Essential oils and metal ions as alternative antimicrobial agents: a focus on tea tree oil and silver

Abstract

The increasing occurrence of hospital‐acquired infections and the emerging problems posed by antibiotic‐resistant microbial strains have both contributed to the escalating cost of treatment. The presence of infection at the wound site can potentially stall the healing process at the inflammatory stage, leading to the development of a chronic wound. Traditional wound treatment regimes can no longer cope with the complications posed by antibiotic‐resistant strains; hence, there is a need to explore the use of alternative antimicrobial agents. Pre‐antibiotic compounds, including heavy metal ions and essential oils, have been re‐investigated for their potential use as effective antimicrobial agents. Essential oils have potent antimicrobial, antifungal, antiviral, anti‐inflammatory, antioxidant and other beneficial therapeutic properties. Similarly, heavy metal ions have also been used as disinfecting agents because of their broad spectrum activities. Both of these alternative antimicrobials interact with many different intracellular components, thereby resulting in the disruption of vital cell functions and eventually cell death. This review will discuss the application of essential oils and heavy metal ions, particularly tea tree oil and silver ions, as alternative antimicrobial agents for the treatment of chronic, infected wounds.

Introduction

The use of metal ions and essential oils as antimicrobial agents is of particular interest in topical wound management. Management of chronic wounds, such as varicose skin ulcers and burns, aims to induce rapid healing and minimise the extent of scarring. Infection of such wounds can not only delay the healing process but also lead to the development of a chronic wound with increased potential to develop into a systemic infection. Topical application of antimicrobial agents is common since effective concentrations may be difficult to achieve with systemic drugs as the effects of the wound trauma may impede delivery of the agent into the wound 1, 2. The reduced concentration may also create selective pressure for antibiotic resistance.

The current problems posed by increasing antibiotic resistance in Gram‐positive bacteria (methicillin‐resistant Staphylococcus aureus, MRSA, Vancomycin‐resistant enterococci, VRE) and Gram‐negative bacteria (New Delhi metallo‐β‐lactamase‐1 (NDM‐1) positive Escherichia coli, multidrug‐resistant Acinetobacter baumannii, ciprofloxacin‐resistant Pseudomonas aeruginosa) 3, 4 have renewed interest in pre‐antibiotic antibacterial agents, such as metal ions and essential oil [tea tree oil (TTO)] 5, 6. Alternative, non‐antibiotic‐based treatments are attractive due to their decreased side effects compared with synthetic drugs and their multiple target sites within the microorganisms, which may contribute to reduced development of resistant strains 7. Despite their usefulness and long history of use, these agents are mainly restricted to topical applications against infected wounds, skin burns, ulcers and fungal infections of the skin. Despite their effectiveness in treating topical infections, the complete healing of wounds, especially slow/non‐healing wounds, may require repeated application or use of high concentrations that may have adverse effects on the patient. Current developments in wound treatment focus on approaches that will allow reduced concentration (using combined agents) or delivery via controlled release delivery systems to minimise potential side effects whilst maintaining bioavailability to achieving therapeutic effects 8, 9, 10, 11.

The useful properties of alternative antimicrobial agents, together with advances in drug‐delivery technologies, may be able to enhance and expand the medical applications of these agents 9, 10, 12. Combining these alternative antimicrobial agents with advanced drug‐delivery systems aims to:

• Promote bioavailability of agent at microbiocidal concentrations.

• Reduce drug concentration to enhance safety and practicality of application.

• Minimise scarring and promote wound‐healing processes.

• Reduce discomfort and pain in consideration of the patient's psychological needs.

• Decrease the frequency of dressing changes.

These aims would increase convenience, provide less opportunity for infection and/or reinfection of the wounds and ultimately reduce treatment costs.

Microorganisms and wound management

In healthy individuals, the skin supports a natural microflora comprising a balanced community of microorganisms, which rarely cause infection. However, a disturbance to the normal ratio of microflora or an exposure of subcutaneous tissue due to trauma may result in the pathogenic invasion by these microorganisms 7. Colonisation of wounds by these opportunistic pathogens is usually polymicrobial 12. The diversity and proliferation of the pathogens is influenced by various factors including the type, depth and location of the trauma as well as the host immune system response 7. The presence of microorganisms at a wound site does not confirm infection 13. Infection only occurs when the host immune system can no longer cope with the virulence factors expressed by the colonising microorganisms, thus triggering a series of systemic responses that delay the healing process 7, 13.

The increasing occurrence of hospital infections and widespread emergence of resistant microorganisms contribute to escalating treatment costs. In addition, hypersensitivity reactions to antibiotics and the lack of access to new treatments within the health care industry makes the provision of sufficient support and care for patients difficult. Modern lifestyles that frequently lack physical activity increase the possibility of developing various life‐long (interconnected) health conditions, for example, diabetes, obesity and hypertension in old age. These underlying health conditions may influence the complexity and severity of wound healing. In addition, the growing size and longevity of the elderly population has increased the prevalence of wounds associated with these conditions, including slow/non‐healing ulcers. The improvement in medical facilities has increased the number of patients surviving from complicated wounds, such as those caused by burns. Although rates of survival have improved, severely burned patients usually require extended stays in hospital, suffer from lowered immunity and extensive loss of skin. Such patients are prone to infection by both common wound pathogens as well as antibiotic‐resistant microorganisms, which may further complicate the treatment regime 1.

