The role of clays in human health has experienced a revival in interest due to advances in modern instrumentation [e.g., transmission electron microscopes (TEM), field emission scanning electron microscopes (FESEM), atomic force microscopy (AFM), and secondary ion mass spectrometers (SIMS)], that allow us to study surfaces of nano-scale minerals in situ within their natural environmental matrix. By identifying the special characteristics that make a particular clay antibacterial, we may elucidate some of the reasons these common nano-minerals not only have potential applications in medicine, but may also contribute to the general understanding of antibacterial mechanisms lending insights to potential cures.
Recent reviews regarding uses of clay in achieving and maintaining human health have focused on the ancient practice of geophagy, which is the practice of eating earth materials containing clay minerals (Wilson, 2003; Ferrell, 2009). The purpose of geophagy is to elicit a healing response in humans through ingesting the easily available materials that may physically soothe an infected and inflamed gastrointestinal lining (Droy-Lefaix et al., 2006). Alternatively, clays have been used topically in mud spas (pelotherapy) to adsorb toxins from skin and provide heat to stimulate circulation for rheumatism treatment (Carretero et al., 2006; Gomes et al., 2007). Historical accounts of humans using ‘healing clay’ began with Aristotle (384-322 BC) (Mahaney et al., 2000) and Pliny the Elder (23–79 AD) later recounted the cure of intestinal ailments by ingestion of volcanic muds (Carretero et al., 2006). Of the many historical accounts of clays, muds, and soils used by people for ‘healing’, the scientific evidence of the action of clays for treating and healing ulcers, tumors, cysts, cancers, osteoporosis, etc, is lacking.
Nonetheless, the observations of humans who have been ‘cured’ of illness by clay applications and the correlating photographic documentation (Brunet de Courssou, 2002; Williams et al., 2004) were the stimuli for our research into the healing mechanism of clays. We provide a general compilation of the descriptions of healing and the common mineralogical attributes of clays and clay minerals used in the past.
In 2002, Line Brunet de Courssou reported to the World Health Organization (WHO), a summary of ~10 years of work in the Ivory Coast of Africa (east Africa), where she documented the use of specific clay minerals as therapeutic treatment of advanced Buruli ulcer disease (Brunet de Courrsou, 2002). However, she was unable to conduct additional experiments of traditional and accepted scientific studies due to lack of financial support. There have been several reports describing the antibacterial properties of natural and synthetic clay minerals (Herrera et al., 2000; Hu et al., 2005; Tong et al., 2005; Williams et al., 2004; Wilson, 2003; Haydel, 2008). Despite these studies and the clinical evidence suggesting that clay minerals promote healing in individuals infected with Mycobacterium ulcerans, the chemical interaction occurring at the clay mineral – bacterial interface, and the mechanism by which the clay minerals inhibit bacterial growth remains unknown.
Our research into the healing process lead us to propose in vitro testing of the effect of ‘healing clays’ on a broad spectrum of human bacterial pathogens. We make an important distinction between ‘healing clays’ and ‘antibacterial clays’. While clays may heal various ailments by their unique physical properties (e.g., high absorbance, surface area, heat capacity, exchange capacity, etc.), we have identified only a few natural clays that kill pathogenic bacteria. Building on the recent literature on healing and antibacterial minerals and compounds, this paper reviews the methods and analytical protocols we have established for investigating the antibacterial properties of clays.
Traditional Uses of Clays for Human Health
Healing practices of ancient cultures, as well as modern society, have depended on clay minerals with powerful adsorptive and absorptive properties to treat a variety of topical maladies. Adsorption is the process of attraction, binding, and accumulation of molecules or particles to a solid surface in a condensed layer. Absorption results when a substance diffuses or penetrates into a liquid or solid forming a transition zone or layer, often with a new composition, adjacent the substrate. Clay minerals are ubiquitous in nature and their adsorptive and absorptive capabilities have been exploited in a variety of cosmetics and pharmaceutical formulations. Traditionally, clay minerals are mixed with water for various periods of time (days to years) to form clay gels or pastes that can be applied externally for cosmetic or skin protective purposes (Carretero, 2002; Carretaro et al., 2007; Gomes et al., 2007). The high adsorption and absorption capacities, cation exchange capacity, as well as the extremely fine particle size of certain clays, e.g. smectites (expandable clay minerals) and kaolin group minerals are important reasons why these minerals are used to remove oils, secretions, toxins, and contaminants from the skin. By adsorbing and absorbing moisture and impurities from the skin, the clays also serve to cleanse and refresh the skin surface and to aid in the healing of topical blemishes, the major selling point for many cosmetics. Although consumers generally consider clays to be safe when applied topically, it is important to recognize that cosmetic firms must substantiate the safety of their products and that the U.S. Food and Drug Agency does not subject these products to pre-market approval.
The intentional consumption of earth materials, such as clays, by humans and animals is known as geophagy (Wilson, 2003). This complex and poorly understood practice is largely attributed to religious beliefs, cultural practices, psychological disorders, dietary/nutritional needs, and medicinal benefits (Abrahams et al., 1996; Aufreiter et al., 1997; Hunter, 1973). Although often viewed as an abnormal behavior by medical practitioners (i.e., pica), geophagy is believed to be an adaptive phenomenon in mammals and primates and a learned behavior in various societal cultures (Klaus and Schmidt, 1998; Krishnamani and Mahaney, 2000). Historically, geophagy is believed to be practiced to remedy a physiological response to mineral nutrient deficiencies, such iron or zinc, to satisfy a dietary craving, and to ease psychosocial problems, including anxiety, stress, and obsessive-compulsive disorder (Lacey, 1990; Sayetta, 1986). However, several studies and medical reports indicate that ingesting large amounts of clay (>200 grams per day), particularly clay with a high cation-exchange capacity, can impede absorption of iron, zinc, and potassium, leading to iron, zinc, or potassium deficiencies (Arcasoy et al., 1978; Cavdar and Arcasoy, 1972; Cavdar et al., 1977a,b; Gonzalez et al., 1982; Mengel et al., 1964; Minnich et al., 1968; Severance et al., 1988; Ukaonu et al., 2003).
The ingestion of dried clay minerals or a clay suspension is commonly used as a source of dietary elements, as a detoxifying agent, and as an allopathic treatment of gastrointestinal illnesses and acute and chronic diarrhea (Carretero, 2002). For example in Ghana, the iron, copper, calcium, zinc, and manganese consumed in clays were in the range of 2 to 15 percent of recommended dietary allowances (Hunter, 1973) and it was concluded that moderate ingestion of clays lacking high cation-exchange capacities could serve as a nutritional supplement for these essential elements. In the acidic environment of the stomach, the clay minerals could bind to positively charged toxins and serve as detoxifying agents to reduce bioavailability interfering with gastrointestinal absorption of the toxin (Hladik and Gueguen, 1974; Johns and Duquette, 1991; Mahaney et al., 1996; Phillips, 1999; Phillips et al., 1995). Over-the-counter pharmaceuticals that originally contained kaolinite, attapulgite, or clay-like substances (i.e. Kaopectate®) represent classic examples of the use of clay minerals by human populations to treat diarrhea and intestinal illnesses (Vermeer and Ferrell, 1985) and soothe gastrointestinal ailments. (Note: Kaopectate was reformulated in 2002 and now contains bismuth subsalicylate instead of kaolinite or attapugite.) Kaolinite has many medically beneficial attributes primarily related to its ability to adsorb lipids, proteins, bacteria, and viruses (Adamis and Timar, 1980; Lipson and Stotzky, 1983; Schiffenbauer and Stotzky, 1982; Steel and Anderson, 1972; Wallace et al., 1975).
Geology and Geochemistry
The study of medicinal applications of minerals requires collaborative efforts by many specialists with diverse expertise and educational backgrounds including, for example, geology, geochemistry, microbiology, environmental science, soils and agriculture, medicine, statistics, and pharmacology to name a few. Understanding the interactions of natural minerals and microbial systems is an immense undertaking, so there is no limit to the efforts of diverse scientific disciplines in these arenas. Because of this it is important to establish a basis of communication among the diverse scientific groups. Here we present some back ground and define some common terms used by geologists/mineralogists and clinical microbiologists in attempt to alleviate misconceptions across disciplinary boundaries.
Clays and Clay Minerals
First and foremost, it is essential to define what clay is and how it differs from mud, soil, and minerals. In the field of geology clay is a size-based term for very fine-grained minerals with an estimated spherical diameter <2.0 µm and approximate density of 2.65 g/cm3 as defined by Stokes Law (Jackson, 1979; Moore et al., 1997). As gravel, sand, and silt are terms for sedimentary grain sizes, clay is the term for the finest fraction of sediments that all consist of accumulations of different minerals (e.g., quartz, feldspar, carbonates, etc.) and organic matter. Clay, when moistened with water, creates a mud-like consistency, comparable to that used in spas for pelotherapy. Soils, often comprise many minerals and especially clay-sized minerals and organic matter (humus), as this medium must be able to support life by exchange of ions through water and gas that fills the spaces between the solid particles (Voroney, 2006). Mud is a slurry of water and sediment dominated by clay and silt sized particles. A mineral is a natural solid with a generally uniform composition and repeating internal crystalline order. When the crystalline domains are nanometers in scale, they are sometimes referred to as disordered or poorly crystalline. Clay minerals exhibit a wide range of order/disorder and crystallinity.
