Medical Center in Pennsylvania. To heighten their awareness
prescribed copper for pulmonary diseases and for purifying of modes of infection, the students were given blood agar
drinking water. Gangajal (holy water taken from the Ganges plates and instructions on their use. The students returned
River) is stored in copper utensils in every Hindu household with bacterial cultures from such diverse sources as toilet
due to copper's anti-fouling and bacteriostatic properties. The bowl water (remarkably clean), salad from the employees’
early Phoenicians nailed copper strips to ships’ hulls to cafeteria (heavily colonized), and doorknobs. Brass (an alloy
inhibit fouling since cleaner vessels are faster and more of 67% copper and 33% zinc) doorknob cultures showed
maneuverable. sparse streptococcal and staphylococcal growth while
By the 18th century, copper had come into wide clinical stainless steel (about 88% iron and 12% chromium)
doorknob cultures showed heavy growth of gram-positive
use in the Western world in the treatment of mental disorders
organisms and an array of gram-negative organisms.
and afflictions of the lungs. Early American pioneers moving
west across the continent put silver and copper coins in large Based on this observation, Dr. Kuhn investigated
wooden water casks to provide safe drinking water for their bacterial growth on metals. Small strips of stainless steel,
long journey. In the Second World War, Japanese soldiers brass, aluminum, and copper were inoculated with broths of
put pieces of copper in their water bottles to help prevent Escherichia coli, Staphylococcus aureus, Streptococcus
dysentery. Copper sulphate is highly prized by some group D, and Pseudomonas species. The broths contained a
inhabitants of Africa and Asia for healing sores and skin very heavy inoculum (~107 bacteria/ml). The strips were
diseases. NASA designed an ionization copper-silver water then air-dried for 24 hours at room temperature, inoculated
sterilizing system for its Apollo flights. onto blood agar plates, and incubated for 24 hours at 37°C.
Copper is one of a relatively small group of metallic The results were striking. The copper and brass strips
showed little or no growth, while the aluminum and stainless
elements which are essential to human health. These
steel strips produced a heavy growth of all the different types
elements, along with amino and fatty acids and vitamins, are
of microbes.
required for normal metabolic processes. The adult body
contains between 1.4 and 2.1 mg of copper per kg of body The test was repeated at varying intervals of 15 minutes,
weight. It is estimated that a human eats and drinks about 1 1, 5, 7, 20 and 24 hours. Brass disinfected itself in seven
mg of copper every day. After nutritional requirements are hours or less, depending on the inoculum size and the
met, excess copper is released into bile and excreted in the condition of the surface of the metal. Freshly scoured brass
feces [3]. disinfected itself in one hour. Copper disinfected itself of
some types of microbes within 15 minutes. Aluminum and
Copper is considered safe to humans as demonstrated
stainless steel produced heavy growths of all isolates after
inter alia by the widespread and prolonged use of copper
intrauterine devices (IUDs) [4,5]. In contrast to the low eight days and growths of most isolates after three weeks.
sensitivity of human tissue (skin or other) to copper [6], In another experiment, stainless steel, aluminum, brass
microorganisms are extremely susceptible to copper. Several and copper strips were covered with seeded agar and
mechanisms have been suggested to explain the toxicity of incubated in culture for 24 hours. Replica plates from the
copper to microorganisms (see Section 2 below). stainless steel and aluminum strips allowed the growth of
bacteria, while replica plates from the brass and copper strips
did not. Scanning electron micrograph pictures of the
*Address correspondence to this author at the Hameyasdim 44, Kfar Gibton surfaces of the metals showed that E. coli was completely
76910, Israel; Tel: 972-546-611287; Fax: 972-8-9491254; E-mail: disrupted on brass while remaining intact on stainless steel.
gadi@cupron.com

