Antimicrobial copper
By Jason Ren
Contents Page:
Abstract……………………………………………………………………………………………………3
Aim…………………………………………………………………………………………………………..3
Background Information……………………………………………………………………………3
Equipment………………………………………………………………………………………………17
Hypothesis………………………………………………………………………………………………17
Variables…………………………………………………………………………………………………18
Controls…………………………………………………………………………………………………..18
Procedure………………………………………………………………………………………………..18
Risk assessment……………………………………………………………………………………….26
Results……………………………………………………………………………………………………..27
Calculations..………………………………………………………………………….…………………28
Analysis……………………………………………………………………………………………...……30
Discussion………………………………………………………………………………………………..34
Conclusion………………………………………………………………………………………………..39
Bibliography……………………………………………………………………………………………..40
Appendix…………………………………………………………………………………………………..43
Date: 9 June- 25 June
Abstract:
Antibiotic resistant bacteria are a prominent issue in modern day society and are becoming more prevalent in many clinical environments such as hospitals. Infections caused by antibiotic and non-antibiotic resistant bacteria are often spread by contact surfaces such as handrails, door handles, taps, and toilet seats. Copper surfaces have been suggested to have antimicrobial properties, killing up to 90% of bacteria and has been proposed as an alternative to stainless steel and plastic surfaces. Cells exposed to copper surfaces have been shown to suffer extensive membrane damage and showed loss of cell integrity.
Aim:
To determine if copper surfaces can be used as an antimicrobial agent to limit bacterial growth and if it has practical applications in clinical healthcare environments such as hospitals
Background Information:
Bacterial cell structure
First seen under a microscope in 1676 by Anton Van Leeuwenhoek, bacterial cells are much smaller then plant or animal cells. As microscope technology has improved, scientists have come to understand bacterial cell structure in more depth. A bacterial cell is made up of different parts such as the capsule, cell wall, plasma membrane and the nucleoid[1]. The cell membrane is the semipermeable membrane that surrounds the cytoplasm of a cell and has numerous roles. These roles encompass a variety of functions such as energy generation and transport of solutes as well as housing many enzyme systems[2]. The plasma membrane is predominantly composed of phospholipids and proteins. Within the cell membrane are the cytoplasm, ribosomes, mesosomes, plasmid and the nucleoid[3]. The cytoplasm is where metabolic processes necessary for life occur. Ribosomes are sites of protein synthesis, i.e. they are involved in the manufacture of proteins. The nucleoid is the area that contains the cell’s DNA. Mesosomes play a role in cellular respiration, which is a process that breaks down food to release energy. The plasmid contains extrachromosomal genetic material. These genes are usually not necessary for the bacterium’s day-to-day survival. Instead, they help the bacterium overcome, for example, situations where it is exposed to antibiotics. In these instances many plasmids, contain genes that when expressed make the bacterium resistant to antibiotics. Other plasmids can also contain genes that help the bacterium kill other bacteria. Without these important structures such as ribosomes and mesosomes, the bacterial cell cannot function properly and may quickly die.
Antibiotics
Antibiotics are medicines used to treat diseases or infections cause by bacteria. Antibiotics differ in how they work and the types of bacteria they work against. The main classes of antibiotics include: penicillins, cephalosporins[4], macrolides, aminoglycosides and fluoroquinolones[5]. Cephalosporins and penicillins kill bacteria by destroying bacterial cell walls. Macrolides and aminoglycosides work by binding to a specific subunit of ribosomes in susceptible bacteria, inhibiting the formation of bacterial proteins[6]. This action in most organisms inhibits cell growth, however in high concentrations it can cause cell death. The protein that the antibiotic aims to inactivate is sometimes referred to as the target protein[7]. Fluoroquinolonies can cause sever side effects in rare cases, and are therefore not used for regular treatment of bacterial infections. They are used for more resistant strains of bacteria and work by blocking DNA replication pathways of bacteria thereby inhibiting bacterial replication.