Most of the clay-sized fraction of sediments consists of clay minerals or phyllosilicates (defined below). Clay minerals are formed by weathering of other silicate minerals on and in the earth’s crust, or they may precipitate directly from a solution. Minerals that form deep in the earth are likely to be unstable under the lower temperature and pressure conditions on earth’s surface. When water and carbon dioxide from the atmosphere and from soil respiration interact with these minerals, they may dissolve often if the waters are acidic (carbonic acid) and the leached components precipitate as a variety of clay minerals (Giese and van Oss, 2002).
Most of the healing clays described in the literature (e.g., Carretero and Lagaly, 2007 and papers therein) are bentonites. However, bentonite is not a mineral but is a generic term for rocks derived from ‘altered volcanic ash beds’. The ash is a layered sedimentary deposit that accumulates during volcanic eruptions. This ash produces mostly disordered solids but glassy particles formed by rapid quenching (cooling) of magma when the liquid material is thrown into the atmosphere. The volcanic ash layers, when compacted and infiltrated with water, alter primarily to kaolinite in acidic environments, smectite in mildly alkaline (seawater) environments, or zeolites in highly alkaline environments (Bohor and Triplehorn, 1993). The mineralogical variations occur depending on the chemical characteristics of the volcanic glass and the local water chemistry (Christidis, 1998).
Smectite is a group of expandable clay minerals with a variety of structural and chemical differences that affect their surface charge and chemistry. Montmorillonite, commonly identified in ‘healing clays’, is one type of smectite. It has a structure of 2 tetrahedral sheets that sandwich a single octahedral sheet (). The 2:1 structure forms layers that stack like cards with the space between cards called the ‘interlayer’. The collective of interlayer, octahedral, and tetrahedral sites of montmorillonite forms a mineral with an ideal chemical formula; R0.33+ (Al1.67 Mg0.33) Si4 O10 (OH)2 (where R represents interlayer cations; Moore and Reynolds, 1997). Many elements can substitute into these various structural sites. In addition water and organic compounds may be found in the interlayer sites and/or adsorbed on the edges and exterior surfaces of the crystal structure. Zeolites have properties similar to clays, but form tubes or cage-like structures that can also incorporate a variety of molecules and ions. Over time, if the bentonite is deeply buried and subjected to an increase in temperature, or if hot volcanic water percolates through the ash layer, the smectites will alter to form illites or other minerals that are stable under the higher temperature conditions. The point here is that a bentonite usually does not consist of a single pure smectite, and each natural deposit is mineralogically variable.
Schematic representation of the basic structure of a clay mineral, showing tetrahedral and octahedral arrangements of atoms in sheets, separated by cations (large spheres) and water in the interlayer between the silicate sheets. Small spheres are H+. (modified from Giese and van Oss, 2004)
Bentonite deposits are found worldwide wherever volcanic eruptions have taken place and preservation of the ash has exceeded erosion. Ten billion tons of bentonite are mined worldwide each year with about 35% produced in the western United States (primarily Wyoming). The second largest producer of bentonite is Greece (Grim and Güven, 1978), however, significant reserves are also found in many countries; Italy (Sardinia), India, China, and Australia to name a few. Many of the clays used in pelotherapy come from these deposits and localities (Cara et al., 2000; Veniale et al., 2007).
While natural clay samples contain a variety of minerals and organic compounds, clay minerals can often be isolated from the sample by selecting the finest size fraction (<0.2 µm). It is very difficult to remove silica from the finest clay fractions, but other detrital minerals (feldspars, micas, carbonates, etc) are usually concentrated in the coarser fractions (1–2 µm) (Jackson, 1979; Moore and Reynolds, 1997).
Clay minerals are phyllosilicates (phyllo is Greek for ‘leaf’ or layers) consisting of layers of silicates arranged in tetrahedral and octahedral sheets (). The clay silicate layers consist of sheets containing hexagonal rings of SiO4 (silicate tetrahedra) stacked on octahedral sheets containing primarily Al, Mg, and/or Fe bound to two planes of closest packed oxygen atoms and/or hydroxyl groups. The edge sharing octahedra may be filled with two trivalent (di-octahedral) or three divalent (tri-octahedral) cations for a total charge of +6 (Giese and van Oss, 2002). This charge is partially balanced by the −2 charge on oxygens, but the total charge balance depends on the structural arrangement and precise elemental substitutions within the silicate framework.
Kaolinite (the predominant mineral employed for making porcelain) and its polymorphs are 1:1 clay minerals i.e. they have layers consisting of one tetrahedral sheet and one octahedral sheet. Smectite is one example of 2:1 clay minerals with layers containing an octahedral sheet sandwiched between two tetrahedral sheets (). The stack of two tetrahedral sheets to one octahedral sheet, has an interlayer with water, cations, and molecules of highly variable chemical composition. Layers of the 2:1 clay minerals may be stacked in a variety of orientations and held together by electrostatic and van der Waals forces (Brindley and Brown, 1980; Moore and Reynolds, 1997). When trivalent ions (e.g., Al3+) substitute for tetravalent Si4+ in the tetrahedral sheets, or divalent ions (Fe2+) replace Al3+ in the octahedral sheets, the basal surface (bottom plane of the tetrahedral sheets) develops a net negative charge. The magnitude and distribution of this charge can vary depending on where the substitutions take place (Güven and Pallastro, 1992; Johnston, 1996). Smectites are the most common alteration product of bentonites, and many varieties of smectite are present in the antibacterial clays. Cations are attracted to the negative surface of the silicate sheets, whereas anions are attracted to the positively charged sites where bonding take place at the edges of the crystalline structure (Moore and Reynolds, 1997). If this region collapses (water is removed) due to a high surface charge attracting cations (primarily K+) to interlayer sites, it forms an illite.
Chlorite is another group of layered silicates but with hydroxide sheets (e.g., MgOH3) between the 2:1 silicate layers, and are known as 2:1:1 minerals. Giese and van Oss, (2002) present excellent diagrams of the crystallography of the major classes of clay minerals. Most natural clay samples contain mixtures of these four major groups of phyllosilicates; smectite, illite, kaolinite and chlorite. summarizes the basic mineralogical classification of phyllosilicates. Within the 2:1 clays, the layer charge (interlayer cation occupancy) is used to distinguish the major clay sub-categories. Nonetheless, there are vast chemical substitutions and structural re-arrangements in clay minerals that result in the wide variety found in nature.
(antigorite, crysotile, lizardite)
Crystal Growth and Chemical Variability
The growth of clay crystals and changes that may occur in their chemistry and structure is complex (see reviews in Altaner and Ylagan, 1997; Srodon, 1999). However, it has been shown for sedimentary environments that clay minerals precipitate as small particles (<10 nm) and grow in diameter over time as water provides a continuous supply of components or ‘building blocks’ to the structure (Eberl et al., 1998; Nadeau et al., 1984). The crystallite size distribution of clay minerals in most natural samples is log-normal, meaning there are more small crystals than large crystals (Eberl et al., 2002). This distribution has been shown to result from the stunted growth of a large number of nucleated crystals from which a limited number of the clay crystallites continue to grow. Clay crystals can grow in a variety of morphologies depending on the temperatures and pressures of their geologic environment. At low temperatures (~50°C,) the crystallites tend to be irregular flakes. As temperature increases, rectangular laths may be found, and finally hexagonal plates may form at >100°C (Inoue et al., 1988; Lanson and Champion, 1991). The crystallite growth usually incorporates other dissolved elements into the clay structure, therefore a change in the water chemistry over time may be recorded in different sized crystals of clay (Clauer et al., 1997; Srodon, 1999; Williams et al., 2006).
Medicinal Uses of Clays
Most uses and research emphasis on healing clays has focused on the physical characteristics of clay minerals that benefit digestion or protect and cleanse the skin (Carretero, 2002). The adsorptive and absorptive properties of clay minerals have historically been the driving force behind the traditional use of healing and therapeutic clays. Initially, negatively-charged interlayer sites of clays will absorb positively-charged substances to their extensive surface area. Over time, many clay minerals may absorb substances in between the stacked silicate layers of the mineral, allowing for expansion and swelling or contraction. While the physical adsorption of water and organic matter is the most common attribute of healing clays, the geochemical mechanisms controlling antibacterial properties of clays have received significantly less attention.