0929-8673/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

2164 Current Medicinal Chemistry, 2005, Vol. 12, No. 18 Borkow and Gabbay

More recently, the ability of various electroplated [21]. The main sources for contamination are the patient’s
coatings (cobalt, zinc, copper, and cobalt-containing alloys skin flora, the flora on the hands of medical and nursing
of nickel, zinc, chromium, etc.) to inhibit the growth of staff, and contaminated infusion fluids.
pathogenic bacteria was examined [8]. Among the organisms
It has been demonstrated that sheets in direct contact with
used were gram-positive bacteria (Enterococcus faecalis and a patient’s skin and a patient’s bacterial flora are an
methicillin-resistant S. aureus) and gram-negative bacteria
important source of infection [22,23]. Moreover, sheets were
( E. coli, Pseudomonas aeruginosa and K l e b s i e l l a
significantly more contaminated by patients carrying
pneumoniae). The amounts of H2O2 produced and metal ions
infection than by non-infected patients [22]. Therefore, use
dissolved from the surfaces of the various electroplated
of self-sterilizing fabrics in pajamas, sheets, pillow covers,
coatings were measured, and it was found that the inhibitory
and robes in a hospital setting may reduce the spread of
ability of the coatings correlated with the amount of H2O2 microorganisms in hospital wards, which could result in a
produced. The bacterial survival rates on the surfaces of the
reduction of nosocomial infections.
coatings were almost zero when H2O2 was produced in
amounts greater than 10-6 mmol/cm2. It was also noted that An additional potential use of copper-impregnated
the concentration of metal ions dissolved from coatings were fabrics is related to foot ulcerations, a common complication
outside of the bacterial lethal range. In another study, it was of type 1 and 2 diabetes, which afflicts approximately 130
shown that although H2O2 is toxic to bacteria in metal ion- million individuals around the world [24]. In many cases,
depleted media, the lethal H2O2 dose level was sharply these ulcerations can become highly infected due to
reduced if copper salts and reductants were added [9]. cuts/bruises that do not heal, or heal very slowly. Infections
Similarly, it was found that the antibacterial potency of that do not heal have been known to cause the tissue to die
several compounds is significantly higher when they are (gangrene). In severe cases, toes and legs may have to be
complexed with copper [10,11]. Likewise, copper phosphate amputated in order to save remaining healthy body parts.
cement used as a restorative material for treatment of caries Use of socks containing copper-impregnated fibres by
demonstrated the greatest in vitro and in vivo antibacterial diabetics may significantly reduce the risk of foot infection
activity, among several restorative materials tested [12,13]. by rendering the area aseptic. A preliminary clinical trial
Addition of activated copper significantly improved the already indicates that such is the case (Dr. Richard Zatcoff,
antibacterial properties of calcium hydroxide used to kill Upstate Podiatry, 110A Hospital Drive, Simpsonville, SC,
bacteria in dentinal tubules [14]. USA, unpublished data).
The bacteriostatic effectiveness of copper used in paints 1.2. Copper as a Water Purifier
to render surfaces self-disinfecting has also been
Recognition of the bacteriostatic properties of copper has
demonstrated [15]. Nearly all of the copper paints tried were
led to testing its capacity as a water purifier. Copper was
found to reduce the number of tested organisms (S. aureus,
found to be one of the most toxic metals to heterotrophic
E. coli, P. aeruginosa, and E. faecalis) to negligible levels
bacteria in aquatic environments. Albright and Wilson [25]
within 24 hours of exposure. This led to the use of copper in
paints to reduce fouling and microbial biofilm formation in found that sensitivity to heavy metals of microflora in water
was (in order of decreasing sensitivity): Ag, Cu, Ni, Ba, Cr,
ships [16]. Fouling, the growth of barnacles, seaweed,
Hg, Zn, Na, Cd. Sagripanti et al. [26] found that cupric
tubeworms, and other organisms on boat bottoms, produces
chloride inactivated 9 of the 13 bacteria strains that they
surface roughness that increases turbulent flow, acoustic
tested by more than 5 logs within 30 minutes. The other four
noise, drag, and fuel consumption. An average increase of 10
strains were inactivated to a lesser extent.
m in hull roughness can result in a 1% increase in fuel
consumption! In another study [27], water with a suspension of E. coli
was introduced into 50 foot coils of different plumbing
Recently, the use of copper as a bacterial inhibitor in
materials and changes in bacteria viability were periodically
various stages of food processing has been demonstrated. It
determined. While in different types of plumbing material,
was shown that metallic copper surfaces inhibited the growth
including glass, the level of bacteria remained the same or
of two of the more prevalent bacterial pathogens that cause
foodborne diseases, Salmonella enterica and Campylobacter even increased, in the copper loop only 1% of the E. coli
bacteria remained viable after five hours.
jejuni [17]. Similarly, the addition of copper to drinking
glasses has been shown to reduce biofilm formation of Similarly, it was found that water distribution systems
Streptococcus sanguis, reducing the risk of oral infections made of copper have a greater potential for suppressing
[18]. growth and for decreasing persistance of Legionella
A platform technology has recently been developed, pneumophila cells in potable water than distribution systems
constructed of plastic materials or galvanized steel [27]. The
which binds copper to textile fibres from which woven and
use of a continuous culture model system for the growth of
non-woven fabrics can be produced [19]. The ability to
L. pneumophila on copper and other plumbing materials was
introduce copper into textile fabrics may have significant
also examined. It was found that bacteria levels were
ramifications. Copper containing textiles may possibly be
reduced on copper surfaces compared with a glass control
used to reduce healthcare-associated (nosocomial) infections
in hospitals. Nosocomial infections rank fourth among and other plumbing materials at the various temperatures
tested and in the three different waters used [28].
causes of death in the United States, behind heart disease,
cancer and stroke [20]. Nosocomial infections can be of A controlled evaluation was also made of the efficacy of
bacterial, viral, fungal, or even parasitic origin. These copper-silver ionization in eradicating L. pneumophila from
infections are largely device-associated or surgically-related a hospital water supply. Copper-silver ionization units were
Copper as a Biocidal Tool Current Medicinal Chemistry, 2005, Vol. 12, No. 18 2165

installed on the hot water recirculation line of a building From then onwards copper fungicides have become
positive for Legionella. Another building with the same indispensable and many thousands of tons are used annually
water supply served as a control. Legionella species all over the world to prevent plant diseases.
persisted within the system when copper and silver
The discovery that many algae are highly susceptible to
concentrations were < 0.3 and < 0.03 ppm, respectively. copper sulphate led to its use by water engineers to prevent
When copper and silver concentrations were > 0.4 and >
the growth of algae in potable water reservoirs. Copper
0.04 ppm, respectively, there was a significant decrease in
sulphate is also employed to control green slime and similar
Legionella colonization, but the percentage of water fixtures
algal scums in farm ponds, rice fields, irrigation and
testing positive for organisms remained unchanged in the
drainage canals, rivers, lakes and swimming pools. However,
control building. When the ionization unit was inactivated,
copper sulphate may be very toxic to fish. In addition, the
water fixtures continued to be free of Legionella for two sulphate ions tend to combine with hydrogen in an aqueous
additional months [29]. Similar results were obtained in
solution to form sulphuric acid, which is highly corrosive.
other studies [30-33]. According to a report published in
1998, more than 30 hospitals in the USA are now using The environmental hazards resulting from copper build-
copper-silver ionization to control Legionella in their water up in sediments, and the need for high dosages, have led to
distribution systems [34]. the production of compounds that provide the copper in a
chelated form [37-39]. Chelated copper is non-reactive with
The efficacy of a 1:10 silver/copper combination for in
other chemical constituents in the water.
vitro inactivation of Hartmannella vermiformis amoebas and
the ciliated protozoan Tetrahymena pyriformis was also However, the use of copper sulphate for algae control is
studied [35]. Tetrahymena and Hartmannella were still very common [40], primarily because of its low cost and
inactivated by 2 log steps using a combination of 100 + 1000 ease of application. Copper sulphate is also widely used to
g/L Ag + Cu. The investigation clearly showed that levels inhibit timber and fabric decay by rendering them
within the limit of German drinking water regulations (10 + unpalatable to insects and protecting them from fungus
100 g/L Ag + Cu) could not inactivate these protozoa in attack. Copper sulphate has been in use since 1838 for
vitro. It was further demonstrated that in the case of preserving timber and is currently the base for many
Naegleria fowleria, the organism responsible for primary proprietary wood preservatives. Copper is also used as the
amoebic meningoencephalitis, a combination of silver and active ingredient in products that prevent roof moss
copper ions (80 and 800 ug/L, respectively) was ineffective formation [41].
in inactivating the amoebae. However, addition of 1.0 mg/L Similarly to copper sulphate, copper-8-quinolinolate and
free chlorine produced a synergistic effect, with superior some of its derivates have been shown to have fungicidal
inactivation relative to either chlorine or silver-copper used activity against Aspergillus spp. at concentrations above 0.4
alone. A similar synergy was reported for Staphylococcus sp. g/ml [42-45]. Since infection with this fungus is a major
and P. aeruginosa [36]. problem among immunocompromised patients, such as
1.3. Copper as an Algicide, Fungicide, Molluscicide and AIDS patients, this agent has been used to reduce
Acaricide environmental contamination of fungi in hospitals [46].
Copper compounds are most extensively employed in Use of copper by the wider population may also be
agriculture. The first recorded use of copper in agriculture beneficial for more benign conditions. About 15-20% of the
was in 1761, when it was discovered that seed grains soaked population suffers from tinea pedis [47,48]. While there are
in a weak solution of copper sulphate inhibited seed-borne many clinical presentations of tinea pedis, the most common
fungi. Within a few decades, the practice of treating seed is between the toes and on the soles, heels and sides of the
grains with copper sulphate had become so general and foot. Although this fungal infection is not usually dangerous,
effective that today infections of seeds with fungi are rare it can cause discomfort, may be resistant to treatment, and
and are no longer of any economic importance. may spread to other parts of the body or other people.
Affected feet can also become secondarily infected by
The greatest breakthrough for copper salts as fungicides bacteria. Recently, it has been found that copper-
undoubtedly came in the 1880's with the development of a impregnated socks may be useful in preventing and treating
lime-copper formulation by the French scientist Millardet. tinea pedis (Ref. [19] and Dr. Richard Zatcoff, unpublished
He showed that spraying grapes and vines with a mixture of results).
copper sulphate, lime and water renders them remarkably
free of downy mildew. By 1885, his vintner’s spray Another possible use of copper in fabrics is related to
formulation was the fungicide of choice in the U.S. and was allergies and asthma. It is estimated that 15% of the general
given the name of "Bordeaux mixture." Within a year or two population suffer from one or more allergic disorders of
of the discovery of Bordeaux mixture, Burgundy mixture, which allergic rhinitis is the most common [49]. Allergic
which derives its name from the district of France in which it rhinitis affects an estimated 20 to 40 million people in the
was first used, appeared on the scene. Burgundy mixture is US alone. Similarly, nearly 15 million Americans have
prepared from copper sulphate and sodium carbonate and is asthma, including almost 5 million children. Approximately
analogous to Bordeaux mixture. 5,500 persons die each year from asthma [50]. Dust mites are
considered to be an important source of allergen for
Trials with Bordeaux and Burgundy mixtures against perennial rhinitis and asthmatic attacks [51], and copper
various fungus diseases of plants soon established that many impregnated fabrics have been shown to destroy them [19].
plant diseases could be prevented with small amounts of Thus, elimination of house dust mites in mattresses, quilts,
copper applied at the right time and in the correct manner.
2166 Current Medicinal Chemistry, 2005, Vol. 12, No. 18 Borkow and Gabbay