The nature of burn wounds, and varicose ulcers in particular, may involve relatively lengthy treatment with antibiotics, which carries the attendant risk of selecting drug‐resistant bacteria. Various approaches have been conducted to find the best method to treat and overcome this problem. The increasing incidence and broadening spectrum of pathogens resistant to antibiotics has refocused scientific interest on the use of alternative antimicrobial compounds 6, 14. Alternative, non‐antibiotic‐based treatments are attractive because of their decreased side effects compared with synthetic drugs and their multiple target sites within the microorganisms, which may contribute to reduced development of resistant strains 7. Despite their usefulness and long history of use, these agents are mainly restricted to topical applications against infected wounds, skin burns, ulcers and fungal infections of the skin.

Microorganisms and the wound environment

In healthy individuals, the skin supports a natural microflora comprising a balanced community of microorganisms, which rarely cause infection. However, a disturbance to the normal ratio of microflora or an exposure of subcutaneous tissue because of trauma may result in pathogenic polymicrobial invasion by these microorganisms 7. The diversity and proliferation of the pathogens is influenced by various factors, including the type, depth and location of the trauma as well as the host immune system response 7. The presence of microorganisms at a wound site does not confirm infection 13. Infection only occurs when the host immune system can no longer cope with the virulence factors expressed by the colonising microorganisms, thus triggering a series of systemic responses that delay the healing process 7, 13. Besides patient microflora, other sources of infection include those acquired directly or indirectly via air, other infected patients, health care workers, contaminated medical devices, hospital environment and external sources, such as visitors 15.

Wound healing mechanism

Wound healing is a complex cascading event involving cellular, enzymatic and biochemical pathways, spanning from the time the skin is damaged until the wound is completely healed 16, 17. Maintenance of healthy wound site homeostasis is thus important to maximise wound healing capacity, especially in immuno‐compromised patients or those with underlying chronic health conditions. The events in wound healing have been divided into four main stages 10, 18, 19:

1. Exudative – Haemostasis and clot formations occur, release of inflammatory mediators including cytokines, chemokines and growth factors

2. Resorptive – Inflammation occurs, signs of redness, swelling, heat, pain and possible loss of function, immune system reacts by sending neutrophils and macrophages into the wound cite to remove any foreign body (e.g. bacteria) and inflammatory debris to facilitate conditions for angiogenesis

3. Proliferative – Wound closure is initiated by the migration of epithelial cells from the wound edge, angiogenesis occurs, simultaneously, a provisional matrix is formed and granulation tissue is also deposited

4. Regenerative – Re‐epithelialisation occurs to allow full wound closure, development of scar, tissue remodelling occurs to re‐establish functionality

In general, wound healing follows this sequence but, being non‐linear, the stages may move forwards and backwards, based on intrinsic and extrinsic factors as well as the severity of the wound 19. The rate of wound healing differs between individuals and is influenced by factors such as type and depth of wound, age, nutrition, immune status, underlying health conditions as well as local microbial burden.

Microbial infection in particular retards wound healing by increasing the bio‐burden at wound sites, thus stalling the normal process at the inflammatory phase. It is common for chronic wounds to become contaminated and colonised by microorganisms. Such colonisation by microorganisms, especially common skin commensals such as Staphylococcus epidermidis and Corynebacterium species, does not usually impair wound healing, provided the population remains below the critical threshold of approximately 10⁵ cells per gram of wound tissue 20. Their presence and production of proteolytic enzymes may help to stimulate the activity of neutrophils, thereby encouraging an inflammatory response which may facilitate wound healing 21. Critical colonisation is defined as a condition in which bacterial infection exceeds the ability of the host's immune system to eliminate further proliferation and colonisation (20).

Cutaneous wounds offer a favourable (moist, warm, nutritious) environment to support bacterial growth and proliferation. Heavy microbial infection (above critical colonisation) retards wound healing by increasing the bio‐burden at wound sites, which stalls the normal process at the inflammatory phase. When acute wounds become infected and reach critical colonisation by pathogenic microorganisms, the stimulated pro‐inflammatory environment (due to microbial production of toxins, proteases or pro‐inflammatory molecules) will stop the process of wound healing, and the site develops into a chronic wound 7, 10, 12, 20.

Alternative antimicrobial agents

Although conventional antibiotics are regarded as effective antimicrobial agents, there is concern about their side effects and the increasing incidence of microbial resistance to them 22. Antimicrobial agents are only effective until resistant strains of the target microorganisms begin to emerge 6, 23. With conventional antibiotics, the emergence of resistance is mainly because of their action against a single target. This has led to re‐examination of the use of other antimicrobial agents, such as metal ions and plant extracts, which often attack multiple target sites 6, 22, 24, 25.

The application of essential oils to reduce bacterial growth and prevent decay is not a new idea. Plants synthesise aromatic secondary metabolites to protect against predation and prevent colonisation by plant pathogens 24. These aromatic compounds are divided into classes, including essential oils (primarily phenolics and/or terpenoids), alkaloids, lectins, polypeptides and polyacetylenes, all of which have different mechanisms of antimicrobial activity 24. Some examples of essential oils or plant extracts commonly used for their antimicrobial properties are TTO, ylang ylang, betel pepper, manuka, eucalyptus, arnica, lemon verbena, rosemary, green tea extract and cadendula 23, 24. Although extensively practiced since ancient times, the use of natural extracts from plants as antimicrobial compounds declined after the development of synthetic antibiotics.

Metals (especially heavy metals) were used as disinfecting agents since ancient times. Silver, copper and gold, for example, have been used to treat diseases, disinfect wounds and water. Examples of metals commonly used in these applications include zinc, iron, bismuth, cobalt, magnesium, titanium, copper and the more extensively used heavy metal, silver. Despite having useful properties, treatment using metals is limited because excessive concentrations of metals, especially heavy metals, are toxic to human cells. Amongst the heavy metals, silver has a long history of use as an antimicrobial agent because of its relatively lower toxicity to human cells 26, 27. Recently, in response to issues surrounding antibiotic resistance, topical application of silver compounds has increased in popularity 28.