It is well known that metallic ions, such as silver, copper, and zinc, have strong inhibitory and bactericidal effects on a broad spectrum of bacteria (Berger et al., 1976; Domek et al., 1984; Gordon et al., 1994). Various forms of silver ions have been used to treat burn wound infections, osteomyelitis, urinary tract infections, and central venous catheter infections (Becker and Spadaro, 1978; Davenport and Keeley, 2005; Fox, 1968; Fox and Modak, 1974; Jansen et al., 1994; Liedberg and Lundeberg, 1990). In low concentrations (4 µg/ml), silver ions produced inhibitory and bactericidal effects with no obvious toxic effect on human blood cells (Berger et al., 1976). Although required by most living organisms at low concentrations, elevated levels of copper can inhibit the growth of some microorganisms and exhibit bactericidal activity (Domek et al., 1984; Gordon et al., 1994). The use of copper-coated products or copper alloys has been proposed for surfaces exposed to human contact to reduce the transmission of infectious microbial agents. Other metallic oxides, including zinc oxide, magnesium oxide, and calcium oxide, have antibacterial activity with demonstrated effectiveness against E. coli and S. aureus (Sawai, 2003). The nanometer particle size of these oxides, as well as titanium and silicon dioxide (Yamamoto et al., 2000; Yamamoto, 2001; Adams et al., 2006), have proven to be important for antibacterial activity. Zinc oxide has been used in a variety of dental composites to treat or prevent dental caries and as an endodontic sealer (Turkheim, 1955; Moorer and Genet, 1982; Siqueira and Goncalves, 1996). Nonetheless, the antibacterial mechanism has not been identified.
The high cation exchange capacity of different clay minerals has been targeted in the creation of inorganic antibacterial materials. Synthetic antibacterial clay minerals are prepared by exchanging their native ions with known antibacterial ions such Ag (Ohashi, 1992; Ohasi et al., 1998; Marini et al., 2007). The rational being that the novel exchanged ions will be gradually released from the synthetic clay for long-term antibacterial effectiveness. Thus far, silver-loaded clays have been pursued more aggressively than other antibacterial chemical ion options, although copper-loaded mineral substrates have been recently investigated (Gant et al., 2007).
The mineral group zeolites, also have vast adsorptive and absorptive capabilities through a rigid, porous, three-dimensional channel or tube in their basic cryalline structure. They are compositionally similar to clay minerals and also have a high cation exchange capacity (Baerlocher et al., 2007). With strong affinity for oxidized silver ions (Ag+), up to 40% (w/w), silver-exchanged zeolites have demonstrated antibacterial effectiveness against aerobic and anaerobic Gram-negative and Gram-positive bacterial pathogens, including Pseudomonas aeruginosa, Porphyromonas gingivalis, Prevotella intermedia, Staphylococcus aureus, Streptococcus mutans, and Streptococcus sanguis and have been used in various dental applications (Hotta et al., 1998; Kawahara et al., 2000; Matsuura et al., 1997). Another study investigated the mechanism of action of silver-exchanged zeolite and determined that physical contact with the bacterial cell, silver transfer into the cell, and/or the generation of reactive oxygen species attributed to the bactericidal activity of silver zeolite (Matsumura et al., 2003). However, while these inorganic products have antibacterial activity and low toxicity, issues related to the reduction of silver ions to elemental silver and subsequent loss of antibacterial effectiveness could be problematic (Li et al., 2002). Therefore, an oxidized form of Ag appears to be important for bactericidal activity of silver-exchanged zeolites.
Vermiculite is another clay mineral that has been targeted for medicinal uses. Vermiculite is a smectite-like mineral, but with a high layer charge, thus a high attraction for cations (Mathieson and Walker, 1954; Mathieson, 1958). As an alternative to silver, it is speculated that copper-loaded vermiculite could offer reduced costs, improved stability, and better antifungal activity (Li et al., 2002). As demonstrated by antimicrobial testing, a copper-exchanged vermiculite (containing 5.5% Cu2+), as well as 200°C– and 400°C-heated copper-exchanged vermiculite, inhibited growth of E. coli (Li et al., 2002).
Natural Antibacterial Clays
Unlike the synthetic clays and chemical manipulations of clay minerals manufactured to kill certain types of environmental and clinical bacteria, we focus now on natural, untreated clay minerals that are effective at killing a broad spectrum of human pathogens. The detailed results have been published elsewhere (Haydel et al., 2008; Williams et al., 2008), but herein we outline the approach we use to evaluate the antibacterial activity of clays. schematically summarizes our approach to investigating the antibacterial properties of natural clays. We evaluated the effect of various clays and clay minerals on a broad spectrum of bacteria and determined whether the bactericidal effect was via a physical or chemical attack on the various pathogens. Again, our overarching goal is to understand how clay minerals kill bacteria in order to create new topical treatments for antibiotic-resistant skin infections and for large, necrotic skin ulcers. If we can identify the mechanism by which natural clay minerals kill a wide range of bacterial species, then it is possible that either natural clay deposits could be located and made available as an inexpensive treatment modality for topical infections or that materials with similar properties could be designed to provide a safe alternative to current antibiotics and antimicrobials.
General scheme for evaluating and testing antibacterial clays.
Clinical Observations of Natural Clays Healing Patients with Buruli ulcer
Within the past decade, the incidence of Buruli ulcer has dramatically increased in several countries in sub-Saharan Africa, Australia, Asia, Mexico, and Peru, leading the WHO to declare this disease a global health threat in 2004 (WHO, 1998). Despite being the third most common mycobacterial disease in immunocompetent humans after tuberculosis and leprosy (Weir, 2002), Buruli ulcer, considered a “neglected tropical disease” by the WHO, does not garner the attention given to other infectious diseases. Although the exact prevalence and burden of the disease are difficult to determine, Buruli ulcer is endemic in central and western Africa with more than 40,000 Buruli ulcer cases recorded in the African countries of Ivory Coast, Ghana, and Benin from 1978 to 2006 and some villages reporting rates as high as 16–22% (Amofah et al., 1993; Marston et al., 1995; WHO, 2007). The WHO estimates that the incidence of Buruli ulcer will surpass that of leprosy and could become more problematic than tuberculosis in some African regions (van der Werf et al., 1999; WHO, 2007).
M. ulcerans is a slow-growing environmental mycobacterium with an unknown source or natural reservoir (Dobos et al., 1999). Human transmission is believed to occur via the skin by direct inoculation or an insect vector (Portaels and Meyers, 1999; Weir, 2002). Most individuals infected with M. ulcerans initially develop a small, painless, pre-ulcerative skin nodule or plaques with larger areas of indurated skin and edema (van der Werf et al., 1999). As the disease progresses over 1–2 months, the infected skin will begin to ulcerate with characteristic necrosis of the subcutaneous fatty tissues, deeply undermined edges, and vascular blockage. These necrotic ulcers can lead to very extensive skin loss, damage to nerves, blood vessels, and appendages, and deformity and disability, particularly in children (van der Werf et al., 1999; Weir, 2002). One study reported that 26% of patients with healed Buruli ulcers suffered from chronic functional disability (Marston et al., 1995).
Currently, treatment of Buruli ulcer, depending on the size of the lesion, includes antibiotic therapy and/or surgical excision of the ulcerative lesion. Thirty years ago, variable successes in uncontrolled trials were demonstrated with heat treatment and hyperbaric oxygen treatment (Krieg et al., 1975; Meyers et al., 1974). However, the impracticality and costs of these treatments abrogate its usefulness in endemic and underprivileged populations. Phillips et al. (2004) used topical aqueous creams releasing nitrogen oxides to decrease the size of ulcers with minimal adverse side effects. Recently, Chauty et al. (2007) determined that a combination of rifampin (taken orally) and streptomycin (injected intramuscularly) successfully treated 47% of the cases and was more effective against small Buruli ulcer lesions (<5 cm in diameter). In addition, rifampin and streptomycin treatment converted early M. ulcerans lesions (nodules and plaques) from culture positive to culture negative (Etuaful et al., 2005). According to WHO guidelines (2004), combined use of streptomycin and rifampin is the recommended treatment of early Buruli ulcer lesions (nodules, papules, plaques, and ulcers less than 5 cm in diameter). For treatment of ulcerative lesions greater than 5 cm in diameter, combined antibiotic therapy for four weeks, followed by surgical excision of the lesion and another four weeks of antibiotic treatment is recommended (WHO, 2004). While surgery is a standard treatment for large ulcerative lesions, antibiotic therapy reduces the extent of surgical excision and infection recurrence (WHO, 2004). Since surgical treatment is often not available or practical in rural, endemic regions and possibly subjects patients to other infections, development of an effective and affordable drug treatment and new treatment modalities is a research priority for the control of Buruli ulcer (WHO, 2007).
The treatment of Buruli ulcer by Line Brunet de Courssou employed two clay samples provided by different suppliers of ‘French green clay’ (Brunet de Courrsou, 2002). These clays are thought to be altered volcanic ash deposits from Central France. The dry clay is mixed with water and applied as a paste directly to the ulcerated lesions and extended healthy skin of infected patients. The course of treatment is to remove and renew the clay packs at least once a day. Within days of initiating treatment with clay poultices, the therapeutic properties of the clay minerals were demonstrated, with the initiation of rapid, non-surgical debridement of the destroyed tissue. Extended treatment with the clay poultices resulted in continued debridement of the ulcer, regeneration of healthy tissue, and wound healing. After several months of daily clay applications, the Buruli ulcer wounds healed with soft, supple scarring, allowing return of normal motor function (Brunet de Courrsou, 2002; Williams et al., 2004). These observations are highly relevant since antibiotic treatment is only effective for small early lesions (nodules and plaques <5 cm in diameter) and has generally been unsuccessful as the sole treatment for larger, ulcerative forms of Buruli ulcer disease (Chauty et al., 2007; van der Werf et al., 1999).