carpets and pillows would be an important step in improving use of copper in free flow filters that deactivate HIV-1 and
the quality of life of those suffering from dust-mite related West Nile Virus has recently been reported. The copper
allergies [19]. filters reduced the infectious titers of both viruses by 5 to 6
logs [19].
Copper sulphate has been found to be a potent
molluscicide [52]. Control of snails may be an important Wong et al. [61] reported a 106-fold reduction in
strategy in fighting some human diseases, such as bacteriophage R17 infectivity due to RNA degradation in the
bilharziasis. This disease is caused by a trematode parasite, presence of both ascorbate and Cu2+. A study published in
Schistosoma mansoni, which uses snails and humans as 2001 reported the inactivation of poliovirus and
hosts. The International Copper Research Association bacteriophage MS-2 in copper pipes containing tap water as
screened 23 copper compounds, in addition to copper a result of a synergistic effect between copper and free
sulphate, for their effectiveness in killing snails in water of chlorine [62]. It was found that the log reduction/hour of the
low- and high-alkalinity, with and without high levels of bacteriophage MS-2 in the presence of 400 g/L of leached
suspended solids. One compound, cupric chloride-bis-n- copper was 0.385, in 20 mg/L free chlorine 7.605 and with
dodecylamine, was considerably superior to copper sulphate both copper and chlorine, 10.906. This suggests that an
under all test conditions. A bivalent, organic complexed, oxidizing agent such as chlorine or hydrogen peroxide is
copper nitrate was marginally superior to copper sulphate necessary to break open the virus protein coat and allow the
[52]. copper to bind to and denature the nucleic acid.
1.4. Copper Antiviral Activity The International Copper Association, Ltd. investigated
the effects of copper on the survival of waterborne viruses
In 1964, Yamamoto and colleagues [53] reported the [63]. It found that poliovirus was completely inactivated by
inactivation of bacteriophages by copper. In 1971, Jordan copper sulphate (20 mg/L) in the presence of hydrogen
and Nassar [54] showed that copper inactivated infectious peroxide (10 M), confirming observations of a synergistic
bronchitis virus. In 1974, Totsuka and Ohtaki [55] showed effect of copper ions in the presence of oxidizing agents. The
that the effect of copper sulphate on poliovirus RNA is effect was reduced by the presence of a protective agent, L-
proportional to its concentration, and that most amino acids histidine. Other proposed protective agents, disodium
except cysteine had a protective effect against copper as did hydrogen orthophosphate, bovine serum albumin and
Fe2+ and Al3+. In 1974, Coleman and colleagues [56] catalase, were found to be relatively ineffective. Similarly,
reported that herpes simplex virus (HSV) type I was the Association found that copper reduced coxsackie virus
sensitive to silver. In 1992, Sagripanti and colleagues [57,58] types B2 & B4, echovirus 4 and simian rotavirus SA11
found that cupric and ferric ions were by themselves able to infectivity by over 98%. It concluded that there does not
inactivate five enveloped or nonenveloped, single- or appear to be any significant difference between the capacity
double-stranded DNA or RNA viruses (phi X174, T7, phi 6, of copper to inhibit the different types of virus tested. The
Junin, and HSV). The metals were even more effective than polio, coxsackie and echo viruses may be expected to behave
glutaraldehyde in inactivating the viruses. The metal similarly as they are of a similar size and are all members of
virucidal effect was enhanced by the addition of peroxide, the enterovirus group. However, the rotaviruses are
particularly for Cu2+. In every case, the viruses were more considerably larger (75 nm diameter as opposed to 28 nm)
resistant to iron-peroxidase than copper-peroxidase on a and belong to the reovirus group, a completely different
metal concentration basis. The inactivation of HSV by family of viruses. This suggests that whatever the
copper was enhanced by reducing agents. mechanism removing or inactivating the viruses, it is not
The protective effects of metal chelating agents and dependent on subtle properties associated with the surface of
catalase, the lack of effect of superoxide dismutase, and the the viruses.
partial protection conferred by free-radical scavengers In another study, the efficacy of copper and silver ions,
suggest that the mechanism of copper-mediated HSV generated electrolytically until reaching final concentrations
inactivation is similar to that reported for copper-mediated of 700 g copper and 70 g of silver per liter, in
DNA damage [59]. combination with low levels of free chlorine, was evaluated
Sagripanti and Lightfoote [60] reported that Human for the disinfection of hepatitis A virus, human rotavirus,
Immunodeficiency Virus Type 1 (HIV-1) was inactivated by human adenovirus, and poliovirus in water [64]. There was
either cupric or ferric ions when the virus was free in little inactivation of hepatitis A virus and human rotavirus
solution and also three hours after cell infection. Fifty under all conditions. Poliovirus showed more than a 4 log
percent inactivation of cell-free HIV-1 was achieved with titer reduction in the presence of copper and silver combined
Cu2+ at concentrations between 0.16 and 1.6 mM, or by Fe3+ with 0.5 mg of free chlorine per liter or in the presence of 1
at concentrations between 1.8 to 18 mM. Thus, the dose to mg of free chlorine per liter alone. Human adenovirus
inactivate 50% of infectious HIV-1 (IC50) by Cu2+ or Fe3+ is remained active longer than poliovirus after undergoing the
higher than that reported for glutaraldehyde (0.1 mM), for same treatment, although it remained active significantly less
sodium hypochlorite (1.3 mM), and for sodium hydroxide than hepatitis A virus and human rotavirus. The addition of
(11.5 mM). It is, however, significantly lower than that 700 g of copper and 70 g of silver per liter did not
required for HIV-1 inactivation by ethanol (360 mM). enhance the inactivation rates after exposure to 0.5 or 0.2 mg
Treatment of infected cells for 30 minutes at 20°C with 6 of free chlorine per liter, although on some occasions it
mM Cu2+ or Fe3+ completely inhibited the formation of produced a level of inactivation similar to that induced by a
syncytia and the synthesis of virus-specific p24 antigen in higher dose of free chlorine alone. These data indicate that
HIV-infected cells, while still preserving cell viability. The copper and silver ions alone in water systems may not
Copper as a Biocidal Tool Current Medicinal Chemistry, 2005, Vol. 12, No. 18 2167