Antibiotic resistant bacteria
Antibiotic medications are used to kill harmful bacteria, which can cause disease and illness. They have made a major contribution to human health, however, some bacteria have become resistant to commonly used antibiotics, an example being Methicilim-resistant Syaphylococcus Aureus, better known as MRSA[8]. Antibiotic resistance is a current public health problem and can cause serious widespread disease. Some bacteria are naturally resistant to some antibiotics. For example benzyl penicillin has very little effect on most organisms found in the human digestive system. The first step in the emergence of resistance in bacteria is a genetic change[9]. There are various ways this can happen; two methods include spontaneous mutation in the bacterium’s DNA and transfer of antibiotic-resistance genes. Many antibiotics, e.g. Penicillin, work by inactivating an essential bacterial protein. Not only can a genetic change can remove that protein, mutations in the target protein can prevent the antibiotic from binding, or if its does bind; prevent it from inactivating the target protein[10]. To prevent the antibiotic from binding with the target, some bacteria change the structure of the target so that the antibiotic can no longer recognize it or bind to it. Genetic change can also lead to increased production of the target enzyme of an antibiotic, so that there are too many for the antibiotic to inactivate. Alternatively the bacterium may produce an enzyme that inactivates antibiotics. An example is an enzyme called beta-lactamases that “inactivate” penicillin. In addition, to stop antibiotics form entering the cell, the bacterium may alter the permeability of its cell membrane. The second method for a bacterium to gain resistance is by the transfer of an antibiotic-resistance gene from one bacterium to another bacterium. Antibiotic resistance not only spreads due to the transfer of antibiotic-resistance genes, but through the movement of bacteria form one host to another, either indirectly or directly. In humans, when a course of antibiotics is taken there is always the chance that there will be some bacteria with resistance, as well as the fact that often the full course of antibiotics is not taken. Those not killed are now free to multiply without any competition form weaker strains. Friendly bacteria can also be wiped out by antibiotics, which would otherwise compete with the resistant strain for resources.
In modern day society, bacteria are gradually increasing its resistance to antibiotics. The world currently has a demand for a new antibiotic, but finding one is proving to be a great challenge. However copper may be the key to killing antibiotic resistant bacteria such as MRSA as well as improving general health by limiting bacteria growth.
What is copper?
Copper (Cu) is one of the best electrical conductors of metals. Light red in colour and easily oxidized to a green hue, copper can be formed and drawn to serve many purposes from water pipes and circuit boards to jewellery and architecture, and in the case of this project to possibly replace common contact surfaces made out of materials such as plastic, to limit bacteria growth, in hospitals.
Possible property of copper that kills bacteria
Properties of copper thought to kill bacteria include its high conductivity as well as the release of copper ions when contact between bacteria and the metallic surface occurs.
What differentiates copper from antibiotics?
In the context of killing bacteria, copper does not kill bacteria via “conventional” methods used by antibiotics. In stead it uses other methods such as in causing holes to appear in the cell membrane. The antibiotic penicillin causes a similar reaction in bacteria to kill bacteria. However it does so by targeting proteins and enzymes in the membrane. Resistance to this action has already emerged and can be spread easily. On the other hand a proposed method of how copper creates holes is that copper essentially “Short circuits” the cell membrane[11]. This is an example of how copper attacks bacteria from “another direction”, i.e. it kills bacteria in in a different way, when compared to antibiotics. Another, perhaps more important factor that differentiates copper form antibiotics, is that copper can kill drug resistant “superbugs”. In other words, copper can kill some antibiotic resistance bacteria. Studies conducted have shown that copper surfaces can kill E.Coli, Clostridium difficile, Influenza A, Adenovirus, and perhaps the most infamous of them all Methicillin-resistant Staphylococcus aureus, better known as MRSA. In 2004 the University of Southampton research team conducted an experiment that demonstrated copper inhibiting the growth and replication of MRSA. This study found that “Faster antimicrobial efficacies were associated with higher copper alloy content”, and that “stainless steel did not exhibit any bacterial benefits”. Furthermore, in 2008, the United States Environment Protection Agency (EPA)[12], after evaluating a wide body of research, granted a registration approval that certified “copper alloys kill more that 99.9% of MRSA within two hours”[13].