The mechanism by which natural clay minerals kill bacteria should be understood in order to search for the least expensive cure modalities and specifically to maximize effective antibacterial agents. Therefore, we have been investigating the antibacterial properties of several different natural clays, including the two French clays used by Brunet de Courssou, referred to as CsAr02 and CsAg02. We have tested these clays on a series of Gram-negative, Gram-positive, and mycobacterial pathogens using the technique of broth culture susceptibility testing. The cell envelopes of these bacteria greatly differ and these differences could influence their vulnerability and response to inorganic materials. Compositionally, the three groups of bacteria, are similar with a cytoplasmic membrane and a rigid polysaccharide-peptide cell wall called peptidoglycan (). However, the Gram-positive cell wall is considerably thicker than the Gram-negative cell wall. The thinner Gram-negative cell wall is covered by an outer membrane, composed of phospholipids and lipopolysaccharides, and has a periplasmic space between the cell membrane and the outer membrane (). Genetically, mycobacteria are Gram-positive bacteria. However, the mycobacterial cell envelope is more structurally similar to Gram-negative bacteria. In addition to having a thinner cell wall than traditional Gram-positive bacteria, the mycobacterial peptidoglycan layer is linked to a waxy lipid coating by an arabinogalactan layer. This outer waxy layer is predominantly composed of special lipids called mycolic acids, lipoarabinomannan, and glycolipids (). The different cell types have distinct cell envelopes that play a significant role in the effectiveness of various antimicrobial agents
Schematic representation of the cell envelopes of Gram-positive bacteria, Gram-negative bacteria, and mycobacteria. LPS, lipopolysaccharide. SOURCE?
The use of antibiotics and chemotherapeutic agents during the past century represents one of the greatest advances in human health and has led to a remarkable reduction of morbidity and mortality related to bacterial infections. In modern medicine, antibacterial, antimicrobial, and chemotherapeutic agents are terms used to describe chemical agents effective at treating infectious diseases. Most of these agents are antibiotics, which by definition are low molecular weight byproducts of microorganisms that kill or inhibit the growth of other and susceptible microorganisms. The term ‘antibiotic’ is often incorrectly used to describe antibacterial or chemotherapeutic agents that are synthetically manufactured or modified by chemical processes, independent of microbial activity, to optimize their activity. Although the antibacterial clay minerals discussed herein are natural substances, they are not produced by microorganisms and are not considered antibiotics. In an ideal situation, antimicrobial agents disrupt microbial processes or structures that largely differ from those of the host. The majority of known antimicrobials function by affecting cell wall synthesis, inhibiting protein and nucleic acid synthesis, disrupting membrane structure and function, and inhibiting key enzymes essential for various microbial metabolic pathways.
Chemotherapeutic or antibacterial agents can be either bacteriostatic or bactericidal. A bacteriostatic agent reversibly inhibits microbial growth and microorganisms will resume growth upon removal of the bacteriostatic agent. Since bacteriostatic antimicrobials do not kill the bacteria, elimination of the infection is dependent on the host’s resistance and immune response. When administered at sufficient levels, a bactericidal agent kills the targeted bacterial pathogen. However, it is important to realize that the effectiveness of the antimicrobial is largely dependent on the targeted bacterium. An antimicrobial agent that is bactericidal for one particular bacterial species may be bacteriostatic for another. Moreover, various antibacterial agents vary considerably in their range of effectiveness. A narrow-spectrum antibacterial is effective against a limited number of pathogens, usually Gram-positive or Gram-negative bacteria, but not both. A broad-spectrum antimicrobial is generally effective at destroying or inhibiting the growth of a wide range of Gram-positive and Gram-negative bacteria.
Effects of two natural clay minerals on bacterial growth
The extensive use of antibiotics has led to an increase in antibiotic resistance in many pathogenic and clinically-relevant bacteria, including Mycobacterium tuberculosis, S. aureus, Enterococcus faecalis, and Streptococcus pneumoniae (Menichetti, 2005; Shah, 2005; Sharma et al., 2005; Zetola et al., 2005). Therefore, modern and innovative research approaches are needed to identify and generate new antimicrobials for treating infections that are resistant to existing antibiotics or for which there is no known effective therapeutic agent. Using antibiotic-sensitive and antibiotic-resistant bacterial strains obtained from the American Type Culture Collection (ATCC) and a local diagnostic laboratory, we have been investigating the antibacterial properties of the two clays, CsAg02 and CsAr02, used to treat Buruli ulcer patients. The CsAr02 mineral promoted or had no effect on bacterial growth (Haydel et al., 2008). In contrast, CsAg02 exhibits an extraordinary ability to kill pathogenic E. coli, Salmonella enterica serovar Typhimurium, P. aeruginosa, ESBL E. coli (which is resistant to 11 antibiotics), and Mycobacterium marinum (a species genetically closely related to M. ulcerans that also causes a cutaneous infection) as well as inhibit the growth of pathogenic S. aureus, penicillin-resistant S. aureus (PRSA), methicillin-resistant S. aureus (MRSA; and also resistant to 10 antibiotics) (Haydel et al., 2008). During the course of her observations, Brunet de Courssou suggested that the CsAg02 clay was not as effective in killing M. ulcerans as the CsAr02 clay (although this was not demonstrated microbiologically), but was more suited for promoting skin granulation after the mycobacteria were killed (Brunet de Courrsou, 2002). The scientific basis of the therapeutic differences or the healing characteristics of these two clay minerals is still under investigation.
Broad-spectrum in vitro Susceptibility Testing
In vitro broad-spectrum antimicrobial activities of clay minerals were tested against bacterial strains that are recommended by the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) as quality control strains for laboratory testing of antimicrobials (NCCLS, 2004). Bacteria are grown overnight in common laboratory liquid media and diluted with fresh medium to achieve an approximate initial density of ~107 bacteria per ml. Adjustment of each bacterial inoculum is performed using a spectrophotometer since the experimental microorganisms exhibit varying replication times in liquid media. To confirm the initial bacterial counts, serially-diluted bacterial cultures are plated on appropriate agar plates and colony-forming units are counted after incubation at 37°C. Before use in any susceptibility testing, all clay mineral samples are sterilized by autoclaving at 121°C (at 15 psi) for 1 h to assure removal of airborne, inherent, or contaminating microbes. To achieve a consistency similar to the hydrated clay poultices used to treat Buruli ulcer patients, 200 mg of clay minerals is added to 400 µl of the initial inoculum of bacteria in the appropriate growth medium. After the addition of the clay minerals, the bacteria - clay mineral mixtures are incubated in capped test tubes at 37°C in ambient air for 24 hours (NCCLS, 2004) with constant rotary agitation to ensure contact with the clay minerals and to prevent sedimentation. Positive controls for growth of bacteria in the absence of clay minerals are included in each series of independent experiments. To ensure that the clay samples were sterilized after autoclaving and maintained sterilization during storage, negative control growth experiments with clay minerals in liquid media are performed several times throughout the course of the study. These conditions revealed complete killing of E. coli, S. typhimurium, P. aeruginosa, and M. marinum by CsAg02 (Haydel et al., 2008).
Given that the clays were hydrated with water for treating Buruli ulcer lesions and are generally hydrated with water for therapeutic use, we have initiated “use-derived” antibacterial testing of the clay minerals whereby the clay and the bacteria are incubated together in a sterile, deionized water solution. After growth in a liquid medium, bacteria are washed twice and suspended (~107 CFU) in 1 ml of sterile deionized water before the addition of clay minerals and subsequent incubation at 37°C. Depending on the type of clay, poultice consistencies used for therapeutic purposes are generally ratios of 1:2 or 1:3 (clay:water). To determine the effect of quantity, various amounts of the clays can be added to one ml aliquots of the aqueous bacterial suspension. Bacteria-clay mineral suspensions are incubated rotating at 37°C for 24 hours at which time serial dilutions of all samples are plated to determine bacterial viability. Minimal bactericidal concentration (MBC) is defined as the lowest concentration of a particular antibacterial agent that kills ≥99.9% of the bacterial population in a liquid medium.
Mineralogical and Geochemical Assessment of Antibacterial Clays
Initial testing of the two French green clays (in 2003) showed that only one of the clays (CsAg02) killed a nonpathogenic, laboratory strain of E. coli even though the two clays, CsAg02 and CsAr02, were very similar in general mineralogy with the dominant portion of the sample consisting of clay minerals as set out in along with quartz, calcite, and feldspar (Williams et al., 2004). Subsquent testing of CsAg02 and CsAr02 against a pathogenic strain of E. coli revealed similar results (Haydel et al., 2008) and their mineralogical composition was very similar. The clay fraction was dominated by smectite, a group of expandable clay minerals, a common component of all of the antibacterial clays we have investigated. However, a substantial amount of illite is also present in the French clays. Notably, the illite-smectite crystals in the French clays are of extremely small size relative to natural illite and smectite reference materials from the Clay Minerals Repository (www.clays.org) at Purdue University (Fig. 4).
Comparison of illite and smectite textures and crystal size. The standard reference materials (top) are much coarser than the French clays (containing both illite and smectite). The very small crystal size may play a role in the antibacterial mechanisms of clays (SEM images by Lynda Williams).
