Rose CooperCentre for Biomedical Sciences, Department of Applied Sciences, Cardiff School of Health Sciences, Cardiff Metropolitan University, Cardiff, Wales, UK
22.1
Introduction
At one time all our medicines and preservatives were derived from natural products. Their use relied on anecdotal observations of efficacy, without knowledge of their respective mechanisms of action. Infections were feared and outcomes with traditional rem-edies were often unsuccessful. The discovery of antiseptics, fol-lowed by the discovery of antibiotics, allayed fears about most infectious diseases. Complacency was even expressed by the US Surgeon General who in 1969 considered it possible “to close the book on infectious disease” [1]. Extensive use, misuse and abuse of antibiotics resulted in the selection of antibiotic-resistant microorganisms and the emergence of strains with multiple drug resistance has compounded the problem. More recently the rec-ognition of an association between the presence of a bacterial biofilm and persistent infection has been significant [2], espe-cially as the susceptibility of infective agents to antimicrobial agents was found to be diminished in species living within bio-films [3]. Difficulties in treating osteomyelitis, cystic fibrosis, urinary tract infections, prostatitis, gingivitis, sinusitis and infec-tions associated with medical implants with conventional antibi-otics are examples that suitably illustrate this predicament.
Failure to discover enough new antibiotics during the last 30 years and difficulties in effectively treating biofilms have made the need to find novel antimicrobial agents urgent [1]. Advances in
genome technology have made it possible to search for distinct microbial target sites [4] and re-evaluating former remedies is bringing new insight to the potential of products derived from natural products. In some of the communities in Asia and Africa up to 80% of the population rely on traditional medicine, and in developed countries increasing numbers are turning to alterna-tive or complementary medicine [5]. There are concerns about the efficacy and safety of some of these products and tighter controls are imminent [6], yet products based on earlier remedies are beginning to reach modern medicine. The natural products with existing or projected commercial potential that are described here have been divided into three broad sections depending on their origin: bacterial, plant or animal. Most are formulated into products that are protected by patents, but some are not yet well developed in a commercial sense. Although many natural prod-ucts exert multiple effects on human health, antimicrobial activity will be emphasized here. Because that activity is sometimes a function inherent to the defense system of the producer species, a short preface on host defense peptides has been included.
Host defense peptides (antimicrobial peptides)
Most organisms produce antimicrobial peptides (AMPs) as part of their innate immune defense mechanisms. Over 700 examples have been described and those that have attracted most attention
Russell, Hugo & Ayliffe’s: Principles and Practice of Disinfection, Preservation and Sterilization, Fifth Edition. Edited by Adam P. Fraise, Jean-Yves Maillard,
peptides), 550Natural products of bacterial origin, 551Natural products of plant origin, 552
Natural products of animal origin, 556Antimicrobial peptides of animal origin, 559Conclusions, 560References, 560
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inwards and become an integral part of the pore (Figure 22.1.1b). In the carpet model, AMPs smother a membrane in a linear fashion and at a critical concentration act like a detergent to form micelles of part of the membrane, which then detach and leave relatively large AMP-lined channels across the membrane (Figure 22.1.1c).
In higher organisms AMPs also play a complex role in coordi-nating immune responses, but the mechanisms are not yet fully elucidated. Examples of AMPs selected for further discussion here are bacterial AMPs, cathelicidin (human), melittin (bee venom), bee defensin-1 or royalisin (honey and royal jelly) and magainin (frog).
Natural products of bacterial origin
Other than conventional antibiotics, the range of bacterial prod-ucts currently used in controlling microorganisms is limited to AMPs and enzymes.
Bacterial antimicrobial peptidesAlthough bacteria do not possess an immune system in the classic sense, they do produce peptides that exhibit antimicrobial activity. The best-known bacterial AMPs are bacteriocins and lantibiotics.
BacteriocinsBacteriocins belong to a diverse group of proteins that are pro-duced by bacteria to inhibit closely-related bacteria. Their range
come from humans, fish, insects, amphibians, plants and bacteria. Typically AMPs are relatively small, cationic, amphipathic mole-cules comprised of less than 100 amino acids and a molecular mass below 5 kDa. AMPs represent a heterogeneous group in terms of structure and function. This diversity has given rise to the development of several schemes of classification. In one example five classes have been proposed [7]:1. Anionic peptides requiring zinc as a cofactor (e.g. dermcidin).2. Linear cationic α-helical peptides of less than 40 amino acids with a distinctive, looped, three-dimensional structure (e.g. cecropins, magainin, melittin).3. Linear peptides without cysteine residues (e.g. bacteriocins, abaecin, indolicidin).4. Charged peptides that are derived from larger peptides (e.g. lactoferricin, cathelicidins).5. Peptides containing six cysteine moieties as three disulfide bridges and β-sheets (e.g. plant and arthropod defensins, β-defensins of birds, reptiles and mammals).
Generally AMPs display broad-spectrum activity against bac-teria, fungi, protozoa and viruses [8]. The mechanism of action of AMPs is not universal, but many exert their effect on microbial species by disrupting membrane structure and function by forming pores. Several models explain how this is achieved. In the Barrel–Stave model, AMPs insert themselves vertically across a membrane with their hydrophobic regions facing membrane lipid molecules and their hydrophilic regions facing the lumen of the resulting pore (Figure 22.1.1a). In the toroidal pore model, AMPs orientate themselves in a similar way, but the phospholipid components of the membrane of the target organism curve
Figure 22.1.1 Different models explaining the mode of action of antimicrobial peptides (AMPs).
a
AMP + membrane
a+
bBarrel−Stave pore model
cc
Toroidal pore modelp
Carpet pore model
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Many lantibiotics are bactericidal against Gram-positive bacte-ria either by inducing pore formation in target cell membranes, or by inhibiting peptidoglycan biosynthesis. Both mechanisms of inhibition involve binding of the lantibiotic to lipid II within the bacterial cytoplasmic membrane of the target species [13, 14]. Reduced efficacy against Gram-negative bacteria may be due to restricted movement of lantibiotics across the outer membrane.