Plant products as antimicrobial agent

Plants with medicinal properties have been used for the treatment of various diseases both in traditional and modern medicine. When faced with microbial invasion or attack leading to infection, plants have their own defence mechanisms, which rely on the production of compounds that interfere with the cellular and intracellular structure of microorganisms 29. These antimicrobial plant compounds that show effective antimicrobial activity are often secondary metabolites formed in aromatic plants. These aromatic compounds give the plants their characteristic strong odour 29 that can repel insects or herbivores. In addition, certain compounds give plants pigment or flavour; these may be irritants to other organisms and may flow out from injured plants to prevent colonisation by microorganisms 30, 31. These secondary metabolites are mainly present as volatile compounds such as phenols or their oxygen‐substituted derivatives, which are categorised into five major classes detailed in Table 1 24. All plant organs, including buds, flowers, leaves, stems, seeds, fruits, twigs and branches, are able to synthesise these compounds.

Table 1.

Summary of the major classes of active antimicrobial compounds from plants [summarised from references 24, 29, 39, 99, 100, 101, 102]

Class and description	Subclass	Example	Mechanism	Structure
Phenolics • Simple phenolic ring structures that may exist in their highest oxidation state. For example, cinnamic and caffeic acid, which are phenylpropene‐derived compounds. • Number and site of hydroxyl group(s) present on the compound may indicate the relative toxicity to microorganisms. • Highly oxidised phenols or those with increased hydroxylation have increased microbial inhibitory effects. • Active against various types of bacteria, viruses and fungi. • May cause iron deprivation, reaction with sulphydryl groups, protein‐related polyamide polymers via non‐specific interaction and hydrogen bonding to inactivate proteins or enzymes in the cell.	Simple phenol and phenolic acid	Catechol, epicatechin and caffeic acid	Substrate deprivation and membrane disruption
	Quinone	Hypericin	Binds to adhesins, complex with cell wall and inactivate enzyme
	Flavonoids	Chrysin	Binds to adhesins
	Flavones	Abyssinone	Complexes with cell wall and inactivates enzymes
	Tannins	Ellagitannin	Binds to proteins, adhesins, inhibits enzymes, causes substrate deprivation, complexes with cell wall, membrane disruption and metal ion complexation
	Coumarins	Warfarin	Interactions with eukaryotic DNA and has antiviral properties
Terpenoids • Actively disrupts various membranes of bacteria, fungi, protozoa and enveloped viruses via its lipophilic properties.	Monoterpenes	Terpinen‐4‐ol	Membrane disruption
Alkaloids • Secondary plant metabolites that have antimicrobial activity.	N/A	Berberine and piperine	Intercalate into cell wall or DNA, differentially inhibit sterol and chitin biosynthesis
Polyacetylenes • Has antibacterial, antimycobacterial, anti‐inflammatory, anti‐platelet‐aggregatory effects.	N/A	Falcarinol	Mechanism unknown
Lectins and polypeptides • Inhibition of membrane function and integrity via interaction with proteins, ion channels and displacement of lipids. • Presence of cholesterol and increasing ionic strength in the target membrane reduces the antimicrobial activity due to stabilisation of the lipid bilayer as well as the weakened electrostatic interaction to encourage binding to target membrane. Note: interaction with membrane peptides or weakening of electrostatic charge interactions required for antimicrobial activity.	N/A	Mannose‐specific agglutinin, fabatin, defensins and thionins	Inhibit adhesion and fusion interactions between virus and host cell. May form ion channels in microbial membranes or reduce adhesion of microbial proteins to host receptors via competitive inhibition

The amount of active compound present in botanical extracts varies depending on the main adaptation of the plant to its environment, harvesting period, the extraction process, dehydration procedures, purification and storage methods 25. The extraction of secondary metabolites from plants is usually by distillation (water or steam) to produce a volatile essential oil. Essential oils are aromatic compounds synthesised by plants as secondary metabolites and are well known for their antibacterial, antifungal and antiviral properties 6, 29, in addition to anti‐cancer, anti‐diabetic, anti‐inflammatory and antioxidant activities 29, 32. Essential oils are multi‐component compounds, usually with terpenes and their derivatives (terpeniods) as the major components. After extraction, they present as clear, almost colourless volatile liquids, soluble in organic solvents and lipids in addition to some hydrophilic components 29. Due to the versatility and wide‐ranging properties of essential oils, they are used not only in the pharmaceutical industry but also incorporated into the cosmetics, agriculture, sanitation, disinfection, food preservation and manufacturing industries 25, 29. Examples of the various uses of essential oils are summarised in Table 2.

Table 2.

Common essential oils extracted from plants and their medicinal and/or antimicrobial activity