Comparison of the mineralogy of the two French clays
|intermediate Microcline feldspar
||intermediate Microcline feldspar
|Albite feldspar (Cleavelandite)
||Albite feldspar (Cleavelandite)
|1Md illite (+ dioct mica & smectite)
||1Md illite (+ dioct mica & smectite)
|1M Illite (R>2; 88%I)
||1M Illite (R>2; 88%I)
||Phlogopite (2M1 )
Size Fractionation of Clay Minerals
In order to eliminate the possibility that other mineral phases were responsible for killing the bacteria, we separated sequentially smaller size fractions of the mineral mixture using centrifugation (Jackson, 1979). Evaluating the antibacterial effect of the coarse (1.0 –2.0 µm), medium (0.2–1.0 µm) and fine (<0.2 µm) size fractions independently against E. coli allowed us to eliminate detrital minerals (quartz, carbonate, feldspar) from the clays as potential participants in the antibacterial effect. We found that only the finest fraction (<0.2 µm) was effective against E. coli, while the coarser fractions had no effect on bacterial growth (Haydel et al., 2008; Williams et al., 2007). Furthermore, X-ray diffraction analyses of the finest clay fraction confirmed that the coarser detrital mineral phases had been largely eliminated. Smectite dominated the <0.2 µm fraction. shows two different morphologies of crystals in the <0.2 µm fractions of the French clays. The antibacterial clay has crystals as small as 20 nm, and this enormous relative surface area, controls the water chemistry when wet.
The antibacterial French clay (CsAg02) has two clay crystal morphologies; 200nm diameter hexagonal plates (left sample analyzed by SEM, gold coated by L. Williams) and 20nm × 40nm rectangular laths (right sample uncoated on carbon planchet; by L. Garvie). The uncoated sample allows imaging of the very finest crystallites in the clay.
Others have shown that particle size effects antibacterial activity for various oxides (e.g., ZnO, MgO), such that bactericidal activity increases with decreasing particle size (to 0.1 µm) (Yamamoto, 2001; Sawai, 2003). Adams et al. (2006) showed that nanoparticles of TiO, SiO, and ZnO are photosensitive, with the light promoting formation of reactive oxygen species. Further evaluation is needed to determine the potential role of oxides associated with the clay mineral surfaces in chemical reactions that could influence the survival of pathogens. One way to do this is to selectively chelate metal oxides out of the sample and evaluate the resulting antibacterial effect, or to remove hydroxyl and superoxide radicals before testing. Mossbaüer spectroscopy is another useful tool for evaluating oxide versus clay mineral–metal bonds in clay samples (Stucki et al., 1996).
Next, we need to determine if the fine clay fraction was killing E. coli by a physical or chemical effect. Were the clays physically impeding a metabolic process of the bacteria by surface attractive forces causing the clay to wrap around, penetrate, or otherwise destroy the cell walls? Or, was the clay producing a toxic chemical that precipitates on the cell walls or that enters the bacterial cell inducing cellular death? By inserting a dialysis tube filled with an antibacterial clay suspension into a bacterial population in liquid growth media, Metge et al. (2007) showed that the bacteria were killed without physically contacting the clay mineral surfaces. Given this observation, we leached the antibacterial clay with distilled, deionized water. Two grams of the antibacterial clay was ultrasonified (using a Braun microtip ultrasonic disaggregator) in 40 ml water, then shaken for 24 hrs to equilibrate. The clays were removed from suspension by centrifuging for 3 hrs at 20,000 rpm (SS-34 rotor). The leachate was then tested against bacteria. Compared to control samples of E. coli in distilled water, the clay leachate also killed E. coli. This evidence suggested that the antibacterial clay killed by providing a toxin. Nonetheless, after water leaching, the clay sample continued to kill E. coli. In fact, separation of the different size fractions of the clay also required multiple washings and centrifugation cycles in deionized water, which did not eliminate the bactericidal effects of the minerals. These results indicate that the antibacterial agent is not highly soluble.
Cation Exchange of Clay Minerals
The next test was to evaluate if the antibacterial agent(s) could be removed by cation exchange. Ions and molecules can be bound to the interlayer surface of the expandable clays or adsorbed on the edges. Cation exchange is performed by soaking the clay fraction of interest in a concentrated (1M) solution containing ‘preferred cations’ or ions with the best fit in ionic radius and charge for the interlayer exchange sites (Jackson, 1979). One can remove any chemical species that are not tightly bonded (fixed) in the clay structure. This is the commonly used procedure to determine the ‘cation exchange capacity’ of clay (Moore and Reynolds, 1997), but the process also removes ionic or molecular species that might be involved in the antibacterial mechanism. After cation exchange, we found that the smectite-rich clay samples (<0.2 µm fraction) no longer killed E. coli (Haydel et al., 2008), indicating that the antibacterial agent is linked to ions that are presumably in the exchangeable sites of the clay. However, the cation exchange will also affect the surface energy of the clay mineral and may affect the pH, of the clay surface (zero point of charge), so we attempted to evaluate these complicating factors.
One common way to remove interlayer water and hydroxyl groups from clays is by progressive heating. In general, the clay becomes dehydrated, as the interlayer water is removed, by heating to 200°C. At 550°C or more (depending on chemical bonding), most iron-rich smectites dehydroxylate, meaning that all hydroxyl bonds in the octahedral layer are broken (Heller-Kallai and Rozenson, 1980). However, it is important to note that the temperature for dehydroxylation varies depending on the clay mineral structure and composition (Bish and Duffy, 1990). Upon heating to 900°C, the clay mineral structure is effectively destroyed, leaving only the oxide components.
Progressive heat treatments were applied to the French antibacterial clay in order to eliminate some of the possible bactericidal elements from consideration. Heating to 200°C (24 hrs in air) removes volatile elements in addition to water, (including H, O, N, F, Cl, Br, I). Although it is important to consider how these elements are bound in the mineral structure, certainly their presence in the hydrated interlayer would be affected by temperatures leading to volatilization. Tests of the clay after heating to 200°C show that it still killed E. coli (Williams et al., 2008; Haydel et al., 2008). This heating step provides evidence that the antibacterial agent in the clay is not an inherent microorganism, as they would certainly be killed at this temperature, even if the clay had previously physically protected them.
Heating to 550°C (24 hrs in air) showed significant oxidation of the French antibacterial clay as it turned from green to orange-red due to oxidation of Fe in the octahedral sites of the clay (Williams et al., 2008). However, the clay still killed E. coli (Haydel et al., 2008). This high temperature would be expected to volatilize elements including S, P, and Hg if they were not tightly bound in the phyllosilicate structure. Furthermore, this step of the process verifies that the antibacterial agent is not an organic compound as they are certainly eliminated at this high temperature.
Heating to 900°C highly oxidized the French antibacterial clay (CsAg02) turning it a deep red and caused the clay to lose its antibacterial effectiveness (Williams et al., 2008; Haydel et al., 2008). The lost antibacterial effect could point to elements lost above 550°C as the active antibacterial agents, however the highly oxidized state may alter the toxicity of remaining elements, or recrystallization may change the availability of elements that were bactericidal before heating. The major oxides of both clay samples including Si, Al, Ca, Fe, Mg, Mn, and Ti remain, but the clays lose their coherent crystalline structure. This temperature leaves many refractory elements in oxidized form, but those lost between 550°C and 900°C include Na, K, As, Se, Rb, Cd, and Cs. Table 3 compares the abundances of the refractory elements in the two French clays before heating.
Comparison of refractory elements in the antibacterial and non-antibacterial French clay samples. Highlighted elements are those removed by heating above 550°C.
The conclusion from this heating analysis is that the more volatile elements or compounds are not necessary for the antibacterial action displayed by this particular clay. The experiments do not conclusively identify a toxic substance, but are a method for eliminating some of the variables from consideration.
All of the tests and chemical manipulations of the French antibacterial clay (CsAg02) leave elemental components in abundances that are well below the minimal inhibitory concentrations (MIC) reported to be toxic to E. coli (Dopson et al., 2003; Nies, 1999; Wackett et al., 2004) and other bacteria tested (Haydel et al., 2008). However, MICs are usually tested at a neutral or near neutral pH, which does not attest to the fact that the pH and oxidation state of the metals must also be considered. Metal speciation is critical to their bioavailability and the subsequent interaction with bacteria (Reeder and Schoonen, 2006). Therefore, in the future we aim to establish what chemical species are soluble under the pH and oxidation state buffered by the clay mineral surfaces in order to evaluate the element mobility of antibacterial clays (Tateo and Summa, 2006). The whole process of transferring elements from a clay surface through water to a cell membrane involves numerous chemical reactions and variables that can be affected not only by the source clay and water chemistry, but also by the surface complexation of chemical species on the bacteria (Barrok et al., 2005).