EnzymesThe opportunities that peptidoglycan provides as a target site for novel inhibitors have not escaped attention [15]. Enzymes capable of hydrolyzing peptidoglycan are lysozyme, autolysins and virolysins, which are synthesized by eukaryotes, bacteria and bacteriophages, respectively. Although no commercial products containing bacterial cell wall hydrolases have yet been developed, the potential to inhibit bacteria is apparent.
Enzymes have potential in controlling wound infections. A combination of glucose oxidase and lactoperoxidase was bacteri-cidal for planktonic cells of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Pseudomonas fluorescens and model biofilms prepared on steel and polypropylene disks, but biofilm was only removed when polysaccharide-hydrolyzing enzymes were added to the mixture [16]. A similar approach has been employed in the development of two novel wound dressings. In one product, glucose oxidase and lactoper-oxidase have been incorporated into an alginate hydrogel [17]. In the other, a hydrogel sheet containing glucose is placed in contact with a hydrogel sheet containing glucose oxidase in the wound [18]. Glucose oxidation releases low levels of hydrogen peroxide that inhibits bacteria. Additionally, low levels of iodine are gener-ated in the sheet dressing by a further reaction [18].
Natural products of plant origin
From ancient civilizations to the present, plant products have played an important role in medicine. Plants produce a wide range of secondary metabolites which are used for protection against invasion by microbes, insects and herbivores. They also confer colors, odors and flavors to plants. Various parts of the plant have been utilized for medicinal purposes, including flowers, fruits, seeds, stems, leaves, roots, bark and wood; bioactive materials may be found in several locations in some plants. Most often dried plant material was extracted with hot water, but occasionally alco-holic extracts or tinctures were used. Variation in the quality of plant preparations is common and there is a need to standardize extraction methods and laboratory evaluations because published studies often provide conflicting observations [19].
A large number of antimicrobial components have been iso-lated from plants, and their chemistry is complex. They were divided into the several major groups by Cowan in 1999 [19] and additional subclasses include polyketides, polyamines, iso-thiocyanates, sulfides, thiosulfinates, glycosides, phenanthrenes and stilbenes [20]. Selected examples are given in Table 22.1.1.
of activity is generally narrower than that of conventional antibi-otics and the genes that encode bacteriocins are normally located on a plasmid or transposon. The names of bacteriocins often reflect the organism that produced them, for example colicin from Escherichia coli. Bacteriocins can be divided into two groups depending on whether they are produced by Gram-positive or Gram-negative bacteria, with the bacteriocins of Gram-positive bacteria further subdivided into four subgroups. They contain a wide range of proteins with modes of action that vary from membrane permeabilization to the interruption of protein syn-thesis or the degradation of DNA.
Many of the commensals that naturally colonize humans produce bacteriocins. The role of these organisms in preventing invasion by opportunist pathogens became evident when they were eradicated following the long-term administration of sys-temic antibiotics. By inference, the role of bacteriocins in coloni-zation resistance has been deduced and their therapeutic use as probiotic and bioprotective agents has been recognized [9]. Bac-teriocins of lactic acid bacteria and of E. coli have attracted most research to date. Their potential in rearing animals and fish without infection and in preventing human enteric, oral, respira-tory or vaginal infection is as yet unrealized.
LantibioticsThe lantibiotics are another type of bacteriocin. Lantibiotics are unusual peptides synthesized by Gram-positive bacteria that inhibit Gram-positive bacteria. They are composed of between 19 and 38 amino acids and contain a thioether amino acid which is either lanthionine or 3-methyl lanthionine. Their name is derived from the term “lanthionine-containing antibiotics”. The first one was described in 1928 [10] and more than 50 have since been discovered. It has been suggested that they are formed as part of the stress response during the late exponential or early stationary phase of growth.
The precursor peptides of lantibiotics are biosynthesized on ribosomes and then modified by dehydration of either serine or threonine residues, followed by intramolecular addition of cysteine with cyclization to form either lanthionine or methyl-lanthioine bridges, respectively. Removal of a leader sequence is necessary for biological functionality to be realized.
Lantibiotics can be categorized into three classes, based on post-translational mechanisms and their diverse biological functions [11, 12]. The best-known lantibiotic is nisin, which has been used as a food preservative for more than 40 years. Despite the extensive use of this safe additive, there is little evidence of nisin resistance. Nisin is synthesized by Lactobacillus lactis and added to processed cheeses, liquid egg, cold meats and some drinks as a preservative. It exhibits broad-spectrum activity against Gram-positive bacteria other than lactobacilli at low concentrations and it inhibits Gram-negative bacilli in the presence of chelating agents. The antibacte-rial properties of lantibiotics offer potential for treating lung infections in patients with cystic fibrosis, as well as for oral use in curing halitosis or preventing dental caries. Their ability to affect virulence or to act as biosurfactants has yet to be exploited [12].
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consists of 10–12 cloves which are each bound by a parchment-like skin. The bulb has been used in the human diet as a vegetable and condiment since ancient times and it has been cultivated for at least 4500 years. Much has been written about the medical use of garlic [24]. It was valued by each of the ancient Egyptian, Chinese, Hebrew, Greek, Indian, Japanese and Roman civiliza-tions and garlic bulbs were included in Tutankhamen’s tomb in 1352 bc. During the Middle Ages garlic was eaten to protect against bubonic plague and during World Wars I and II it was used to treat infections sustained by soldiers. Garlic has been used therapeutically for a wide range of conditions, but only its anti-infective use will be included here.
Garlic is now cultivated in many countries, but the largest producers are China, India, Pakistan and Bangladesh. Many forms of garlic are available commercially: fresh, frozen, pickled and dehydrated. For experimentation, fresh aqueous extracts, freeze-dried extracts and oil obtained by steam distillation of garlic have been mainly utilized.