Fig. (1). Mechanisms of Toxicity of Copper to Microorganisms.

provide a reliable alternative to high levels of free chlorine manifests itself as leakage of mobile cellular solutes, such as
for the disinfection of viral pathogens. It should be pointed potassium ions, and cell death. For example, exposure of
out that the USA Environmental Protection Agency (EPA) intact Saccharomyces cerevisiae to Cu2+ (100 M CuCl2 in a
has determined that drinking water should not contain more buffer of low ionic strength) caused a loss of the
than 1.3 mg copper per liter of water (1.3 mg/L) [3]. permeability barrier of the plasma membrane within two
minutes at 25ºC. The release of amino acids was partial, and
2. MECHANISMS OF TOXICITY OF COPPER TO the composition of the released amino acids was different
MICROORGANISMS
from the composition remaining in the cells. Primarily
Metals at high concentrations are toxic to glutamate was released, while arginine was retained in the
microorganisms. Toxicity occurs through several cells. Cellular K+ was released rapidly after the addition of
mechanisms (Fig. 1), such as displacement of essential CuCl2, but 30% of the total K+ was retained in the cells.
metals from their native binding sites or through ligand These and other observations suggest that Cu2+ caused
interactions. In general, nonessential metals bind with selective lesions in the permeability barrier of the plasma
greater affinity to thiol-containing groups and oxygen sites membrane but did not affect the permeability of the vacuolar
than essential metals. Toxicity also results from changes in membrane. These selective changes were not induced by the
the conformational structure of nucleic acids and proteins other divalent cations tested [66].
and from interference with oxidative phosphorylation and Similar effects reported in higher organisms have now
osmotic balance. The redox properties that make some been largely attributed to the redox-active nature of copper
metals, such as copper, essential elements in biological and the ability of copper to catalyze the generation of free
systems, may also contribute to their inherent toxicity. For radicals that promote membrane lipid peroxidation [67-70].
example, as shown in Fig. (1), redox cycling between Cu2+ For example, Cu2+ uniquely catalyzed peroxidation of rat
and Cu1+ can catalyze the production of highly reactive erythrocyte membrane lipid in the presence of 10 mM H2O2,
hydroxyl radicals, which can subsequently damage lipids, while several other transition metal ions had no significant
proteins, DNA and other biomolecules. effect [70]. Thus, a copper-oxygen complex may be directly
2.1. Copper Mediated Cell Membrane Damage involved in the initiation of lipid peroxidation.
Copper’s initial site of action is thought to be at the Extensive metal-induced disruption of membrane
plasma membrane [65-67]. It has been shown that exposure integrity inevitably leads to loss of cell viability. However,
of fungi and yeast to elevated copper concentrations can lead even relatively small alterations in the physical properties of
to a rapid decline in membrane integrity. This generally biological membranes can elicit marked changes in the
activities of many essential membrane-dependent functions,
2168 Current Medicinal Chemistry, 2005, Vol. 12, No. 18 Borkow and Gabbay