Cellular processes important for metabolism, nutrient transport, movement, and cell division are localized at the cell membrane, which is the reactive surface controlling chemical accommodation, and may vary with environment (Konhauser, 2007; Lalonde et al., 2008 a,b). The ability of a given bacterial species to modify its surface chemistry in order to adapt to various environmental stresses depends on the growth phase, regulatory networks, metabolic pathways, and chemical variables in the environment (Warren and Ferris, 1998; Lalonde et al., 2008 a,b). There are three general mechanisms by which bacteria can accommodate high concentrations of ions that may be toxic to the species (i) the ions may be expelled from the cell by efflux (Nies and Silver, 1995); (ii) the metal ions may complex into non-toxic molecules such as thiols (S-compounds) in the surrounding solution, or (iii) the metal ions may be reduced to a less toxic oxidation state in the cell (Nies, 1999). Unwanted reduced metal ions usually are eliminated from cells by an efflux system. However by application of antibacterial clay or its soluble compounds to the bacteria, there is the potential for precipitation of compounds that would inhibit efflux of toxins from bacteria. A geochemically-induced gradient in pH or oxidation state, imposed by the presence of the antibacterial clay mineral could clearly be damaging to the functions of even the most adaptive bacteria. Reactive oxygen species are another frontier for investigation of the possible inhibitors active against bacteria. Fenton-mediated reactions, for example, drive the oxidation of mineral-bound Fe2+ to generate hydroxyl radicals (Fenton, 1894; Schoonen et al., 2006; Cohn et al., 2006) that can damage cells. Furthermore, during active infections, it is important to consider the complexity of host-pathogen interactions, largely influenced by in vivo chemistry, in addition to the chemical interactions identified in vitro (Sahai and Schoonen, 2006). If we can integrate analytical protocols in the study of bacterial metabolism, adaptation, and pathogenesis with methods of mineral and chemical manipulation and characterization (see reviews in Banfield and Nealson, 1997; Reeder and Schoonen, 2006), the forward motion of identifying new antibacterial agents or processes will be much better served.
In this era when bacteria are developing antibiotic resistance to existing pharmacological agents, the potential for discovery of new broad-spectrum antibacterial agents, such as natural clay minerals, to combat pathogenic bacteria would be particularly advantageous. Analysis of the chemical interaction occurring at the clay mineral – bacterial interface is being explored and will be pertinent in understanding the mechanism by which the clay minerals can inhibit bacterial growth. Initial investigations indicate that particular natural clay minerals can have striking and very specific effects on microbial populations. These effects can range from enhanced microbial growth to complete growth inhibition, and these opposite effects can occur with clay minerals of similar structure and bulk crystal chemistries. The key antibacterial agent is likely a trace element or transition metal groups stabilized by the ability of particular clay minerals to buffer the aqueous speciation of those elements involved in the antibacterial process.
During the past 25 years, approximately 70% of newly discovered drugs introduced in the U.S. have been derived from natural products (Newman and Cragg, 2007). Topical treatments by clay minerals have considerable advantages over surgery or generalized antibiotic therapy due to the practical simplicity of the application in the area specifically affected, rather than ingestion of drugs with potential side effects. The broad-reaching impacts of antimicrobial mineral research with applications in topical antimicrobial dressings, wound care management, personal care, and animal care markets are obvious. The discovery that natural minerals harbor antibacterial properties should provide impetus for exploring terrestrial sources for novel therapeutic compounds. Often natural products, such as clays, which are heterogeneous in chemical composition and physical character, are rejected as therapeutic agents by regulatory agencies. Nevertheless, in comparison to organic antimicrobial agents, inorganic minerals are likely to be considerably more stable and heat resistant, making the development and use of inorganic antimicrobial agents particularly advantageous. Combining the availability of natural, potentially bioactive, resources with powerful combinatorial chemistry optimization methodologies could result in the development of new antibacterial agents to fight existing antibiotic-resistant infections and diseases for which there are no known therapeutic agents, such as advanced M. ulcerans infections.
Clearly understanding the antibacterial mechanism of natural clay is complex, and it is possible that no single mechanism or set of reaction pathways is uniquely responsible for the observed bactericidal activity. Our future work will focus on identifying general themes displayed by the interaction presented herein between problematical human pathogens (e.g., MRSA) and the natural clay minerals that have now been shown to kill such bacteria (Williams et al., 2008). To progress towards understanding how antibacterial clays can be effective for treating bacterial infections will require integrated mineralogical, chemical, and microbial studies
The work of Konhauser (2007) and his co-workers describe novel methods for studying environmental bacteria and their chemical interactions with minerals in sedimentary environments. Similar methods must be employed in clinical microbiological research on antibacterial minerals. For example, the forces of attraction between bacterial species and mineral surfaces (Bank and Giese, 2007), the formation of membrane vesicles and other mechanisms for accommodating environmental stresses (Mashburn-Warren et al., 2008), the response of various bacteria to oxidative and reductive variables, in addition to pH, should be evaluated. This new focus in medical geomicrobiology should grow exponentially, just as environmental microbiology has developed exponentially over the last three decades (Konhauser, 2007). Although the use of clays in human health has been promoted empirically and traditionally, perhaps since the beginning of mankind, our knowledge of natural mineral impacts on human pathogens is in its infancy. New technology for determining physical and chemical interactions in situ on the nano-scale will provide the keys to opening these doors (Skinner, 1997).
We thank Catherine Skinner who invited our contribution to this special issue. This paper benefited greatly from careful reviews by Paul Schroeder, John Smoglia and Catherine Skinner. This work would not have been possible without collaborative efforts on the part of all researchers involved. Funding was made possible by the NIH-National Center for Complementary and Alternative Medicine. We also thank Line and Thierry Brunet de Courssou who brought the antibacterial clays to our attention; students and assistants, including Amanda Turner, Christine Remenih, Tanya Borchardt; and technical support from the Center for Solid State Science and School of Life Sciences, in particular Lawrence Garvie and Dave Lowry who assisted with electron microscopy.
- Abrahams PW, Parsons JA. Geophagy in the tropics: a literature review. The Geographical Journal. 1996;162:63–72.
- Adamis Z, Timar M. Investigations of the effects of quartz, aluminum silicates and colliery dusts on peritoneal macrophages in vitro. In: Brown RC, Gormley IP, Chamberlain M, Davies R, editors. The in vitro effects of mineral dusts. London: Academic Press; 1980. pp. 13–18.
- Adams LK, Lyon DY, McIntosh A, Alvarez PJ. Comparative toxicity of nano-scale TiO2, SiO2 and ZnO water suspensions. Water Science and Technology. 2006;54:327–334. [PubMed]
- Altaner SP, Ylagan RF. Comparison of structural models of mixed-layer illite-smectite and reaction mechanisms of smectite illitization. Clays and Clay Minerals. 1997;45:517–533.
- Amofah GK, Sagoe-Moses C, Adjei-Acquah C, Frimpong EH. Epidemiology of Buruli ulcer in Amansie West district, Ghana. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1993;87:644–645. [PubMed]
- Arcasoy A, Cavdar AO, Babacan E. Decreased iron and zinc absorption in Turkish children with iron deficiency and geophagia. Acta Haematologica. 1978;60:76–84. [PubMed]
- Aufreiter S, Hancock RGV, Mahaney WC, Stambolic-Robb A, Sanmugadas K. Geochemistry and mineraology of soils eaten by humans. International Journal of Food Sciences and Nutrition. 1997;48:293–305.
- Baerlocher C, McCusker LB, Olson DH. Atlas of zeolite framework types. Amsterdam: Elsevier; 2007. p. 404.
- Banfield JF, Nealson KH, editors. Reviews in Mineralogy. v. 35. Mineralogical Society of America; 1997. Geomicrobiology: Interactions between microbes and minerals; p. 448.
- Barrok D, Turner BF, Fein JB. A universal surface complexation framework for modeling proton binding onto bacterial surfaces in geological settings. American Journal of Science. 2005;305:826–853.
- Becker RO, Spadaro JA. Treatment of orthopaedic infections with electrically generated silver ions. Journal of Bone and Joint Surgery. 1978;60:871–881. [PubMed]
- Berger TJ, Spadaro JA, Chapin SE, Becker RO. Electrically generated silver ions: quantitative effects on bacterial and mammalian cells. Antimicrobial Agents and Chemotherapy. 1976;9:357–358. [PMC free article] [PubMed]
- Bish DL, Duffy CJ. Thermogravimetric analysis of minerals. In: Stucki JW, Bish DL, editors. Clay Minerals Society Workshop Lectures: Thermal Analysis in Clay Science. Boulder, CO: The Clay Minerals Society; 1990. pp. 95–157.
- Brindley GW, Brown GC, editors. Crystal structures of clay minerals and their X-ray identification.London: Mineralogical Society; 1980. Pages.
- Brunet de Courrsou L. Study Group Report on Buruli Ulcer Treatment with Clay, 5th WHO Advisory Group Meeting on Buruli Ulcer. Geneva, Switzerland: 2002.
- Cara S, Carcangiu G, Padalino G, Palomba M, Tamanini M. The bentonites in pelotherapy: chemical, mineralogical and technological properties of materials from Sardinia deposits (Italy) Applied Clay Science. 2000;16:117–124.
- Carretero MI. Clay minerals and their beneficial effects upon human health. A review: Applied Clay Science. 2002;21:155–163.
- Carretero MI, Gomes CSF, Tateo F. Clays and human health. In: F. Bergaya F, Theng BKG, Lagaly G, editors. Handbook of Clay Science. Elsevier Ltd; 2006. pp. 717–741.
- Carretero MI, Lagaly G. Clays and health: an introduction. Applied Clay Science. 2007;36:1–3.