Proposed methods of how copper kills bacteria
Research has been conducted in the area of how copper kills bacteria, however results are not certain and are subject to disagreement. The most conclusive and evidence backed property of copper that kills bacteria is through contact killing. Although the mechanism of contact killing is still not fully understood, recent studies suggest that copper surfaces kill bacteria by a three-pronged attack: damage of the bacterial membrane, extensive intracellular damage, and DNA degradation. The sequence of these events is still under debate and may be different depending on the microorganism. For many organisms, copper as a trace element is an essential nutrient. Furthermore, in respiration, copper serves as a cofactor and is thus needed for aerobic metabolism. However, when copper is in excess and in high concentrations, it is highly toxic. Every cell’s membrane including both multicellular and single celled organisms like bacteria contains a stable electrical micro-current, often called “trans membrane potential” and is essentially the voltage difference between the inside and outside of a cell. It is suspected that when bacterium comes into contact with a copper surface, a short-circuiting of the current in the cell membrane can occur. This may be due to the high conductivity of copper. This short-circuiting weakens the membrane and creates holes. Another method that holes may be opened in the membrane is through the interaction of copper ions with lipids causing their peroxidation[14]. The opening of holes in the cell membrane can compromise the integrity of the cell, which can cause the leakage of essential solutes resulting in a desiccating effect. Once the cell membrane has been breached, there is essentially an unopposed stream of copper ions entering the cell. It is at this stage that most of the damage is done to the cell. Copper readily catalyses reactions that result in the production of hydroxyl radicals through Haber-Weiss and Fenton reactions. Hydroxyl radicals can damage virtually all types of macromolecules; some examples include nucleic acids, amino acids and lipids. The formation of radicals can also inactivate viruses. Highly reactive oxygen intermediates causes lipid peroxidation and oxidation of proteins. In other words, in this process, lipids and proteins are degraded by oxidation. Copper ions inactivate proteins by damaging Fe-S clusters as well as by replacing the respective metals in metalloproteins with copper. Copper ions may also disrupt enzyme structures and functions by binding to sulphur. To put it simply, copper ions entering cells puts vital processes inside the cell in danger. Copper can obstruct cell metabolism as well as stopping enzymes inside the cell from performing vital functions. This occurs when the copper ions make molecular bonds with these enzymes. For Escherichia coli (E-Coli) in particular, copper damage to the respiratory chain in E-Coli cells has been linked with impaired cellular metabolism. Recently in 2014, live/dead staining performed in various studies indicated that cell membrane damage occurred in cells on copper surfaces but not steel surfaces. These findings suggest that metallic copper does not kill via DNA damage. In contrast, membranes are most likely the Achilles heel of cells exposed to copper. Although copper may not kill via DNA damage it still may degrade DNA when “attacking” cells.
Further studies of antimicrobial properties of copper found that the surface structure of copper as well as the environment that it was in affected the rate at which bacteria was killed[15]. The importance of the release of copper ions in the killing of bacteria implies that the surface structure of a copper surface is a factor in the rate at which bacteria is killed. A study performed by the National Centre for Biotechnology Information (NCBI) found that contact killing of bacteria in copper is essentially supressed if bacterial contact with metal is prevented[16]. This is an important issue as over time, copper corrodes to develop a green verdigris (or patina). This layer of verdigris may inhibit the ability of copper to kill bacteria. On the other hand, the more contact a copper surface has with bacteria, the higher the rate at which bacteria is killed. A possible explanation for this is that the more contact the copper surface has with bacteria the faster it corrodes. Faster corrosion rates have been correlated with faster inactivation of microorganisms. The rational behind this is that a higher corrosion rate means a increased availability of cupric ions (copper ions with a +2 charge), which is believed to be one of the factors responsible for copper’s antimicrobial action.
Bacteria-metal contact can also be prevented by a build up of dead bacteria. This dead bacteria can act as a barrier between the “healthy and alive” bacteria and the copper surface. As discussed above, if bacterial-metal contact is prevented then the killing of bacteria is supressed. However, the barrier of dead bacteria will eventually be broken down and new bacteria will come into contact with the copper surface. Dead bacteria can also be removed by cleaning the copper surface. Furthermore, dry copper surfaces killed bacteria faster and in larger numbers then moist copper surfaces. An explanation is that the intake of copper ions is faster form dry copper than from moist copper[17]. In addition, moist surfaces promote bacterial growth, whereas dry surfaces do not.
Bacterial growth
Bacterial growth is generally dependent on the existence of water, however there are some parameters for optimum bacterial growth.
- Supple of suitable and retrievable nutrients- the nutrients present should be in a form that allows the bacterial cell to passively or actively intake them[18]
- Existence of water- as mentioned above, bacterial growth is strongly dependent on water. Dry surfaces doe not promote bacterial growth
- Presence of a source of carbon or other forms of energy- all life forms that exist take up some form of energy to survive. For example, microorganisms that perform photosynthesis and receive primary energy from sunlight, require the gas carbon dioxide as a carbon source
- Existence of appropriate temperature- different microorganisms have different requirements regarding temperature for optimum growth. E-Coli falls into the category of mesophiles and has an optimum growth temperature of 370C. Mesophiles are bacteria that can grow and divide between 100C-450C[19]
- Appropriate pH of the environment- most microorganisms including E-Coli, grow best when the pH is around 7(neutral pH)
Methods of sterilisation and disinfecting