Common name	Scientific name	Active compound	Activity or therapeutic properties	Application/product	References
Aloe	Aloe barbadensis, Aloe vera	Anthraquinones (aloin, emodin and resistanol), β‐sitosterol and gibberellins	• Antimicrobial • Increases wound healing • Induces hypoglycaemic effect • Lowers blood cholesterol	• Used on burns and wounds • Treatment of psoriasis and genital herpes • Mediate insulin production	42, 103, 104, 105, 106, 107
Arnica	Arnica montana	Sesquiterpene and lactones	• Anti‐inflammatory	• Applied to bruises and swelling • Osteoarthritis treatment	108
Basil	Ocimum basilicum	Linalool, methylchavicol, epi‐α‐cadinol, α‐bergamotene, γ‐cadinene and germacrene D	• Antimicrobial • Antioxidant	• Antimicrobial activity in food preservation and packaging industry	109, 110
Calendula (Marigold)	Calendula officinalis	Triterpenoid	• Antimicrobial • Anti‐inflammatory • Anti‐tumorogenic	• Herbal antimicrobial mouthwash • Treatment against athletes foot, ringworm and candidal infections	111, 112, 113
Cinnamon	Cinnamomum zeylanicum	Cinnamaldehyde	• Analgesic • Antiseptic	• Inhibits biofilm formation by clinical strains of Staphylococcus epidermidis • Active against common food spoilage microbes	114, 115
Clove	Syzygium aromaticum	Eugenol	• Antibacterial • Antifungal	• Used to treat toothache and skin sores	104, 116, 117
Eucalyptus	Eucalyptus globulus	1,8‐cineole, Tannin	• Antimicrobial • Insect repellent • Skin penetration enhancer for drugs	• Herbal antimicrobial mouthwash • Inhibits growth of food pathogen • Fragrant in pharmaceuticals, soaps, detergents and food • Natural pesticide/insecticide	118, 119, 120, 121
Garlic	Allium sativum	Allicin	• Antimicrobial • Potential anti‐cancer agent • Lowers cholesterol and triglycerides	• In vitro antimicrobial activity against Helicobacter pylori • Food preservative • Reduce risk of hypertension	32, 104, 122, 123
Green tea extract	Camellia sinensis	Catechins, tannins, caffeine	• Anti‐inflammatory • Antimicrobial • Anti‐cancer • Antioxidant	• Reduces dental caries • Lowers risk of Helicobacter infection	111, 124, 125, 126
Lemon verbena	Aloysia triphylla or Lippia citriodora	Verbacoside	• Antimicrobial, • Anti‐cancer agent • Antioxidant • Anti‐inflammatory • Antispasmodic • Antipyretic	• In vitro antimicrobial activity against H. pylori, Escherichia coli, Mycobacterium tuberculosis, Staphylococcus aureus and Candida albicans • Treatment of asthma, cold, fever and indigestion	127, 128, 129, 130
Manuka	Leptospermum scoparium	α‐terpineol, cineole, β‐triketone	• Antimicrobial • Anti‐inflammatory	• Aids wound healing • Relieve fevers, inflammation and pain • Respiratory ailments	23, 111
Oregano	Origanum vulgare	Thymol, carvacol, ρ‐cymene, γ‐terpinene, Thymoquinone	• Antimicrobial • Antioxidative	• Used in food preservation	131, 132
Rooibos	Aspalathus linearis	Flavonoids, aspalathin, iso‐orientin, orientin and rutin	• Antimicrobial • Antioxidant activity	• In vitro antimicrobial and antifungal activity against Bacillus cereus, Micrococcus luteus and C. albicans, • In vitro anti‐HIV activity	104, 133, 134, 135
Rosemary	Rosmarinus officinalis	α‐pinene, bornyl acetate, eucalyptol, camphor, 1,8‐cineole	• Antimicrobial • Antioxidant • Anti‐cellulitis	• In vitro antifungal activity, particularly against Malassezia (Pityrosporum) spp. • Food preservation	131, 136, 137, 138, 139
Tea Tree Oil	Melaleuca alternifolia	Terpinen‐4‐ol, α‐terpineol, 1,8‐cineole	• Antimicrobial • Anti‐cancer • Anti‐inflammatory	• Treatment of wounds • Pharmaceutical cosmetics and cleaning product applications	32, 33, 111

Tea tree essential oil as an antimicrobial

History, background of use and production of TTO

Bundjalung Aborigines of northern New South Wales traditionally used crushed tea tree leaves as a treatment for coughs and colds (inhalation), a herbal infusion for sore throats and also sprinkled them directly onto skin wounds to promote healing 33. The use of TTO as an antimicrobial agent became a common practice after Penfold published reports on its antimicrobial properties in the 1920s and 1930s 33. In modern society, the useful properties of TTO are made commercially available, either as the essential oil per se or alternatively formulated into various products, including antiseptic creams, soaps, shampoo, anti‐acne creams, toothpaste and household cleaning agents.

TTO is recognised as having broad spectrum antibacterial, antifungal, antiviral, antimycoplasmal and antiprotozoal activity as well as anti‐inflammatory and anti‐cancer properties 33, 34. There are over one hundred different components in whole TTO, of which its major component, terpinen‐4‐ol, is primarily responsible for its active antimicrobial properties 33. TTO is extracted from the leaves and terminal branches of the Melaleuca alternifolia (M. alternifolia) plant via steam distillation and condensed to yield a pale yellow oil 33. The potential variation in the composition of TTO has lead to the classification of six chemotypes of M. alternifolia, based on the amount of terpinen‐4‐ol (one type), terpinolene (one type) and 1,8‐cineole (four types) 33. Commercially acceptable grade of TTO are classed in the terpinen‐4‐ol chemotype and have to comply with ISO4730:2004 standards for 'Oil of Melaleuca, terpinen‐4‐ol type', which defines the maximum and minimum level for the 14 major components of the essential oil, including terpinen‐4‐ol 33.

Mode of action

The physicochemical properties of the oil include those from hydrophilic hydrocarbon compounds with sufficient lipophilicity and allow the oil to partition preferentially into biological membranes, causing bilayer expansion 33. Thus, TTO components diffuse easily through the hydrophobic lipid bilayer of the microbial cell membrane, causing disruption to integrity and function, increased fluidity, loss of permeability and inhibition of embedded membrane enzymes. Consequently, the cell loses essential metabolites and repair enzymes, ultimately resulting in cell death 33, 35. The microbiocidal properties of active monoterpenes in particular can mainly be attributed to disruption of the cell membrane's barrier function; cells are thus unable to establish control over membrane‐coupled energy‐transducing processes, solute transport, regulation of metabolism and maintenance of turgor pressure 35. However, when a compound is highly lipophilic, its low solubility in aqueous media hinders its ability to contact with and permeabilise cell membranes 36.