- Cavdar AO, Arcasoy A. Hematologic and biochemical studies of Turkish children with pica: A presumptive explanation for the syndrome of geophagia, iron deficiency anemia, hepatosplenomegaly and hypogonadism. Clinical Pediatrics. 1972;11:215–223. [PubMed]
- Cavdar AO, Arcasoy A, Cin S. Pica with zinc deficiency. Lancet. 1977a;1:761. [PubMed]
- Cavdar AO, Arcasoy A, Cin S. Zinc deficiency in Turkey. American Journal of Clinical Nutrition. 1977b;30:833–834. [PubMed]
- Chauty A, Ardant MF, Adeye A, Euverte H, Guedenon A, Johnson C, Aubry J, Nuermberger E, Grosset J. Promising clinical efficacy of streptomycin-rifampin combination for treatment of Buruli ulcer (Mycobacterium ulcerans disease) Antimicrobial Agents and Chemotherapy. 2007;51:4029–4035. [PMC free article] [PubMed]
- Christidis GE. Comparative study of the mobility of major and trace elements during alteration of an andesite and a rhyolite to bentonite in the islands of Milos and Kimolos, Agean, Greece. Clays and Clay Minerals. 1998;46:379–399.
- Clauer N, środoń J, Francu J, Sucha V. K-Ar dating of illite fundamental particles separated from illite-smectite. Clay Minerals. 1997;32:181–196.
- Cohn CA, Mueller S, Wimmer E, Leifer N, Greenbaum S, Strongin DE, Schoonen MAA. Pyrie-induced hydroxyl radical formation and its effect on nucleic acids. Geochemical Transactions. 2006:11. Open Access http://www.geochemicaltransactions.com/content/7/1/3. [PMC free article][PubMed]
- Davenport K, Keeley FX. Evidence for the use of silver-alloy-coated catheters. Journal of Hospital Infection. 2005;60:298–303. [PubMed]
- Dobos KM, Quinn FD, Ashford DA, Horsburgh CR, King CH. Emergence of a unique group of necrotizing mycobacterial diseases. Emerging Infectious Diseases. 1999;5:367–378.[PMC free article] [PubMed]
- Domek MJ, LeChevallier MW, Cameron SC, McFeters GA. Evidence for the role of copper in the injury process of coliform bacteria in drinking water. Applied and Environmental Microbiology. 1984;48:289–293. [PMC free article] [PubMed]
- Dopson M, Baker-Austin C, Koppineedi PR, Bond PL. Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic microorganisms. Microbiology. 2003;1490:1959–1970.[PubMed]
- Droy-Lefaix MT, Tateo F. Clays and Clay Minerals as Drugs. In: Bergaya F, Theng BKG, Lagaly G, editors. Handbook of Clay Science. Elsevier Ltd; 2006. pp. 743–752.
- Eberl DD, Drits VA, Srodon J. Deducing growth mechanisms for minerals from the shapes of crystal size distributions. American Journal of Science. 1998;298
- Eberl DD, Kile DE, Drits VA. On geological interpretations of crystal size distributions: Constant vs. proportionate growth. American Mineralogist. 2002;87:1325–1334.
- Etuaful S, Carbonnelle B, Grosset J, Lucas S, Horsfield C, Phillips R, Evans M, Ofori-Adjei D, Klustse E, Owusu-Boateng J, Amedofu GK, Awuah P, Ampadu E, Amofah G, Asiedu K, Wansbrough-Jones M. Efficacy of the combination rifampin-streptomycin in preventing growth of Mycobacterium ulcerans in early lesions of Buruli ulcer in humans. Antimicrobial Agents and Chemotherapy. 2005;49:3182–3186. [PMC free article] [PubMed]
- Fenton HJH. Oxidation of tartaric acid in the presence of iron. Journal of the Chemical Society. 1894;65:899–910.
- Ferrell RE. Medicinal clay and spiritual healing. Clays and Clay Minerals. 2008;56:751–760.
- Fox CL. Silver sulfadiazine - a new topical therapy for Pseudomonas in burns. Archives of Surgery. 1968;96:184–188. [PubMed]
- Fox CL, Modak SM. Mechanism of silver sulfadiazine action on burn wound infections. Antimicrobial Agents and Chemotherapy. 1974;5:582–588. [PMC free article] [PubMed]
- Gant VA, Wren MWD, Rollins MSM, Jeanes A, Hickok SS, Hall TJ. Three novel highly charged copper-based biocides: safety and efficacy against healthcare-associated organisms. Journal of Antimicrobial Chemotherapy. 2007;60:294–299. [PubMed]
- Giese RF, van Oss CJ. Colloid and Surface Properties of Clays and Related Minerals. New York: Marcel Dekker; 2002.
- Gomes CSF, Silva JBP. Minerals and clay minerals in medical geology. Applied Clay Science. 2007;36:4–21.
- Gonzalez JJ, Owens W, Ungaro PC, Werk EE, Wentz PW. Clay ingestion: a rare cause of hypokalemia. Annals of Internal Medicine. 1982;97:65–66. [PubMed]
- Gordon AS, Howell LD, Harwood V. Response of diverse heterotrophic bacteria to elevated copper concentrations. Canadian Journal of Microbiology. 1994;40:408–411. [PubMed]
- Grim RE, Güven N. Bentonites: Geology, Mineralogy, Properties and Uses. New York: Elsevier Publishing Co.; 1978. Pages.
- Güven N, Pollastro RM. CMS Workshop Lectures 4. Boulder, CO: The Clay Minerals Society; 1992. Clay-Water interface and its rheological implications.
- Haydel SE, Remenih CM, Williams LB. Broad-spectrum in vitro antibacterial activities of clay minerals against antibiotic-susceptible and antibiotic-resistant bacterial pathogens. Journal of Antimicrobial Chemotherapy. 2008;61:353–361. [PMC free article] [PubMed]
- Heller-Kallai L, Rozensen I. Dehydroxylation of dioctahedral phyllosilicates. Clays and Clay Minerals. 1980;28:355–368.
- Herrera P, Burghardt RC, Phillips TD. Adsorption of Salmonella enteritidis by cetylpyridinium-exchanged montmorillonite clays. Vet Microbiol. 2000;74:259–272. [PubMed]
- Hladik CM, Gueguen L. Geophagy and mineral nutrition in the wild primates. Comptes Rendus de l'Académie des Sciences. 1974;279:1393–1396. [PubMed]
- Hotta M, Nakajima H, Yamamoto K, Aono M. Antibacterial temporary filling materials: the effect of adding various ratios of Ag-Zn-zeolite. Journal of Oral Rehabilitation. 1998;25:485–489. [PubMed]
- Hu CH, Xu ZR, Xia MS. Antibacterial effect of Cu2+exchanged montmorillonite on Aeromonas hydrophila and discussion on its mechanism. Veterinary Microbiology. 2005;109:83–88. [PubMed]
- Hunter JM. Geophagy in Africa and in the United States. Geographical Review. 1973;63:170–195.
- Inoue A, Velde B, Meunier A, Touchard G. Mechanism of illite formation during smectite-to-illite conversion in a hydrothermal system. American Mineralogist. 1988;73:1325–1334.
- Jackson ML. Soil Chemical Analysis--Advanced Course. Madison, WI: Jackson, M. L.; 1979.
- Jansen B, Rinck M, Wolbring P, Strohmeier A, Jahns T. In vitro evaluation of the antimicrobial efficacy and biocompatibility of a silver-coated central venous catheter. Journal of Biomaterials Applications. 1994;9:55–70. [PubMed]
- Johns T, Duquette M. Traditional detoxification of acorn bread with clay. Ecology of Food and Nutrition. 1991;25:221–228.
- Johnston CT. Sorption of organic compounds on clay minerals: a surface functional group approach. In: Sawhney BL, editor. Clay Minerals Society Workshop Lectures: Organic pollutants in the environment. Boulder, CO: The Clay Minerals Society; 1996. pp. 2–44.
- Kawahara K, Tsuruda K, Morishita M, Uchida M. Antibacterial effect of silver-zeolite on oral bacteria under anaerobic conditions. Dental Materials. 2000;16:452–455. [PubMed]
- Klaus G, Schmid B. Geophagy at natural licks and mammal ecology. A review: Mammalia. 1998;62:481–497.
- Konhauser K. Introduction to Geomicrobiology. Oxford: Blackwell Publishing; 2007. p. 425.
- Krieg RE, Wolcott JH, Confer A. Treatment of Mycobacterium ulcerans infection by hyperbaric oxygenation. Aviat. Space Environ. Med. 1975;46:1241–1245. [PubMed]
- Krishnamani R, Mahaney WC. Geophagy among primates: Adaptive significance and ecological consequences. Animal Behaviour. 2000;59:899–915. [PubMed]
- Lacey EP. Broadening the perspective of pica: Literature review. Public Health Reports. 1990;105:29–35. [PMC free article] [PubMed]
- Lalonde SV, Smith DS, Owttrim GW, Konhauser KO. Acid-base properties of cyanobacterial surfaces I: Influences of growth phase and nitrogen metabolism on surface reactivity. Geochimica et Cosmochimica Acta. 2008a;72:1257–1268.