Allium produces cysteine sulfoxides, which confer its charac-teristic odor. Its antimicrobial properties are related to its complex sulfur compounds, which depend on species, growth conditions and extraction methods. The principal antimicrobial component of garlic was identified as allicin (allyl-2-propene thiosulfinate), which is not detectable in intact bulbs but is rapidly produced from a microbiologically inactive precursor alliin by alliinase on crushing the bulb [25]. The enzyme and precursor are separated within the intact bulb as alliinase is contained within vacuoles. Allicin is highly unstable and decomposition leads to the forma-tion of ajoene, allyl alcohol, diallyl disulfide (DADS) and diallyl
Production of these substances may be a response to environmen-tal triggers, such as stress or potential invasion/attack. Plant defense chemicals can also be divided into phytoanticipins and phytoalexins on the basis of the timing of their production. The former are synthesized constitutively in an inactive form, for example glycosides, and the latter are inducible.
Of the diverse array of phytochemicals, flavonoids (also known as polyphenols) have significant medicinal potential and have attracted extensive scientific investigations. They are pigments synthesized from phenylalanine, which are ubiquitously distrib-uted in plants, honey and propolis [19, 21]. The mechanism of antimicrobial activity of some of the flavonoids has been reviewed [22], but their ability to also inhibit specific eukaryotic enzymes, mimic hormone action and to scavenge free radicals supports their use in the treatment of some human diseases not caused by infective agents [23]. Mechanisms by which bacteria are inhibited by flavonoids include inhibition of nucleic acid synthesis, inhibi-tion of cytoplasmic membrane function and inhibition of energy metabolism [22]. In general, phytochemicals are less potent than antibiotics in inhibiting microorganisms. However, human cyto-toxicity is normally relatively low and the possibility of structural modification of bioactive components offers the promise of dis-covering or designing more effective agents [22]. Garlic, green tea and an essential oil, tea tree oil, are examples of natural products derived from plants that are included here.
GarlicGarlic (Allium sativum) is a bulbous, perennial plant of the family Liliaceae that is indigenous to Central Asia. Its underground bulb
Table 22.1.1 The major groups of plant compounds with antimicrobial activity (adapted from [19]).
Group Subgroup Examples Plant sources
Phenolic compounds Phenols *Catechola Many fruits and vegetables, e.g. bananaPhenolic acids Caffeic acid
Caffeic acid phenethyl ester (CAPE)CoffeePropolis
Quinones Hypericin St. John’s wortFlavonoidsa Catechins
proteinases are important virulence factors of E. histolytica and have been found to be strongly inhibited by allicin, and the ability of the protozoan to destroy monolayers of baby hamster kidney cells was also inhibited by allicin [33].
Garlic also inhibits a range of fungi including dermatophytes and cryptococci. Most antifungal studies concern the effects of garlic on Candida albicans and different breakdown products of garlic induce different intracellular effects. Growth inhibition studies and structural changes observed by electron microscopy showed that fresh garlic was more effective than garlic powder extract in inhibiting C. albicans [34], whereas allyl alcohol and fresh garlic extract induced oxidative stress [35].
The antimicrobial effects of garlic are largely due to its reaction with thiol groups in enzymes such as alcohol dehydogenase, thioredoxin reductase and RNA polymerase [36]. Proteomic analysis of a commercial garlic preparation produced in China demonstrated that it caused the expression of 21 proteins to be altered in H. pylori. Multiple effects in energy metabolism and processes involved in the biosynthesis of amino acids, proteins, mRNA and fatty acids were indicated [27]. Whole garlic extract and allyl alcohol removed the transmembrane electrochemical potential of Giardia membranes [32]. Permeability of allicin through cytoplasmic membranes allows relatively easy access to intracellular compartments and has been linked to its biological activity [37]. In C. albicans, compounds derived from garlic
trisulfide (DATS) (Figure 22.1.2). The latter two are also potent antimicrobial agents. Two important metabolites of garlic are allyl alcohol and allyl mercaptan. Additional antiprotozoal compo-nents found in garlic are kaempferol and quercetin, which are polyphenols.
Garlic exhibits antibacterial, antifungal, antiprotozoal and antiviral effects. Much of the research into the antibacterial prop-erties of garlic extracts concerns the inhibition of foodborne spoilage and pathogenic bacteria, such as S. aureus, Salmonella spp., E. coli, Listeria monocytogenes and Helicobacter pylori [26, 27]. Garlic oil also inhibits many enteric bacteria [28] and oral bacteria [29]. Aqueous allicin extract and a novel gel formulation were shown to be bactericidal for Lancefield group B streptococci, and a role in treating and preventing vaginal and neonatal infec-tions during the perinatal period has been suggested [30].
One of the breakdown products of allicin, DATS, has been synthesized in China. Following in vitro inhibition of Trypanosoma spp., Entamoeba histolytica and Giardia lamblia by this syn-thetic product, its potential in treating several important human parasitic infections was described [31]. Further breakdown prod-ucts of garlic extract, especially allyl alcohol and allyl mercaptan, were shown to be effective in inhibiting Giardia intestinalis [32]. In this study extensive morphological changes were observed in trophozoites, but the effects of whole garlic extract and allyl alcohol differed to those induced by allyl mercaptan. Cysteine
Figure 22.1.2 Components with antibacterial activity that are derived from garlic. Courtesy of Ana Filipa Henriques.
S
O
Allicin (unstable)
S
S
O NH2
H2C
H2C
H2C
H2C
H2CH2C
H2C
CH2
CH2
CH2CH2
OH
OAlliin (inactive)
alliinase
S
O
ajoene
S
S
OH
allyl alcohol
S
diallyl disulfide (DADS)
S
S
diallyl trisulfide (DATS)
S
S
SH
allyl mercaptan
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grown in the presence of ECG had reduced resistance to oxacillin, thickened cell walls, disrupted cell division and reduced d-alanyl esterification of teichoic acid. Since teichoic acids influence the phase transition profile of phospholipids bilayers, it is possible that this may impact on the function of enzymes within the bacte-rial cell wall and membrane [50].