Cox et al. (2000) examined membrane disruption of E. coli, S. aureus and Candida albicans by TTO via the leakage of potassium ions, materials that absorb at 280 nm (proteins) and uptake of fluorescent nucleic acid stain, propidium iodide 35. All three microorganisms showed decreased microbial viability (inhibition of respiration), increased uptake of propidium iodide and increased leakage of 280 nm absorbing material. Leakage of potassium ions was prominent in E. coli and S. aureus but less so in C. albicans 35, 37. Carson et al. (2002) assessed the release of 260 nm absorbing materials (nucleic acids) from S. aureus after treatment with whole TTO, terpinen‐4‐ol, 1,8‐cineole and α‐terpineol 38. These results showed significant leakage of nucleic acids, suggesting extensive damage to the cytoplasmic membrane.

Applications

Preparations containing TTO are commonly used as antiseptic agents with antimicrobial, cleansing, healing and itch relieving properties 39. For example, creams containing 5% TTO have been used to treat acne and toenail onychomycosis. A 6% gel formulation was shown to have antiherpetic effects; and TTO has been used as an antiseptic agent in handwash soaps and mouthwashes as well as for the treatment of microbial infections such as folliculitis and vaginitis 39, 40, 41, 42, 43. Whole TTO essential oil applied over 12 days successfully and permanently removed skin warts, whereas salicylic acid (12% w/w) and lactic acid (4% w/w) only resulted in temporary removal of the warts 44. Treatment with 4% TTO nasal ointment together with a 5% TTO body wash performed better at eradicating MRSA from patients than 2% mupirocin nasal ointment together with 2% triclosan body wash 45. Similarly, a 10% TTO cream and 5% TTO body wash was more effective at clearing MRSA on skin compared with 4% chlorhexidine gluconate soap and 1% silver sulfadiazine cream 46. In contrast, treatment of MRSA in the nasal cavity alone, even with 10% TTO cream, showed only 47% eradication 46.

TTO has been widely used in the management of wounds because of its antimicrobial and therapeutic properties. Burnaid^® (Mundipharma Pty Limited, Sydney, Australia) is a commercial hydrogel dressing impregnated with TTO for the treatment of burns 47. Other studies have reported the enhancement of antimicrobial activity when using TTO in combination with other antimicrobial agents such as chlorhexidine 8, 23, tobramycin 48 and silver ions 34.

Wound‐healing benefits

In addition to antimicrobial activity, TTO also plays a role in wound healing and modulation of the inflammatory response 6, 49, 50. Water soluble components of TTO, especially terpinen‐4‐ol, contribute to inflammatory regulation by suppressing monocyte production of superoxide ions 50 as well as inflammatory‐inducing mediators, for example, TNFα, IL‐1β, IL‐8, IL‐10 and PGE₂ 49. This in turn limits further production of other inflammatory cytokines 49 and reduces oxidative damage to cells 6, thereby enhancing wound healing.

The aromatic vapours and analgesic properties of TTO may promote wound healing by providing temporary relief to patients 47, 51. Burnaid^® reduces skin temperature at the burn site by approximately 2°C within 20 min, providing localised soothing and cooling effects 47, and may improve the patients' ability to cope with the treatment. Using TTO to treat patients with malodorous skin ulcers showed a significant reduction of the malodour as well as of infection and pus secretion 51, 52. The TTO compounds therefore improved the patient's well‐being by reducing social isolation associated with the malodour.

Resistance

Despite the popularity of TTO in many applications, concerns of microorganisms developing resistance have not been totally neglected. In vitro exposure of S. aureus to stepwise increasing concentrations of TTO resulted in a selection of TTO‐resistant sub‐populations 53.

Although there is little evidence of cross‐resistance to conventional antibiotic‐resistant strains with TTO 33, mutant strains of S. aureus with reduced susceptibility to household cleaning agents containing plant extracts were less susceptible to TTO 54. Based on the available data, the activity of TTO may not favour spontaneous development of resistance; however, it is still important to minimise exposure to sublethal concentrations to limit the possibility of resistance development 33, 53.

TTO resistance is also noted in gram‐negative bacteria because of the nature of the outer membrane, which is composed of lipopolysaccharide, proteins and phospholipids. This membrane provides a hydrophilic permeability barrier, which is an essential factor in the tolerance of P. aeruginosa to membrane‐damaging agents, such as TTO 36.

Toxicity concerns

As with most drugs, overdose or extended exposure induces toxic side effects. Evidence from several reports has shown that toxicity of TTO when ingested is rarely, if ever, fatal 33. In general, the symptoms of oral toxicity of TTO vary according to age and dose ingested. Reported symptoms arise from effects on the central nervous system, resulting in changes in respiration rate, oxygen saturation levels, pupil reactivity, electrolyte and blood glucose concentration, development of systemic hypersensitivity, ataxia, muscle weakness, unconsciousness and hallucinations 55, 56. Similar toxic symptoms including lack of coordination, muscle tremors, dehydration, hypothermic, ataxic effects and, in more severe cases, death have been observed in animals 57.

Dermal toxicity of TTO has been reported to cause irritation and allergic reactions 33. The localised cooling effect on treated burn wound sites may lead to triggering hypothermia when using TTO‐based dressings on large areas of the skin 47.