including transport protein activity [71], phagocytosis [72], [89]. The binding of copper to DNA shows an unexpected
and ion permeability [71]. The physical properties of a high specificity when studied in the presence of other
membrane are largely determined by its lipid composition, metallic ions. The relative efficacy of several different
one of whose important factors is the degree of fatty acid divalent cations to antagonize Cu2+ binding was: Ni = Cd =
unsaturation. Microbial membrane fatty acid composition is Mg >>> Zn = Hg > Ca > Pb >>> Mn, while Cr6+ enhanced
highly variable and is influenced by both environmental and Cu2+ binding to DNA [89].
intrinsic factors. For example, the unsaturated fatty acid
Guanine-specific binding of Cu2+ in dsDNA was
content of microorganisms generally increases at low
demonstrated following crystallization (1.2-A resolution) of
temperatures [71,73]. In addition, some variation can be
DNA soaked with cupric chloride [90,91]. Covalent Cu2+
attributed to inherent differences in fatty acid composition
bonded to guanine occurred at the N-7 position. This
between microbial groups [74]. The relationship between preferential Cu2+ binding to guanine bases in dsDNA may
plasma membrane fatty acid composition and copper toxicity
explain the observed specificity of Cu2+-induced oxidative
was studied in S. cerevisiae. It was found that copper-
DNA damage near guanine residues [92,93]. Hay and Morris
induced plasma membrane permeabilization and whole-cell
[94] proposed that copper may stabilize the helix via a
toxicity increased markedly in cells enriched with
charge transfer complex formed when copper, acting as an
polyunsaturated fatty acids [75].
electron acceptor, intercalates between two adjacent G-C
In another study [9], the bactericidal potency of copper pairs which act as electron donors. Indeed, copper has been
towards several bacteria (Streptococcus lactis, E. coli and P. shown to bind preferentially to G-C pairs [94].
aeruginosa) with different cell envelope structures was
DNA strand breakage is proportional to incubation time,
found to be similar. The authors suggested that the Cu2+ ion-
temperature, and Cu2+ and H2O2 concentrations [92]. DNA
mediated killing appears to be related to the bacterial plasma
strand breakage is inhibited by metal chelators, catalase, and
membrane. Their conclusion was based upon the observation by high levels of free radical scavengers implying that Cu2+,
that the loss of metabolic functions localized at the
Cu1+, H2O2, and OH radicals are involved in the reaction.
membranes paralleled cellular death, while oxidation of
This, together with the specificity of copper binding to DNA
susceptible biomolecules within the cytosol required
(see above), has been interpreted to mean that nucleic acid
considerably more extensive oxidative degradation of the
degradation mediated by copper involves site-specific
cells.
Fenton reactions [95-97]. Thus, subsequent to the specific
Many antibacterial and antifungal compounds are more binding of copper to nucleic acids, repeated cyclic redox
active as copper salts [43,45,76-84]. It has been suggested reactions generate several OH radicals near the binding site
that some compounds, such as diethyldithiocarbamic acid causing multiple damage to the nucleic acids [67,98].
(DDC), which form chelates with copper and whose
Cu2+ may also generate radicals and co-ordinate with
microbiocidal effectiveness is greatly enhanced by small
other toxic molecules such as adriamycin, hydroquinone, and
amounts of copper, are cytocidal by virtue of concentrating reduced mitomycin C [99-102]. For example, the addition of
in the lipid bilayer and, perhaps, by forming amphipathic
cupric acetate with adriamycin in the Ames salmonella
complexes which disrupt membrane integrity [84].
mutagenicity test increases the mutagenicity of adriamycin
2.2. Copper Interaction With Nucleic Acids by more than 700% [99]. This supports the contention that
drug-metal ion-DNA associations might contribute to
The study of radiation effects on diverse biological genotoxicity.
processes has led to the finding that microwave radiation at
nonthermal levels produces single- and double-strand breaks Glutathione, a chelating agent, was shown to inhibit free
in purified DNA. Interestingly, however, it was found that radical formation by copper ions in the presence of hydrogen
this microwave-induced damage to DNA depends on the peroxide, ascorbate and DNA [103]. The protective effect of
presence of small amounts of copper. Moreover, it was glutathione was attributed to its ability to stabilize copper in
found that cuprous, but not cupric, ions were able to mimic the Cu1+ oxidation state, preventing redox cycling and
the effects produced by microwaves on DNA [85]. This generation of free radicals.
finding has led to more detailed investigations into the in It has been shown that the DNA damage, which is the
vitro effects of copper on DNA [88]. Today it is clear that main cause of cell death in E. coli cultures treated with H2O2,
Cu2+ has a specific affinity for DNA and can bind and occurs through iron-mediated Fenton reactions [104,105].
disorder helical structures by crosslinking within and However, under conditions of low iron availability, copper
between strands [86,87]. Cu2+ reversibly denatures DNA in ions take part in the genotoxicity of H2O2 in E. coli [106].
low ionic strength solutions competing with hydrogen This phenomenon only occurs at high concentrations of
bonding present within the DNA molecule [88]. Kinetic H2O2 (20 mM), suggesting that copper ions only become
studies showed that the DNA double helix contains at least available in the intracellular environment in the absence of
two kinds of binding sites for copper [89]. One site is present iron and under severe oxidative stress [106].
in every four nucleotides and has high affinity for copper.
The other is an intercalating site for copper that is present in DNA damage induced by many carcinogens is dependant
every base pair. This site is saturable and has a dissociation on the presence of Cu2+ but not on the presence of other
constant (Kd) for Cu2+ of 41 M. In single-stranded DNA metals. The main modifications in the DNA occur at guanine
(ssDNA), such as that found in many DNA viruses, a copper and thymine residues, resulting mainly in the formation of 8-
binding site was found on average in every three nucleotides hydroxy-2'-deoxyguanosine and piperidine-labile sites at
with lower affinity than in double stranded DNA (dsDNA)
Copper as a Biocidal Tool Current Medicinal Chemistry, 2005, Vol. 12, No. 18 2169