A tea polyphenol extract containing five catechins at sub-MIC levels was also found to reverse oxacillin resistance in 13 clinical strains of MRSA. Proteomic investigation of extracellular pro-teins recovered from cultures of MRSA exposed to this extract showed differential expression of 17 proteins. Three were upregu-lated and 14 were downregulated [51]. A similar study with E. coli found that 17 proteins were altered by green tea polyphenols, nine upregulated proteins were involved in cell defense mechanisms and eight downregulated proteins were involved in carbon and energy metabolism and the biosynthesis of amino acids [52]. Green tea, therefore, has multiple cellular effects on bacteria.
Essential oilsEssential oils have long been used in food, cosmetics and thera-peutic preparations because of their antimicrobial properties. Anti-inflammatory, sedative, analgesic and antioxidant properties extend their therapeutic potential to treating and preventing tumors and cardiovascular disease [53]. Essential oils, also known as volatile or ethereal oils, are extracted from aromatic plants by various methods, including expression, fermentation or distilla-tion. They are complex mixtures of up to 60 components, but mainly terpenes and terpenoids, with aromatic constituents such as aldehydes, alcohols and phenols present in minor proportions [54]. Essential oils are produced from many different species and almost 3000 have been described, of which only 10% are com-mercially important. Reviews of the chemistry and antimicrobial, therapeutic and preservative properties are available [53–55]. Because essential oils usually contain multiple components, it has been difficult to attribute specific cellular effects to individual components, but their hydrophobicity is thought to facilitate membrane permeabilization.
Undesirable effects, such as mammalian cytotoxicity, photo-sensitization, irritation, mutagenicity and carcinogenicity, have been associated with a small number of essential oils [54]. The use of essential oils in foods, cosmetics and medicines is therefore regulated in many countries. Perhaps the largest amount of infor-mation available concerning the antimicrobial effects of an essen-tial oil relates to tea tree oil.
Tea tree (Melaleuca alternifolia) oilMelaleuca alternifolia or tea tree is a small tree indigenous to Australia. The native Aborigines are thought to have understood the curative properties of M. alternifolia because they included its leaves in poultices and they used the water in which leaves had rotted for various remedies. Knowledge of the therapeutic prop-erties of tree tea only spread further afield after Australia was colonized by Europeans [56]. Tea tree oil (TTO) is a light-yellow-colored oil with a distinctive smell; it is extracted from the leaves
caused several events that triggered cell death by apoptosis [34]. In particular, allyl alcohol targeted alcohol dehydrogenases, two of which were in the cytosol and one in the mitochondria [35], and DADS induced thiol depletion and oxidative stress that led to impaired mitochondrial function [38].
Cell-to-cell communication or quorum sensing in bacteria influences the expression of some of the genes that contribute to virulence and biofilm formation. The development of assays to detect for compounds that interfere with quorum sensing [39, 40] has allowed a wide range of natural products to be screened. Garlic has been demonstrated to inhibit quorum sensing in P. aeruginosa [41, 42] and its potential in treating lung infections in cystic fibrosis patients has been recognized. Susceptibility for tobramycin and polymorphonuclear leukocytes grazing on P. aeruginosa biofilms exposed to garlic extract have been dem-onstrated to increase in vitro, as well as improved clearance of P. aeruginosa from pulmonary infections in mice [41]. Using microarray-based transcriptome analysis this garlic extract was shown to repress the expression of 167 genes, of which 92 were quorum-sensing-regulated genes in P. aeruginosa [40].
Green teaTea is produced from the leaves of Camellia sinensis, which is predominantly grown in China, India, Sri Lanka, Indonesia and Malaysia. Green tea is produced by steaming fresh leaves, unlike black tea which involves an oxidation stage. It has a complex chemistry and contains polyphenols, especially flavonoids such as catechins, catechin gallates and proanthocyanides. The bioactive components of green tea are largely associated with the catechins such as epicatechin (EC), epicatechin gallate (ECG), epigallocate-chin (EGC) and epigallocatechin gallate (EGCG).
The antimicrobial properties of tea against a broad range of microorganisms are well documented, with Gram-positive bacte-ria demonstrating greater susceptible than Gram-negatives [43]. EGCG in sub minimum inhibitory concentrations (MICs) inhib-ited slime production and biofilm formation in 20 cultures of staphylococci isolated from patients with eye infections [44]. Much of the recent research has focused on the effects of green tea components on S. aureus and methicillin-resistant S. aureus (MRSA). Virulence of S. aureus was reduced by ECG due to inter-ference in the secretion of coagulase and α-toxin [45], and cell division in MRSA (but not in S. aureus) was disorganized with selective inhibition of penicillin-binding proteins by a compo-nent of tea [46].
A component isolated from a crude extract of green tea polyphenols called compound P was found to reverse methicillin resistance in S. aureus [47]. It also prevented the synthesis of penicillin-binding protein 2′ and β-lactamase [47] and caused cell division in MRSA to be disorganized, with the formation of large clumps of unseparated cells possessing thickened internal cross walls but normal external cell walls [46]. Synergy between EGCG and β-lactams provided confirmation of peptidoglycan as a common target site [48] and the altered cell wall architecture in S. aureus has been confirmed to be due to ECG [49]. MRSA
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importance today include honey, propolis, royal jelly and bee venom. Many therapeutic claims have been made for these prod-ucts, but only their antimicrobial properties will be included here. The composition of all of these products is influenced by their botanical origin, geographical location, climate, species of bee, harvesting processes and storage conditions. Variations between each of the hive specimens tested and the differing methods employed, makes evaluating available data difficult.