Heavy metals as antimicrobial agents

Heavy metals have a density of at least 5 g/cm³ and are located centrally in the periodic table, along with all the other transition elements, because of their ability to form complexes via their partially filled d‐orbitals 58. These d‐orbitals allow the heavy metals to accept electrons to form complexes, either by redox or other biochemical reactions in the cell. At low concentrations, some heavy metals may serve as essential trace elements to maintain normal cell function. However, at high concentrations, cell toxicity may result from the formation of unspecified complexes within the cell 58, 59. Three classes of heavy metals have been proposed by Nies, 1999 58:

1. low toxicity and play an important role as trace elements, for example, iron (Fe), molybdate (Mo), manganese (Mn),

2. toxic but high to moderate importance as a trace element, for example, zinc (Zn), nickel (Ni), copper (Cu), vanadium (V), cobalt (Co), tungsten (W), chromium (Cr); and

3. toxic with little or no beneficial action, for example, arsenic (As), silver (Ag), antimony (Sb), cadmium (Cd), mercury (Hg), lead (Pb) and uranium (U).

Amongst the various biological functions of heavy metals, their antimicrobial activity will be discussed in further detail. The sequence of antimicrobial toxicity is reported as follows: Ag > Hg > Cu > Cd > Cr > Pb > Co > Au > Zn > Fe > Mn > Mo > Sn 60 The mechanism of antimicrobial action of heavy metal cations varies because of differences in chemical characteristics and their effect on different biochemical pathways in the cell (Table 3). These effects result in disruption of the microorganisms' normal cell function, leading to irreversible damage and cell death 58, 61.

Table 3.

Examples of heavy metals with antimicrobial properties

Metal cations	Activity towards microorganism	References
Cadmium, Cd2+	Reacts with sulphdryl groups on various intracellular proteins	140
Copper, Cu2+	Membrane‐bound copper ions may undergo Cu(I) to Cu(II) redox cycle, catalysing formation of highly toxic hydroperoxide radicals (R‐OOH)	58, 141, 142
Cobalt, Co2+	Competes with zinc for the active site of urease, thus inhibiting growth of Helicobacter pylori	143
Silver, Ag+	Interacts with intracellular components to suppress expression of enzymes and proteins essential for ATP production and condenses DNA, thus impairing replication	73
Zinc, Zn2+	Inhibits nutrient uptake, acid production and glycolysis in oral pathogens and rhinoviral replication. Interferes with proton transfer	65, 144, 145

Metal cations interact with ionisable intracellular groups such as carboxylates and phosphates in the lipopolysaccharide layer of gram‐negative bacterial cells, peptidoglycan and teichoic acids of gram‐positive bacterial cells 62. Metal cations may get incorporated into the cell membrane, causing loss of fluidity, followed by further transport into the cell cytoplasm to inhibit vital biochemical processes, hence disturbing the normal growth 58, 63. This transport is usually achieved via an unspecified system using a chemiosmotic gradient between the cell and its environment or, alternatively, an ATP‐dependant specific transport system 58, 61.

Within the cytoplasm, metal cations may affect various cell processes 62; for example, they may bind to sulfhydryl or thiol groups, leading to inhibition of various enzymes 58. Some examples of toxic activities of metal cations towards microorganisms are detailed in Table 3.

Despite their usefulness as antimicrobial compounds, the intensity of exposure (duration and concentration applied) should be carefully considered. Cobalt, cadmium and mercury are too toxic to be used clinically as antimicrobial agents. Copper, zinc and silver are less toxic to human cells. These are often incorporated into antiseptic creams or cleaning agents as well as surfaces of medical devices surfaces, including hospital taps and door handles 64, 65. Examples of applications using heavy metal ions and their mechanism of action are detailed in Table 4.

Table 4.

Applications of heavy metals with lower toxicity to human cells

Heavy metal ions	Antimicrobial mechanism and uses	References
Copper, Cu2+	• Inhibition of bacterial growth or bactericidal activity at 50–250 ppm. • Substitutes essential ions, blocks protein functional groups, inactivates enzymes, weakens membrane integrity and produces hydrogen peroxide‐free radicals when membrane bound. • For example, antimicrobial hand rub, coated contact surfaces (copper toilet seats, brass taps, door handles, door push plates) and cleaning materials (ultra‐microfibre cloths and mops).	64, 146, 147, 148, 149
Silver, Ag+	Microbiocidal activity at 0·05 ppm in phosphate‐buffered saline or at >50–60·5 ppm in complex biological fluids. Interact strongly with thiol groups, inhibits bacterial enzymes, interferes with electron transport, binds to DNA, thus inhibiting normal cell replication. Formulated as creams and incorporated into dressings for the treatment of burns and wound infections. Also incorporated into polyalkenoate dental cements, textiles, water filters, kitchen appliances and medical devices (catheters).	5, 28, 65, 150, 151
Zinc, Zn2+	• Inhibits replication of rhinovirus at <0·1 mmol/l (equivalent to <6·539 ppm). At 0·01–0·1 mM (equivalent to 0·6539–6·539 ppm), inhibits acid production by oral plaque bacteria, effective against plaque and gingivitis when combined with trichlosan. Inhibits nutrient uptake, proton transfer and sugar transport. • Incorporated into polyalkenoate dental cements, stainless steel surface coatings, air conditioning ventilation, intake and exhaust ducts.	65, 144, 145, 150, 152, 153

Silver as an antimicrobial

History and background of the uses of silver

The antimicrobial properties of silver, including its use as an active water disinfectant, have been documented since 1000 B.C. 66. The use of silver as an antimicrobial has been reported since ancient Greek and Roman times, in which silverware was used to store perishable food and drinks. By the 19th century, the use of silver ions, Ag⁺ (as silver nitrate), in medical applications became more widespread, with records of it being used to treat venereal diseases, salivary gland fistulae, bone and perianal abscesses and removal of granulation tissue prior to epithelialisation 66, 67. After the discovery of penicillin and the rapid expansion in the number and use of antibiotics 54, 66, 68, the antimicrobial use of silver declined. Recent widespread emergence of antibiotic‐resistant microorganisms has resulted in Ag⁺‐based agents regaining popularity as their multi‐target action in microbial cells is less likely to lead to silver resistance 60, 69.