thymine residues. There is a clear association between these phosphatase that in conjunction with protein tyrosine kinases
modifications and DNA strand breakage [107]. regulates cell growth and differentiation. Among the various
metal ions (Fe3+, Cu2+, Zn2+, and Cd2+) examined for their
It has been shown that in the cases of most carcinogens,
inactivation effect on VHR, Cu2+ was found to be the most
copper-induced DNA damage occurs through the formation
of H2O2 by the carcinogens [108-113], and that the reactive potent inactivator. The efficacy of Cu2+ as VHR inactivator
was about 200-fold greater than that of H2O2. Cu2+ also
species generated by the reaction of H2O2 with Cu1+
inactivated other protein tyrosine phosphatases, including
participates in the DNA damage. In some cases the DNA
PTP1B and SHP-1. The Cu2+ -mediated inactivation was a
damage is greatly enhanced by the reduction of the oxidized
consequence of the oxidation of a cysteine residue at the
product through the addition of beta-nicotinamide adenine
active site [123].
dinucleotide (NADH) [114-117]. These results suggest that
an intermediate, derived from the reaction of Cu1+ with H2O2, The reaction of a histidine-containing peptide
participates in Cu2+-dependent DNA damage, and that (angiotensin I) with Cu2+/ascorbate under physiological
NADH enhances the DNA damage via a redox cycle. conditions has been studied [124]. In the presence of a
catalytic amount of Cu2+, ascorbate mediated the oxidative
A similar conclusion was reached by studying the
damage to the peptide via selective loss of the histidine
mechanism of carcinogenesis caused by methylhydrazines
[118]. Methylhydrazines in the presence of Cu2+ induce residue. It was shown that the reaction of Cu2+/ascorbate
occurs specifically at the C-2 position of the imidazole ring
DNA cleavage at thymine residues only. The order of Cu2+-
of the histidine residue within a peptide. In addition, the
mediated DNA damage was not correlated with the order of
capacity of copper to mediate free radical attack of proteins
methyl free radical (CH3) generation during Cu2+-catalyzed
was investigated using proteins biosynthetically labeled with
auto-oxidation. It was therefore suggested that the Cu1+-
radioactive proline or histidine [125]. It was found that
peroxide complex rather than the CH3 plays a more
important role in methylhydrazine plus Cu2+-induced DNA protein-bound histidine was substantially converted into
aspartate, and while much of the proline was modified
damage. Mutations in DNA after exposure to copper ions
during radical attack, it was not converted into glutamate.
were found to be clustered and predominantly single-base
Thus, histidine and proline are important sites of protein
substitutions [119]. Electron spin resonance spin-trapping
attack mediated by copper radical formation. Such protein
investigations demonstrated that hydrogen peroxide interacts
modification may result in cleavage. For example, a facile
with Cu1+ to generate the reactive species responsible for
DNA damage, which is either the hydroxyl radical or a cleavage of peptide bonds of apolipoprotein B (apoB) by
radical reaction was found when human low-density
species of similar reactivity [120].
lipoprotein (LDL) cholesterol was subjected to oxidative
An important host defense mechanism for dealing with damage using Cu2+ [126]. When human serum was treated
invading bacteria involves the production of reactive oxygen with Cu2+, a similar cleavage pattern of apoB was also
species, such as superoxide, hydrogen peroxide and observed [126]. Similar selective attack at particular residues
hypochlorous acid, by phagocytic cells. Several antibacterial due to oxidative damage using Cu2+ was shown with bovine
antibiotics have been shown to participate with transition serum albumin (BSA), histones, cytochrome C, lysozyme
metal ions in chemical reactions leading to the formation of and protamine [127].
reactive oxygen species. The production of reactive oxygen
It was suggested in a study of the biochemistry of copper
by redox cycling antibacterial antibiotics has led some
[128] that the biocidal effects of copper are achieved mainly
investigators to suggest that a 'phagomimetic' contribution
may also be made in vivo. Indeed, they showed that adding by the interaction of Cu2+ with SH- moieties present in the
cell membrane and within cells. This interaction results in
copper salts to four structurally different antibacterial
the formation of thiol compounds and Cu1+ ions. The
antibiotics (beta-lactam, tetracycline, bacitracin and
following redox reaction
rifamycin) resulted in oxidative modification to bases in the
bacterial DNA and enhanced antibacterial potency [121]. 2Cu2+ + 6GSH 2Cu1(GS)2 + GSSG ; G= glutathione
Epinephrine, norepinephrine or dopamine in the presence was suggested to occur when an excess of Cu2+ ions interact
of non-lethal concentrations of Cu2+ kill washed or growing with living cells. This interaction leads to inactivation of 3
E. coli cells. This effect is enhanced by anoxia and by SH-groups per copper ion. This assumption was based on an
sublethal concentrations of H 2O2. The rate of cell mortality equilibrium analysis of the Cu1+ - glutathione and Cu1+ -
was proportional to the rate of catecholamine oxidation. The pencicillamine systems, in which it was found that reduced
copper epinephrine complex was found to bind to E. coli glutathione, which is present in all cells and cell membranes
cells, to induce membrane damage and to deplete the cellular in concentrations from 1 to 6 mM, decreased markedly in the
ATP pool. The cells were partially protected by superoxide presence of copper.
dismutases (SOD) or catalase but not by OH radical
scavengers. The addition of H2O2 to cells, which were 2.4. Mechanisms of Copper Antiviral, Antifungal and
Anti-Algae Activities
sensitized by preincubation with the epinephrine-copper
complex, caused rapid killing and DNA degradation [122]. All of the above copper mediated toxic mechanisms,
elucidated mainly in studies with bacteria, are relevant to
2.3. Copper Mediated Protein Damage
viruses as well. For example, viral inactivation via RNA
Copper can alter proteins and inhibit their biological damage, through the Fenton mechanism, was proposed by
activities. For example, copper was the most potent metal Carubelli and colleagues [129]. They found that incubation
inhibiting the protein tyrosine phosphatase VHR [123], a of the RNA phage Q beta at 37ûC with a mixture of 100 mM
2170 Current Medicinal Chemistry, 2005, Vol. 12, No. 18 Borkow and Gabbay

ribose and 10 M CuSO4 resulted in a complete loss of susceptibility to lead and potassium are highly sensitive to
viable phage after 20 minutes. This cytotoxic effect required silver and copper [141].
both ribose and cupric ions. There was a direct correlation
The mechanisms of tolerance include: exclusion by
between the decrease in phage survival and (a) the length of
permeability barriers, intra- and extra-cellular sequestration
incubation, and (b) the concentrations of both ribose and by cell envelopes, active transport membrane efflux pumps,
CuSO4. The addition of the strong chelator
enzymatic detoxification, and reduction in the sensitivity of
diethylenetriaminepentaacetic acid eliminated the cytotoxic
cellular targets to metal ions (Fig. 2). Genes responsible for
effect. These results are consistent with an initial production
these processes may be encoded by the chromosomes or by
of superoxide free radicals by transition metal catalyzed
plasmids. Since some toxic metals are also essential
autoxidation of ribose and Amadori products (e.g. 1-amino-
micronutrients (i.e. copper, cobalt, zinc, nickel), bacteria
-deoxyketose), followed by dismutation of the superoxide must precisely adjust their uptake and efflux systems to
radicals to hydrogen peroxide and generation of lethal
maintain adequate intracellular levels of these metals. In the
hydroxyl radicals by the Fenton reaction. RNA isolated from
case of metals with no biological function (i.e. cadmium,
phage incubated with ribose and CuSO4 retained its
silver), transport systems must be oriented only to the
infectivity, suggesting that the cytotoxic effect may be
expulsion of the toxic ions. In the last few years, several
mediated by a free radical attack on proteinaceous
bacterial systems dedicated to toxic metal efflux have been
components of the phage through site-specific generation of analyzed at the molecular level resulting in a detailed
hydroxyl radicals on protein-bound transition metal ions.
understanding of the mechanisms. Among these are the
HIV-1 protease, an essential protein for the replication of membrane pathways that expel copper, cadmium, zinc,
the virus, was found to be inhibited by approximately nickel, cobalt and silver cations. Two general mechanisms
stoichiometric concentrations of copper ions. Inactivation by have been found: those involving P-type ATPases, and those
Cu2+ is rapid and is not reversed by subsequent exposure to using proton antiporter systems [135,140].
EDTA or dithiothreitol. The addition of copper to the
Natural resistance may take the form of mutations in
protease at pH 5.5 induces aggregation of the protein,
cellular components that prevent interaction with metals or
providing a possible basis for the inhibitory action of copper.
alterations in cell membrane composition. Microorganisms
Direct inhibition by Cu2+ required the presence of cysteine
can possess one or a combination of several resistance
residue(s) in the protease. Oxygen is not required for
mechanisms. There are six known resistance mechanisms to
inactivation [130,131]. heavy metals in bacteria (see below). Each mechanism is
In contrast to the different mechanisms of resistance to discussed below, with emphasis on resistance to copper, and
copper and other heavy metals found in bacteria, fungi and examples are provided of microorganisms that display the
other microorganisms (see Section 3 below), viruses do not characteristics of each resistance mechanism.
possess resistance or repair mechanisms, making them
highly susceptible to high concentrations of copper ions. 3.2. Metal Exclusion by Permeability Barrier