HoneyFrom fragments of a Sumerian clay tablet it is clear that honey was used in ointments applied to wounds in 2600 bc. It featured prominently in the remedies of the ancient Egyptians, Greeks and to a lesser extent Romans [69]. Hippocrates recommended pale honeys for treating leg ulcers and Plato included honey in a diet aimed to promote good health [70].
Honey is produced by bees from either the nectar of blossoms or exudates released from plants following insect damage. These fluids are carried to the hive or nest in bees’ water sacs and mixed with enzymes from the hypopharyngeal gland when deposited there. Ripened honey is a supersaturated solution of four princi-pal sugars (fructose, glucose, maltose, sucrose) with low concen-trations of oligosaccharides, organic acids, proteins and minerals. Water molecules usually account for approximately 20% by weight of honey and pH ranges between 3.2 and 4.5. These prop-erties ensure that honey rarely spoils on storage, although it may contain bacterial spores [71].
The antibacterial characteristics of honey are not confined to its sugar content. Many raw or unprocessed honeys demonstrate increased antibacterial activity on dilution by the generation of hydrogen peroxide from the oxidation of glucose, following the activation of glucose oxidase, which is an enzyme introduced into honey from bees [72, 73]. Excessive heating during harvesting or storage of honey inactivates enzymes that were derived from bees. A small group of honeys are known as non-peroxide honeys because they possess antibacterial activity additional to hydrogen peroxide. In manuka honey this has been attributed to methylg-lyoxal [74, 75]. Using a honey produced under standardized conditions by bees contained in greenhouses and a series of neu-tralizations to remove successive antibacterial components, it was shown that sugar, hydrogen peroxide, methylglyoxal and an AMP identified as bee defensin-1 all contributed to the antibacterial activity of honey [76].
The broad-spectrum antimicrobial nature of honey is well documented [69, 77]. More than 60 bacterial species have been shown to be inhibited by honey; additionally fungi [78, 79], pro-tozoa [80] and viruses [81] are also susceptible to honey. Many early studies employed poorly characterized honeys and poorly described methods, so inconsistent observations have been reported. Since honey is not a uniform product, the floral origin and potency of a sample should be determined before any experi-mental evaluations are attempted. Chemical markers of floral origin have been established by chromatography [82] and a bioassay for assessing the antibacterial efficacy of honey was
and small branches of tea tree by steam distillation. The com-mercial preparation of an essential oil from tea tree began in 1920 and antimicrobial properties of TTO were discovered then [56]. The TTO industry has seen fluctuations in demand. TTO is cur-rently incorporated into many cosmetic and hygienic products that are available throughout the world. Remedies for acne, vaginal infections and tinea pedis are also produced, and its use in wound care products has been suggested [57].
Insolubility of TTO in bacteriological media has limited inves-tigations of antimicrobial activity. Variations in chemical compo-sition due to differing tree strains and extraction conditions have also introduced inconsistencies between laboratory studies. TTO has a complex chemistry with up to 97 components identified by chromatographic analysis. Terpinen-4-ol is one of the main anti-microbial components; others include α-terpineol and α-pinene [58]. Another constituent of TTO is 1,8-cineole, which is an irri-tant. Animal studies have demonstrated cytotoxicity of TTO so its use is limited to topical application rather than systemic use [59].
A diverse spectrum of activity against MRSA [60], staphyloco-cci and propionibacteria [58], pseudomonads [61] and yeasts has been reported for TTO [62, 63]. Whereas planktonic MRSA and methicillin-sensitive S. aureus (MSSA) were less susceptible to TTO, as determined by MIC, than coagulase-negative staphylo-cocci, the reverse was found with biofilms [64]. A comparison of the efficacy of TTO and mupirocin for eradicating MRSA in vivo showed no statistically significant difference between the two agents [65]. Since mupirocin may select for mupirocin-resistant strains and TTO has not yet selected for TTO-resistant strains, its potential for clinical use is apparent. Yet uptake of TTO for the eradication of MRSA-colonized patients has been slow and con-cerns of toxicity and allergenicity may be a limiting factor [59].
In bacteria and yeasts, exposure to TTO inhibited respiration and increased the permeability of cytoplasmic and plasma mem-branes respectively. Leakage of potassium ions from E. coli and S. aureus was also found [66]. A combination of time-kill, lysis, leakage and salt-tolerance assays combined with electron micro-scopy suggest that TTO and its components inhibit S. aureus by compromising the cytoplasmic membrane [67].
Natural products of animal origin
Ancient peoples certainly utilized animal products, such as bile, blood, butter, cobweb, cochineal, egg white, feces, lard, meat and milk, in their primitive wound remedies [68]. None of these prod-ucts would be used today, although beaten egg white was applied to leg ulcers by British nurses up until the 1970s. Two ancient wound remedies are enjoying a renaissance today: honey and maggots.
Hive productsFrom the earliest times bees have been important to mankind because their colonies have yielded valuable products that have been collected and used as food or medicines. Those of medicinal
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initiation of an infection and biofilm development both rely on adherence to host cells. Fructose contained in honey and royal jelly has been shown to block this event by interference with bacterial attachment to fuctose receptors on the host cell mem-branes [112].
The antibacterial effects of honey are not confined to direct inhibitory effects on bacteria, but seem to be augmented by immunomodulatory effects on cells involved in the phagocytic response. Stimulation of inflammatory cytokine release in the presence of honey [113, 114] and modulation of the oxidative burst as determined by the level of reactive oxygen species suggest that honey has multiple means of influencing the infection process [113, 115].
PropolisThe term for propolis is thought to have been invented by Aris-totle from two Greek words pro (before) and polis (city), to mean “before the city” or “defender of the city (or colony)”. Propolis,
developed in New Zealand [83]. It may be used to distinguish between hydrogen peroxide-generating honeys, inactive honeys and non-peroxide honeys. It seems logical that honeys intended for medical use should possess significant levels of antibacterial activity, as honeys intended for the table do not necessarily possess such activity [84]. Criteria for honeys destined for medical use have been formulated [85].