Silver in its non‐ionised form is an inert metal that does not react with human cells 70. Compared with other heavy metals, Ag⁺ has relatively low toxicity towards human cells at concentrations that are still highly effective against microbial pathogens 71, 72, 73.

The reactivity of Ag⁺, even at concentrations as low as 10⁻⁹ to 10⁻⁶ mol/l (equivalent to 0·000108–0·108 ppm), has shown broad spectrum antibacterial, antiviral, antiprotozoal and antifungal activity 74. In addition, the antimicrobial activity and low toxicity of silver towards human cells is also accompanied by wound‐healing properties 67. Silver is able to treat infections while enhancing wound healing, which could be especially useful in the management of severe wounds such as burns and slow/non‐healing ulcers.

Mode of action

Ag⁺ is classified as highly reactive moieties, which readily bind anions formed by electron donor groups containing sulphur (thiols), oxygen and nitrogen 74. Ag⁺ demonstrates broad spectrum antimicrobial activity at concentrations as low as 0·05 ppm in phosphate‐buffered saline or between >50 and 60·5 ppm in complex organic biological fluids 5, 75, 76, 77. Ag⁺ expresses its antimicrobial activity initially by binding to cell surface proteins and enzymes, resulting in morphological cellular changes, inhibition of cell replication 75, 78, 79 and impairment of solute and electron transport systems, leading to reduced production of vital cell components, such as ATP 80. Subsequent uptake of Ag⁺ into the cell cytoplasm, either via non‐specific or substrate‐specific transport systems, allows Ag⁺ to bind and interfere with the activity of essential intracellular enzymes and DNA 74, 78, 79.

In the presence of oxygen, Ag⁺ also promotes the generation of reactive oxygen species through the inhibition of respiratory enzymes, such as NADH dehydrogenase II 81, or by impairing superoxide‐radical‐scavenging enzymes, such as superoxide dismutase 82. Evidence also suggests that the antimicrobial activity of Ag⁺ may be a consequential result of its ability to bind to essential enzyme sulphydrals groups (thiols), thereby breaking these protein bonds 83. The binding of Ag⁺ to anionic groups, most notably disulfide, amino, imidazole, carbonyl and phosphate residues, results in intracellular and nuclear membrane (in eukaryotes) permeability changes as well as structural modifications of the cell wall 79, 84, 85.

Ag⁺ may also bind to DNA bases, causing condensation and degenerative changes of DNA strands, leading to inhibition of cell replication and cell death 72, 79. In general, the antimicrobial mechanism of action of Ag⁺ is described as a cascade of four steps 80:

• The Ag⁺ binds to receptors (especially in protein residues including sulphydryl, amino, imidazole, phosphate and carboxyl groups) on the microbial cell membrane, resulting in membrane damage.

• After entering the cytoplasm, Ag⁺ binds to other essential enzymes, restricting their activity and the production vital metabolites.

• Binding to microbial DNA follows, thereby impairing cell replication.

• With weakened membrane structure and inhibition of cellular processes, vital components leak out; the bacterial cell cannot maintain normal function, resulting in cell death.

Applications

Formulations containing silver are commonly used to treat a variety of Gram‐positive and Gram‐negative bacteria as well as common highly antibiotic‐resistant microorganisms such as P. aeruginosa 79, 86. Silver‐based pharmaceutical preparation, for example, silver sulfadiazine (Flamazine^®, Smith and Nephew Healthcare Limited, Hull, UK), has been used for the treatment of burn wounds. It has been found that the antimicrobial activity of Ag⁺ depends not only on the amount of bioactive ions present but also on bioavailability, which is influenced by the physical and chemical form of silver, the affinity for moisture, rate of release and distribution 74, 79, 84, 87.

In the skin, silver may form a temporary reservoir by binding to proteins with an estimated half‐life of 10–12 hours 26. Being a highly reactive species, Ag⁺ can readily bind to components present in the wound bed, for example, negatively charged proteins, RNA, DNA and chloride ions 5, thereby limiting the bioavailability of the ions. This ‘quenching’ effect of the host tissues may lead to the need for the application of higher and potentially damaging doses of Ag⁺ for effective treatment.

Recent advances in controlled delivery systems, which incorporate antimicrobial agents such as Ag⁺ into delivery devices or dressings, may play a role in overcoming the potential problems caused by the increased exposure and concentration of active antimicrobial agents. Some examples of silver‐containing wound dressings available for current wound management applications include DuoDERM^® (hydrocolloids, ConvaTec, Skillman, NJ, USA), Aquacel^® Ag (hydrofibre dressings containing antimicrobial silver ions, ConvaTec, Skillman, NJ, USA), Tegaderm™ (films, 3M United Kingdom PLC, Bracknell, UK), Vaseline™ (gauze, Unilever, London, UK), Sorbsan^® (alginates, Bertek Pharmaceuticals, Research Triangle Park, NC, USA), Lyofoam^® C (foam dressing, ConvaTec, Skillman, NJ, USA) and Nu‐Gel™ (hydrogels, Johnson and Johnson Wound Management, Somerville, NJ, USA) 16, 17.