The mechanism of copper antifungal and anti-algae Alterations in the cell wall, membrane, or envelope of a
activity has not been well studied. It has been suggested that microorganism are examples of metal exclusion by a
the copper ions form electrostatic bonds with negatively permeability barrier. This mechanism is an attempt by the
charged regions on the microorganism’s cell walls. These organism to protect metal-sensitive cellular components. A
electrostatic bonds create stresses which lead to distorted cell prominent example is the exclusion of Cu2+ resulting from
walls and increased permeability, while also reducing the altered production by E. coli B of the membrane channel
normal intake of life sustaining nutrients. Once inside an protein porin [142]. This is usually a result of a single
algae cell, copper may attack the sulphur groups in amino mutation in the gene that regulates the permeability of the
acids contained in the proteins used for photosynthesis. As a membrane to metal ions.
result, photosynthesis is blocked and cell lysis and death Bacteria that naturally form an extracellular
occurs [132]. polysaccharide coating demonstrate the ability to bioabsorb
metal ions and prevent them from interacting with vital
3. MICROBIAL RESISTANCE TO COPPER
cellular components. For example, a layer of
3.1. General exopolysaccharide improves the survivability of Klebsiella
aerogenes strains in Cd2+ solutions [143]. Nonspecific
Many microorganisms demonstrate resistance to metals
binding of metals by the outer membrane or envelope, such
in water, soil and industrial waste. Metal resistance systems
as in Klebsiella aerogenes, Pseudomonas putida, and
are present in nearly all bacterial types and may have
Arthrobacter viscosus, however, offers limited metal
developed shortly after prokaryotic life started. Several protection due to the possibility of the saturation of binding
factors determine the extent of metal resistance in a
sites [144,145].
microorganism. These include the type and number of
mechanisms for metal uptake, the role each metal plays in Although not fully proven, it is believed that some forms
normal metabolism, and the presence of genes located on of copper resistance are based on periplasmic binding
plasmids, chromosomes, or transposons that control metal [146,147]. Periplasmic binding of Cu2+ is thought to be
resistance [133-140]. Microorganisms may be resistant to found in Pseudomonas species where resistance is coded for
one metal and not to another. For example, clinical in a plasmid by an operon of four genes: copA, copB, copC,
staphylococcal isolates that demonstrate reduced and copD [148-150]. CopA and copB confer partial
resistance, while the addition of copC and copD provides for
Copper as a Biocidal Tool Current Medicinal Chemistry, 2005, Vol. 12, No. 18 2171

Fig. (2). Mechanisms of Microbial Resistance to Heavy Metals.

full Cu2+ resistance [146]. CopA and copC proteins are nomenclature similar to that of the cop sequestration operon
located between the inner and outer membrane, while copB found in Pseudomonas discussed above. CopA is responsible
is found in the outer membrane and copD in the inner for encoding a Cu2+ uptake ATPase and copB encodes a P-
membrane. CopC can bind Cu1+ and Cu2+ at two different type efflux ATPase. The gene products of copY and copZ
sites, occupied either one at a time or simultaneously. The regulate the cop operon. CopY is believed to be a repressor
two sites are approximately 30 A apart and have little protein that inactivates the operon in the absence of Cu2+.
affinity for the ion in the other oxidation state. Oxidation of When Cu2+ is present it may bind to copY, converting it to a
Cu1+-CopC or reduction of Cu2+-CopC causes migration of DNA-binding repressor. Copper also binds to copZ at higher
copper from one site to the other [149]. In addition, levels and together they form a copY-Cu complex that ceases
periplasmic proteins (e.g. PcoC) have been thought to bind repression. This allows copA and copB to be transcribed
copper ions and to increase the level of resistance to copper [154-157].
ions above that conferred by cop gene products [151-153].
Recently, data has accumulated showing that E. coli
3.3. Active Transport of the Metal Away from the encodes four proteins (CusC/F/B/A) that mediate resistance
Microorganism to copper and silver by cation efflux. All four proteins form a
tetrapartite resistance system, which involves a novel
Active transport or efflux systems represent the largest periplasmic copper-binding protein CusF that directly
category of metal resistance systems. Microorganisms use transports Cu1+ from the periplasm across the outer
active transport mechanisms to export toxic metals from membrane [158,159]. Data supporting a copper efflux
their cytoplasm. These mechanisms can be chromosomal or system in S. enterica serovar Typhimurium has also been
plasmid-encoded. Non-essential metals normally enter the reported [160].
cell through normal nutrient transport systems but are rapidly
exported. These efflux systems can be non-ATPase or 3.4. Intracellular Sequestration of Metals by Protein
ATPase-linked and highly specific for the cation or anion Binding
they export.
Intracellular sequestration is the accumulation of metals
Active transport of the essential metal ion Cu2+ away within the cytoplasm to prevent their exposure to essential
from the bacteria is achieved through an ATPase efflux cellular components. Metals commonly sequestered are Cu2+,
mechanism. The cop operon has been found in the gram- Cd2+, and Zn2+. Two examples exist for this form of metal
positive bacteria Enterococcus hirae and contains four resistance: metallothionein production in Synechococcus sp.
genes: c o p A, c o p B , c o p Z , c o p Y . The operon has and cysteine-rich proteins in Pseudomonas species
2172 Current Medicinal Chemistry, 2005, Vol. 12, No. 18 Borkow and Gabbay