Honey was used topically as a remedy for wounds by diverse ancient civilizations but lost favor in western medicine during the 1970s [86]. The licensing of tubes of sterile Leptospermum honey as a complementary therapy by the Therapeutic Goods Adminis-tration in Australia in 1999 marked the beginning of the reintro-duction of honey into contemporary medicine. CE marked (certified for European conformity) wound dressings were approved for use in the UK in 2004, and in Canada and the United States honey-impregnated dressings were approved in 2008 by Health Canada and the Food and Drug Administration (FDA), respectively. A range of wound care products containing various honeys are now available in Australasia, Europe and North America. They are used on burns, diabetic foot ulcers, leg ulcers, pressure sores, surgical incision sites and trauma wounds. Honey has been used not only to prevent infections, [87] eradicate colo-nization of MRSA [88–92] and clear infections, but to promote wound healing. Much clinical evidence has been published and evaluated by systematic review within the past 10 years [93–98].
Using a sugar syrup made from the four main sugars contained within honey and manuka honey produced in New Zealand, it was demonstrated that inhibition of bacteria isolated from wounds was not due exclusively to the sugars contained in the honey [99–101]. Antibiotic-sensitive strains and their respective antibiotic-resistant strains were equally susceptible [99–101]. The mode of inhibition was shown to be bactericidal [100, 102, 103]. Transcriptome analysis was used to investigate the effects of Leptospermum honey on E. coli and multiple cellular effects were noted. Of the 2% of genes that were upregulated, many involved the stress response; the majority of downregulated genes encoded for products involved in protein synthesis [104]. Proteomic analy-sis of MRSA exposed to manuka honey showed multiple changes in protein expression, and universal stress protein A (which is involved in the stamina response) was found to be downregulated [105]. Structural changes in S. aureus and MRSA induced by manuka honey suggest that inhibition was caused by failure of cells to complete the cell cycle because of an inability to separate during cell division [103, 106] (Figure 22.1.3). However, loss of structural integrity, with marked cell surface changes, were observed in P. aeruginosa exposed to manuka honey [107].
Much of the research concerning the inhibition of wound pathogens by honey has been conducted with planktonic cells. A link between chronic wounds and biofilms has recently been established [108] so the effect of honey on biofilms is pertinent to wound care. Honey has been shown to prevent biofilm forma-tion [109]; it can also disrupt established biofilms, but the con-centration and contact time is critical [110, 111]. P. aeruginosa is an opportunist pathogen in wounds that is difficult to treat. The
Figure 22.1.3 The effect of manuka honey on cell division in Staphylococcus aureus. (A) Exponential cells incubated in 0.05 mM Tris buffer pH 7.2 for 4 h at 37°C. (B) Exponential cells incubated in 0.05 mM Tris buffer pH 7.2 containing 10% (w/v) manuka honey for 4 h at 37°C.
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honey and pollen. It is an acidic, milky white fluid. Limited amounts are fed to all bee larvae by these “nurse” bees and the few larvae selected to become queens are flooded with royal jelly in specialized honeycomb compartments. Royal jelly is comprised of water (60–70%), proteins (12–15%), carbohydrates (10–12%), lipids (3–7%) and trace amounts of minerals and vitamins [126]. Royal jelly has most often been used as a dietary supplement or additive to cosmetics products because of the immunomodula-tory properties associated with its major proteins, even though antimicrobial activity has been demonstrated in some of its pro-teins. Inhibition of Gram-negative bacteria by royal jelly was first described in 1938 [127]. The first antimicrobial component iden-tified in royal jelly of the European honeybee (Apis mellifera) was a fatty acid [128]. In 1990, a bee AMP (which has been named royalisin and defensin-1) with activity against Gram-positive bac-teria at low concentration was isolated and characterized [129]. Several bee antimicrobial peptides have been found in royal jelly [130–132] and a family known as the jelleines has also been studied [133]. Royal jelly is a perishable product with a short shelf-life. Storage at low temperature, freezing, freeze-drying or mixing with honey is needed to preserve its activity. Additive activity between royal jelly and honey and synergistic activity between royal jelly and starch [134, 135] have been demonstrated and an ointment for treating diabetic foot ulcers has been tested in a prospective pilot study [136].
Bee venomThere is extensive anecdotal evidence derived from traditional medicine concerning the benefits of bee venom for the relief of symptoms of chronic diseases linked to immune dysfunction, such as rheumatoid arthritis and muscular dystrophy. However, there is considerably less information available on its antimicro-bial activity. Bee venom is a colorless liquid comprised of a complex mixture of proteins. It can be collected from the sting apparatus of bees, or used directly on patients. Clinical use must be strictly avoided in patients with bee venom allergy.
The principal active component is melittin, which is a 26-residue cationic AMP with significant lytic activity against both eukaryotic and prokaryotic cells [137]. Phospholipase A2 also exhibits antimicrobial effects by altering phospholipids associ-ated with membranes [138]. Melittin binds to membrane lipids and causes perturbations that disrupt membrane integrity which result in cell lysis. Binding of the peptide to lipids in a carpet-like fashion as a detergent has been demonstrated [139]. Since bacte-riolytic and cytolytic effects have been demonstrated, the hemo-lytic effects of melittin limit its therapeutic potential. Analogs of melittin that retain antimicrobial activity without mammalian cytotoxicity have been sought. A broad-spectrum antimicrobial peptide called melimine has been synthesized from a portion of the melittin molecule and protamine. This novel cationic peptide possessed reduced hemolytic activity and when covalently linked to contact lenses retained antibacterial activity [140]. It therefore has potential in preventing infections associated with implanted medical devices.
or bee glue as it is also known, is used by bees as a building mate-rial to seal holes and cracks in the hive. It is also used as a means to reduce microbial contamination within the hive. Propolis is made from resinous materials that are collected by bees from local plants and mixed with wax, but other sticky substances present within that environment can also be included on occasions. Hence the chemical composition of propolis may be variable, standardization of samples is necessary and the importance of using chemically characterized propolis samples in biological research has been emphasized [116]. Extrapolation of experimen-tal data from older studies is therefore not always wise.