Wound‐healing benefits

As discussed above, Ag⁺ helps to promote wound healing by reducing the bacterial load at wound site. Ag⁺ can also enhance wound healing directly by modulating the inflammatory response 88. At wound sites, Ag⁺ is taken into epithelial cells responsible for the regulation of tissue metal homeostasis, heavy metal detoxification and wound healing 5. This uptake induces the synthesis of low molecular weight, cysteine‐rich, metal‐binding proteins called metallothioneins (metallothioneins I and II). This activity of metallothioneins, which protect the healthy cells from the toxic effects of metals, is induced by several other xenobiotic metals such as cadmium, gold and mercury 88. Metallothioneins also play an important role in promoting the uptake of key trace elements, such as zinc and copper, promoting RNA and DNA synthesis, cell proliferation, epithelialisation and tissue repair 5, 88. Rising zinc levels induced by the accumulation of Ag⁺ in wounds increased the activity of the zinc metalloenzymes, thus promoting cell proliferation and re‐epithelialisation in rats 88.

During wound repair and the inflammatory response, matrix metalloproteinases (MMPs) are present within the wound. MMPs function to cause breakdown of the extracellular matrix, autolytic debridement, dissolution of basement membranes, growth promotion of capillary beds, re‐epithelialisation and tissue remodelling 72. Hence, MMPs are essential in wound healing, but excessive levels degrade fibronectin and peptide growth factors needed for optimal re‐epithelialisation 5, 72. Ag⁺ may form stable complexes with MMPs, thus downregulating excessive localised inflammatory responses to promote wound healing 72, 88. Comparative studies revealed that patients had improved epithelialisation on the skin grafts when treated with nanocrystalline silver dressings in comparison to topical antibiotics 85. Results indicated that Acticoat™, a nanocrystalline silver‐containing product, was effective as a dressing that reduced the wound bio‐burden and altered the process of inflammation, thus facilitating the healing process 85, 89. The ability of Acticoat^® to provide prolonged sustained release of silver onto the wound site helps to avoid the development of resistance microorganisms while reducing potentially toxic side effects 85, 89.

Resistance

While this multi‐locus action of silver makes development of resistant microorganisms less likely, such resistance has been observed 90, and with the increasing use of silver, it may be a cause for concern 68, 91. The occurrence of resistance genes has been reported and may be chromosomal‐ or plasmid‐born 92, 93. The identified silver resistance plasmid, pMG101, codes for periplasmic binding proteins (SilE and SilF) and a chemiosmotic efflux pump (SilCBA), which exports Ag + donated from an ATPase efflux pump (SilP) via SilF 90, 92, 93, 94.

The initial mechanism of resistance may be because of periplasmic Ag⁺‐binding proteins, SilE, each of which binds five Ag⁺ in the periplasmic space. Synthesis of SilE is stimulated by the presence of Ag⁺ during growth 90. Although SilE has high affinity for Ag⁺, the actual release mechanism of the bound Ag⁺ has yet to be determined. It is possible that the Ag⁺ may be released to the cell exterior via the SilCBA protein trimer. The SilCBA assembly functions as a transmembrane cation/proton antiporter, moving Ag⁺ from the cytoplasm directly to the cell exterior. It is classed as a member of the resistance, nodulation and cell division (RND) superfamily, sharing homologous sequences with similar resistance mechanisms in metal‐resistant Alcaligenes and multi‐drug resistance mechanisms in E. coli 90, 93, 94.

Silver resistance has been found in clinical isolates of E. coli, Enterobacter cloacae, Proteus mirabilis and Klebsiella pneumoniae in a burns unit. In addition, silver nitrate and SSD treatment has also been associated with resistance in Proteus spp., E. cloacae and miscellaneous Enterobacteriaceae 95. Similarly, resistance to multiple antibiotics and Ag⁺ has been shown in Salmonella species 96. Laboratory exposure of clinical strains of E. coli to stepwise increases of silver nitrate or SSD has been shown to induce cross‐resistance against both compounds 97.

A sil E gene has been detected in MRSA strains, isolated from dogs, and is ≥95% homologous with the sil E from plasmid, pMG101 98. However, these isolates were still sensitive to treatment with silver‐impregnated hydrofibres. The results from this study are compatible with those of Silver, 2003 and suggest that there is low prevalence of silver resistance in MRSA 90. The restricted occurrence of a single sil E gene encoding for resistance is not sufficient to induce significant resistance against silver 97.

Toxicity concerns

In common with many drug treatments, over exposure to the agents causes unwelcome side effects. Long‐term topical exposure to high concentrations of Ag⁺ leads to a build up of Ag⁰ in the dermis, causing an irreversible blue‐grey discolouration of the skin (argyria). This is particularly pronounced in areas exposed to sunlight, which accelerates the photoreduction and deposition of Ag⁰ 27, 70. Some patients treated with silver‐containing dressings reported the occurrence of skin rashes, stinging and burning sensations when treated with silver‐impregnated dressings 26. Other more serious problems associated with topical application can include disturbances in electrolyte concentration, resulting in hyponatraemia or hypochloraemia 80.

Despite the beneficial properties of various silver‐based treatments, the potential toxicity and safety issues of silver use have to be carefully considered. With the increased availability of formulations, administration of silver can be tailored to the patient's condition, thus limiting the potential risk of side effects.

Conclusion

There has been increasing interest in the use of alternative, broad spectrum, pre‐antibiotic antimicrobial agents, such as essential oils and metal ions, to address issues relating to increased antibiotic‐resistant hospital infections. The versatility of alternative agents such as TTO and Ag⁺ against a wide range of different microorganisms because of their multiple target sites impedes the development of resistance and might be useful in improving the current wound treatment strategies. Despite the effectiveness of the agents, the potential development of side effects or toxicity to healthy host cells because of prolonged exposure at higher concentrations should be carefully monitored. Efforts to combine the use of these alternative antimicrobial agents with advances in targeted delivery techniques may help to address the issue of localised overloading and toxicity. Based on the current findings, which showed the efficacy and beneficial therapeutic properties of both Ag⁺ and TTO, the potential advantages of using these agents in wound treatment regimes should be explored further.