[142,157]. The metal resistance system in Synechococcus Chromate reduction by CRB5 is mediated by a soluble
species consists of two genes; smtA and smtB. The gene enzyme that was largely contained in the cytoplasm but also
smtA encodes a metallothionein that binds the metals. This found outside of the cells. Membrane-associated Cr6+
gene is induced by high levels of Cd2+, Zn2+, and Cu2+ and is reduction under anaerobiosis may account for anaerobic
repressed by the gene product of smtB. Cysteine residues in reduction of chromate under nongrowth conditions in the
SmtA metallothionein may act as a sink for excess toxic presence of an organic electron donor [166,167].
cations. Another organism that demonstrates intracellular
Metal ion-specific reducing enzyme systems function in
Cd2+ sequestration is a strain of Pseudomonas putida that
the cytoplasm and cell surface layer of microorganisms
was isolated from sewage. This organism produces three
[168]. These enzymes require NADH or NADPH as an
low-molecular-weight cysteine-rich proteins, which may be
electron donor and flavin adenine dinucleotide (FAD) and
related to metallothioneins. flavin mononucleotide (FMN), as an electron acceptor.
Mycobacterium scrofulaceum also demonstrates Electron transport may be controlled by transplamsa
intracellular accumulation of Cu2+ by sequestering it in the membrane redox systems.
form of a black copper sulphate precipitate [161].
3.7. Decrease in Metal Sensitivity of Cellular Targets
3.5. Extracellular Sequestration
Some microorganisms adapt to the presence of toxic
Metal resistance based on extracellular sequestration has metals by altering the sensitivity of essential cellular
been found in several species of yeast and fungi and has been components. Protection is achieved by mutations that
hypothesized for bacteria [162]. For example, S. cerevisiae decrease sensitivity to a toxic metal but do not alter basic
secretes large amounts of glutathione. Glutathione binds Ni2+ cellular function; alternatively, the mutations may increase
with great affinity. The glutathione-metal complex cannot production of a particular cellular component to keep ahead
traverse the cell membrane, thus conferring the yeast of metal inactivation. The microorganism may also protect
resistance to Ni2+ [163]. Other organisms, such as itself by producing metal-resistant components or alternate
Citrobacter species, form insoluble complexes of cadmium pathways in an effort to bypass sensitive components.
phosphate to confer cadmium resistance [164]. A similar
DNA repair mechanisms may also provide limited
mechanism exists in Cu2+-resistant fungi, which secrete
protection to metal-induced damage of plasmid and genomic
oxalate to form a metal-oxalate complex [140].
DNA. For example, upon exposure to Cd2+, unadapted E.
Interestingly, it has been shown that P. aeruginosa coli demonstrate considerable DNA damage; however, after
biofilms were anywhere from 2 to 600 times more resistant subculture, the same organisms show resistance. The growth
to heavy metals than free-swimming cells. The exterior of lag phase of the organism decreases the longer it is exposed
the biofilm was preferentially killed after exposure to to Cd2+. It is postulated that the extended lag phase is
elevated concentrations of copper, while the majority of cells initially due to a period of induction of DNA repair
that remained alive were in the substratum. A possible mechanisms.
explanation for this phenomenon is that the extracellular Natural resistance can result from normal cellular
polymeric substances that encase a biofilm may be
functions that give the organism a base level of tolerance
responsible for protecting cells from heavy metal stress by
[142]. An example is naturally occurring glutathione, which
binding the heavy metals, retarding their diffusion within the
may offer protection to metal ions like Cu1+, Cu2+, Ag1+,
biofilm [165].
Cd2+, and Hg2+ [169]. Glutathione may offer protection from
3.6. Enzymatic Detoxification of a Metal to a Less Toxic Cu2+ and Fe2+ by suppressing free radical formation [142].
Form There appear to be differences in the ability of gram-
There are several examples of enzymatic detoxification negative and gram-positive bacteria to tolerate certain metal
of metals to less toxic forms in microorganisms. Resistance ions. Gram-negative bacteria (E. coli and Pseudomonas
to mercury may be achieved by enzymatic detoxification in species) are better able to carry on protein synthesis in the
both gram-positive and gram-negative bacteria. A set of presence of Cd2+ than gram-positive bacteria. A species of
genes encodes for the production of a periplasmic binding Pseudomonas was shown to tolerate 5 to 30 times more Cd2+
protein and membrane-associated transport proteins. The in growth media before protein synthesis was reduced by
periplasmic binding protein collects Hg2+ from the 50% in comparison to gram-positive S. aureus,
surrounding environment and transport proteins take it to the Staphylococcus faecium, and Bacillus subtilis [170]. Gram-
cytoplasm where it is neutralized by redox chemistry [140]. positive organisms are able to bind 28 to 30 times more Cu2+
than E. coli [140].
Recently, a gram-negative bacterium, a pseudomonad
very similar to Pseudomonas synxantha (CRB5), was Copper tolerance in fungi has also been ascribed to
isolated from a chromium-contaminated site. It was found to diverse mechanisms involving trapping of the metal by cell-
reduce toxic hexavalent chromium Cr6+ (chromate) to an wall components, altered uptake of copper, extracellular
insoluble Cr3+ precipitate under aerobic and anaerobic chelation or precipitation by secreted metabolites, and
conditions. CRB5 tolerated up to 520 mg of Cr6+ per liter intracellular complexing by metallothioneins and
and reduced chromate in the presence of copper and phytochelatins. Only the metallothionein chelation
arsenate. Under anaerobic conditions it also reduced Co3+ mechanism has been studied in detail [65].
and U6+, partially internalizing each metal. Metal precipitates 4. SUMMARY
were also found on the surface of the outer membrane.
Copper as a Biocidal Tool Current Medicinal Chemistry, 2005, Vol. 12, No. 18 2173

In contrast to the low sensitivity of human tissue to similar application includes the neutralization of
copper [3-6], microorganisms are extremely susceptible to cytomegalovirus (CMV), which is transmitted through
copper. Copper toxicity in microorganisms may occur breast-milk. CMV causes severe maladies especially in
through the displacement of essential metals from their premature or other high-risk infants.
native binding sites, from interference with oxidative In conclusion, there are many potential uses of copper in
phosphorylation and osmotic balance, and from alterations in
new applications that address medical concerns of the
the conformational structure of nucleic acids, membranes
greatest importance. Implementation of even a few of these
and proteins. In most microorganisms, but not in viruses,
possible applications may have a major effect on our lives.
there is an integrated set of proteins that delivers copper to
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