Propolis has many interesting pharmacological characteristics and is used extensively in apitherapy globally, but particularly in Asia, eastern Europe and South America. Aside from its immu-nomodulatory properties, it is mainly used to treat skin, respira-tory and intestinal infections. It has also been used in food preservation and in cosmetic preparations, particularly for oral hygiene. For human use propolis is harvested from bee hives by scraping the internal surfaces to obtain the hard, resinous mate-rial. Biologically-active components are extracted by several tech-niques which can influence chemical composition [117]. Ethanolic extracts of propolis are most frequently tested in vitro.
The antimicrobial properties of propolis are well reported with many inferences that flavonoids are implicated in microbial inhi-bition [118]. Brazilian propolis is active against poliovirus [119]. However, there seem to be other inhibitory component(s) as well. For example, antibacterial, antifungal and antiviral activities of propolis samples originating from differing geographical areas were investigated using S. aureus and E. coli, Candida albicans and avian influenza virus, respectively. Despite differing chemical composition, all samples were active against Gram-positive bac-teria and yeast, and most exhibited antiviral activity. Activity was attributed to flavonoids and esters of phenolic acids in samples collected from temperate regions, but they were not present in samples from tropical zones [120]. The inhibition of 15 strains of MRSA by a propolis sample from the Solomon Islands has been demonstrated and activity was attributed to four prenylflavones fractionated from the sample [121]. Similarly, inhibition of S. aureus by two phenolic components derived from Kenyan pro-polis has been demonstrated [122].
The effects of propolis and selected constituent flavonoids were tested on several bacteria and found to uncouple energy transduction and to inhibit motility [123]. In staphylococci propolis has been found to modulate virulence determinants such as lipase and coagulase [124]. The growth of trophozoites of Giardia duodenalis was inhibited by propolis, cell shape was changed, flagellar movement was reduced, attachment was im -paired and trophozoites detached [125]. Such changes would be expected to impair the binding of trophozoites to host target cell membranes.
Royal jellyRoyal jelly is secreted from the hypopharyngeal glands and man-dibular glands of young worker bees that have been fed with
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in Europe and America is intended to encourage further investigation.
Lactoferrin displays multiple biological functions; its antimi-crobial properties are largely due to its iron-binding capacity. Microbial growth is influenced by the availability of iron, which is an essential nutrient. Lactoferrin is able to simultaneously and reversibly bind two cations (usually Fe3+ ions) and two anions (usually carbonate or bicarbonate). By tightly binding iron at the surface of mucosal membranes, lactoferrin restricts the quantity available for bacteria and exerts a bacteriostatic effect. Iron limita-tion affects the ability of bacteria, fungi and viruses to establish an infection because aggregation and adherence to host cells is compromised. In enteropathogenic E. coli, for example, lactofer-rin has been shown to impair the function of the type III secretory system [143].
Lactoferrin destabilizes the outer membrane of Gram-negative bacteria due to the release of lipopolysaccharide when lactoferrin binds to lipid A [144]. Furthermore, proteolytic cleavage of lacto-ferrin within the host releases a peptide with antimicrobial activity, lactoferricin. The release of lactoferrin from lysosomal secretory granules into phagosomes contributes to bacterial killing.
Iron limitation by lactoferrin has also been shown to prevent biofilm formation [145]. Adoption of a treatment strategy designed to eradicate biofilms from chronic wounds coupled sharp debridement with topical application of a mixture of lacto-ferrin and xylitol. This resulted in improved healing outcomes in patients with ischemic limbs [146].
Maggot therapy or biosurgerySince ancient times, civilizations in widely differing parts of the world have used the larvae of flies to treat wounds. The benefits of maggot therapy or biosurgery for wounds come from the ability of larvae to reduce bacterial load and also to promote wound healing. The infestation of a wound by fly larvae is known as myiasis; it occurs naturally when blowfly eggs present in wounds hatch into larvae and immediately feed upon local tissue. Such occurrences were seen during military encounters during the 18th, 19th and 20th centuries and military surgeons observed that such wounds healed rapidly without infection. The first known clinical use of larvae was by Zacharias during the American Civil War. Maggots were used again during World War I by an orthopedic surgeon who later advocated their use in civil-ians with osteomyelitis [147]. Maggot therapy became relatively popular in North America and Europe until the 1940s, when antibiotics were preferred. Although a few case reports were pub-lished during the 1980s, it was not until the 1990s that maggot therapy was revived for treating chronic wounds [148] and maggot therapy began again in the UK in 1995. Sterile larvae are now routinely used for treating non-healing wounds throughout North America and Europe. The range of wounds suitable for biosurgery includes traumatic wounds, burns, surgical wounds, leg ulcers, diabetic foot ulcers and pressure ulcers. Despite the publication of clinical evidence from many sources over the last 80 years to recommend the use of maggots in wounds, a
Antimicrobial peptides of animal origin
Much has been written about the therapeutic potential of AMPs of animal origin, yet few promises have yet been achieved.
Human defense peptidesHuman AMPs can be considered as three types: defensins, cathe-licidins and histatins. Essentially, defensins usually contain six cysteine residues within three disulfide bridges. Cathelicidins contain catelin at the N-terminal domain and a cationic antimi-crobial component that is activated by post-translational cleavage at the C-terminal domain. Histatins contain a high content of histidine residues and are mainly antifungal agents. One human AMP of special interest is LL-37. It is a cathelicidin that is pre-dominantly found in the secretory granules of neutrophils, kerati-nocytes and mucosal membranes.
MagaininsMagainins are cationic AMPs that were isolated from glands in the skin of the African clawed frog (Xenopus laevis). They are amphipathic peptides with an α-linear structure. Initially two magainins were isolated, but many more have since been found in Xenopus and other amphibians. Magainins exhibit broad-spectrum activity against Gram-positive and Gram-negative bac-teria, fungi and viruses by disrupting membrane permeability. An analog, pexiganan, was produced and antibacterial activity was evaluated in vitro. Bactericidal action was demonstrated against more than 3000 clinical isolates, and MIC90 was 32 µg/ml. Time-kill curves showed rapid inhibition of P. aeruginosa (106 organ-isms eliminated in 20 min) and resistance training failed to recover pexiganan-resistant mutants [141]. Despite the impres-sive in vitro activity of this AMP, it did not perform well in vivo. In a randomized, controlled, double-blinded trial, pexiganan actetate cream was compared to systemic ofloxacin in treating mildly infected diabetic foot ulcers, but significant differences between the interventions were not found, except that pexiganan-resistant strains did not emerge and ofloxacin-resistant strains did [142]. Despite efficacy in laboratory tests, in vivo efficacy has not been remarkable and the levels of pexignan that are required are close to cytotoxic levels. Failure to secure FDA approval will undoubtedly limit clinical use of pexiganan.
LactoferrinLactoferrin is a glycoprotein that is intimately involved in human innate immune defense mechanisms. It is secreted in milk, tears, saliva and mucus, and it is also found in the secondary granules of polymorphonuclear leukocytes. Lactoferrin is a globular pro-tein of 80 kDa that belongs to the transferrin family. Since the lactoferrin gene has been sequenced, recombinant lactoferrin has become available as a therapeutic agent. Of the many different uses that have been suggested, inclusion in infant milk formula and the treatment of cystic fibrosis are prominent. Recently the granting of orphan drug status to lactoferrin as Meveol®
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bacterial activity of larval secretions are most effective against Gram-positive bacteria such as staphylococci and β-hemolytic streptococci and less effective against Gram-negative bacteria such as the enterobacteria and pseudomonads [158, 159]. Biofilms of S. epidermidis were shown to be disrupted and prevented by larval secretions containing a molecule(s) >10 kDa with protease or glu-cosaminidase activity, which affected intercellular adhesion [152]. Larval secretions have long been thought to contain novel antimi-crobial factors. One was recently identified as an insect defensin, lucifensin [150]. This molecule is predicted to be responsible for protecting larvae from infection in grossly contaminated environ-ments, as well as inhibiting pathogenic bacteria within the wound.
Conclusions
The range of natural products that possess properties of potential therapeutic and pharmaceutical value seems to be extensive. Only a few have been described here and most of these have not been accepted unconditionally. In the food industry nisin is an estab-lished preservative; in wound care honey and maggots have been incorporated into treatment plans in some healthcare facilities, but they are rarely the first choice when antimicrobial interven-tion is indicated. Garlic promises to be of value in eradicating persistent infections in which biofilms have been implicated. At present the potential of many natural products is unfulfilled. Modes of action for many of these products have not been eluci-dated and this can affect opportunities for commercial develop-ment. Like conventional antibiotics, the route to acceptance depends on licensing or registration, which in turn depends on demonstrating efficacy in vitro and in vivo and safety in terms of the absence of cytotoxicity, allergenicity and mutagenicity. Devel-opment costs will, therefore, be not inconsiderable and manufac-turing costs are also likely to be high. Devising systems to standardize production and deliver consistent natural products may be challenging. Unlike conventional antibiotics, many natural products seem to offer beneficial effects that are not confined to antimicrobial activity, due to their often complex and undefined chemical composition. Furthermore their propensity to select for resistant strains of pathogens seems to be low, probably because their antimicrobial activity is often not confined to the action of a single active component on a specific target site. Honey illus-trates this point especially well [76, 104].
Since the adoption of evidence-based medicine during the 1990s, the introduction of all new interventions, whether syn-thetic or natural, must be supported by objective evidence to demonstrate that improved clinical outcomes will ensue. Natural products will, therefore, never be cheap.
References
1 Nelson, R. (2003) Antibiotic development pipeline runs dry. New drugs to fight
resistant organisms are not being developed, experts say. Lancet, 362,
1726–1727.
randomized clinical trail (VenUS II) conducted within the UK failed to demonstrate significant advantages of maggots over standard debridement therapy [149]. It did, however, indicate that maggots were no worse than routine sharp debridement.
Not all maggots are suitable for clinical use since some ingest living tissue. The larvae of the greenbottle (Lucilia sericata) are usually preferred because they rapidly ingest necrotic tissue without invading into internal organs. Following rearing under sterile conditions, they are available commercially and dispatched to customers in sterile containers with rapid delivery times. The number of larvae required for each wound depends on its dimen-sions and the quantity of necrotic tissue or slough present. Origi-nally maggots were applied “loose”, directly to the wound and kept in place with secondary dressings. More recently they are con-tained within sealed bags that are placed onto the wound bed. For some patients the thought of maggot therapy is unacceptable.
Larvae produce a complex mixture of bioactive components that contribute not only to bacterial inhibition but also to enhanced wound healing (Table 22.1.2). The most noticeable effect of larvae applied to wounds is their ability to rapidly remove (or debride) non-viable tissue which would normally impede healing. Larvae attach to the wound bed by hooks, where they secrete proteolytic enzymes that degrade extracellular matrix components and they absorb solubilized products, rather than grazing on non-viable tissue [154, 157].
Movement of larvae within the wound creates mechanical stim-ulation that promotes increased exudation, which in turn physi-cally displaces material away from the wound bed. Larval ingestion of bacteria reduces numbers in the wound and the ingested bacte-ria lose viability during passage through the insect hindgut. Larvae also cause wound pH to increase by excreting alkaline waste pro-ducts and this is thought to slow bacterial growth [151]. Anti-
Table 22.1.2 Therapeutic properties of maggots in relation to wounds.