- Lalonde SV, Smith DS, Owttrim GW, Konhauser KO. Acid-base properties of cyanobacterial surfaces II: Silica as a chemical stressor influencing cell surface reactivity. Geochimica et Cosmochimica Acta. 2008b;72:1269–1280.
- Lanson B, Champion D. The I-S to illite reaction in the late stages of diagenesis. American Journal of Science. 1991;v. 291:473–506.
- Li B, Yu S, Hwang JY, Shi S. Antibacterial vermiculite nano-material. JOM-Journal of the Minerals Metals & Materials Society. 2002;v. 1:61–68.
- Liedberg H, Lundeberg T. Silver alloy coated catheters reduce catheter-associated bacteriuria. British Journal of Urology. 1990;v. 65:379–381. [PubMed]
- Lipson SM, Stotzky G. Adsorption of reovirus to clay minerals: Effects of cation-exchange capacity, cation saturation, and surface area. Applied and Environmental Microbiology. 1983;v. 46:673–682.[PMC free article] [PubMed]
- Mahaney WC, Hancock RGV, Aufreiter S, Huffman MA. Geochemistry and clay mineralogy of termite mound soil and the role of geophagy in chimpanzees of the Mahale Mountains, Tanzania. Primates. 1996;v. 37:121–134.
- Mahaney WC, Milner MW, Mulyono H, Hancock RGV, Aufreiter S, Reich M, Wink M. Mineral and chemical analyses of soils eaten by humans in Indonesia. International Journal of Environmental Health Research. 2000;v. 10:93–109.
- Marini M, Bondi M, Iseppi R, Toselli M, Pilati F. Preparation and antibacterial activity of hybrid materials containing quaternary ammonium salts via sol-gel process. European Polymer Journal. 2007;v. 43:3621–3628.
- Marston BJ, Diallo MO, Horsburgh CR, Jr, Diomande I, Saki MZ, Kanga JM, Patrice G, Lipman HB, Ostroff SM, Good RC. Emergence of Buruli ulcer disease in the Daloa region of Cote d'Ivoire. American Journal of Tropical Medicine and Hygiene. 1995;v. 52:219–224. [PubMed]
- Marston BJ, Diallo MO, Horsburgh CR, Jr, Diomande I, Saki MZ, Kanga JM, Patrice G, Lipman HB, Ostroff SM, Good RC. Emergence of Buruli ulcer disease in the Daloa region of Cote d'Ivoire. American Journal of Tropical Medicine and Hygiene. 1995;v. 52:219–224. [PubMed]
- Mathieson A. Mg-vermiculite: a refinement and re-examination of the crystal structure of 14.36 phase. American Mineralogist. 1958;v. 43:216–227.
- Mathieson A, Walker GF. Crystal structure of magnesium-vermiculite. American Mineralogist. 1954;v. 39:231–255.
- Matsumura Y, Yoshikata K, Kunisaki S, Tsuchido T. Mode of bactericidal action of silver zeolite and its comparison with that of silver nitrate. Applied and Environmental Microbiology. 2003;v. 69:4278–4281. [PMC free article] [PubMed]
- Matsuura T, Abe Y, Sato Y, Okamoto K, Ueshige M, Akagawa Y. Prolonged antimicrobial effect of tissue conditioners containing silver-zeolite. Journal of Dentistry. 1997;v. 25:373–377. [PubMed]
- Mengel CE, Carter WA, Horton ES. Geophagia with iron deficiency and hypokalemia. Archives of Internal Medicine. 1964;v. 114:470–474. [PubMed]
- Menichetti F. Current and emerging serious Gram-positive infections. Clinical Microbiology and Infection. 2005;v. 11:22–28. [PubMed]
- Meyers WM, Shelly WM, Connor DH. Heat treatment of Mycobacterium ulcerans infections without surgical excision. American Journal of Tropical Medicine and Hygiene. 1974;v. 23:924–929.[PubMed]
- Minnich V, Okcuoglu A, Tarcon Y, Arcasoy A, Cin S, Yörükoglu O, Renda F, Demirag B. Pica in Turkey. II. Effect of clay upon iron absorption. American Journal of Clinical Nutrition. 1968;v. 21:78–86. [PubMed]
- Moore DM, Reynolds RC. X-ray diffraction and the identification and analysis of clay minerals.New York: Oxford University Press; 1997. p. Pages.
- Moorer WR, Genet JM. Antibacterial activity of gutta-percha cones attributed to the zinc oxide component. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, & Endodontics. 1982;v. 53:508–517. [PubMed]
- Nadeau PH, Wilson MJ, McHardy WJ, Tait JM. Interstratified clays as fundamental particles. Science. 1984;v. 225:923–935. [PubMed]
- NCCLS. Performance standards for antimicrobial susceptibility testing. 14th ed. Wayne, PA: Approved standard M100-S15, National Committee for Clinical Laboratory Standards; 2004.
- Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years. Journal of Natural Products. 2007;v. 70:461–477. [PubMed]
- Nies DH. Microbial heavy-metal resistance. Applied Microbiology and Biotechnology. 1999;v. 51:730–750. [PubMed]
- Nies DH, Silver S. Ion efflux systems involved in bacterial metal resistances. Journal of Industrial Microbiology. 1995;v. 14:186–199. [PubMed]
- Ohashi F. Antimicrobial and antifungal agents derived from clay minerals. J. Antibact. Antifung Agents, Japan. 1993;v. 21(11):591–595.
- Ohashi F, Oya A, Duclaux L, Beguin F. Structural model calculation of antimicrobial and antifungal agents derived from clay minerals. Applied clay science. 1998;v.12:435–445.
- Phillips R, Adjei O, Lucas S, Benjamin N, Wansbrough-Jones M. Pilot randomized double-blind trial of treatment of Mycobacterium ulcerans disease (Buruli ulcer) with topical nitrogen oxides. Antimicrobial Agents and Chemotherapy. 2004;v. 48:2866–2870. [PMC free article] [PubMed]
- Phillips TD. Dietary clay in the chemoprevention of aflatoxin-induced disease. Toxicological Sciences. 1999;v. 52 Suppl:118–126. [PubMed]
- Phillips TD, Sarr AB, Grant PG. Selective chemisorption and detoxification of aflatoxins by phyllosilicate clay. Natural Toxins. 1995;v. 3:204–213. [PubMed]
- Portaels F, Meyers WM. Insects in the transmission of Mycobacterium ulcerans infection. Lancet. 1999;v. 353:986. [PubMed]
- Reeder RJ, Schoonen MAA. Metal speciation and its role in bioaccessibility and bioavailability. Reviews in Mineralogy and Geochemistry. 2006;v. 64:59–113.
- Sahai N, Schoonen MAA, editors. Reviews in Mineralogy and Geochemistry. v. 64. Geochemical Society and Mineralogical Society of America; 2006. Medical mineralogy and geochemistry; p. 332.
- Sawai J. Quantitative evaluation of antibacterial activities of metallic oxide powders (ZnO, MgO and CaO) by conductimetric assay. Journal of Microbiological Methods. 2003;v. 54:177–182. [PubMed]
- Sayetta RB. Pica: an overview. American Family Physician. 1986;v. 33:181–185. [PubMed]
- Schiffenbauer M, Stotzky G. Adsorption of coliphages T1 and T7 to clay minerals. Applied and Environmental Microbiology. 1982;v. 43:590–596. [PMC free article] [PubMed]
- Schoonen MAA, Cohn CA, Roemer E, Laffers R, Simon SR, O'Riordan T. Mineral-induced formation of reactive oxygen species. Reviews in Mineralogy and Geochemistry. 2006;v. 64:179–221.
- Severance J, Harry W, Holt T, Patrone NA, Chapman L. Profound muscle weakness and hypokalemia due to clay ingestion. South Medical Journal. 1988;v. 81:272–274. [PubMed]
- Shah PM. The need for new therapeutic agents: what is the pipeline? Clinical Microbiology and Infection. 2005;v. 11:36–42. [PubMed]
- Sharma R, Sharma CL, Kapoor B. Antibacterial resistance: current problems and possible solutions. Indian Journal of Medical Sciences. 2005;v. 59:120–129. [PubMed]
- Siqueira JF, Jr, Goncalves RB. Antibacterial activities of root canal sealers against selected anaerobic bacteria. Journal of Endodontics. 1996;v. 22:79–80.
- Skinner HCW. Preface to geomicrobiology: Interactions between microbes and minerals. In: Ribbe PH, editor. Reviews in Mineralogy. Mineralogical Society of America; 1997. pp. 1–4.
- Srodon J. Nature of mixed-layer clays and mechanisms of their formation and alteration. Annual Review Earth and Planetary Science. 1999;v. 27:19–53.
- Steel RF, Anderson W. The interaction between kaolinite and Staphylococcus aureus. Journal of Pharmacy and Pharmacology. 1972;v. 24:1–129. [PubMed]
- Stucki JW, Bailey GW, Gan H. Oxidation-reduction mechanisms in iron-bearing phyllosilicates. Applied Clay Science. 1996;v.10:417–430.
- Tateo F, Summa V. Element mobility in clays for healing use. Applied Clay Science. 2006;v. 36:64–76.
- Tong G, Yulong M, Peng G, Zirong X. Antibacterial effects of the Cu(II)-exchanged montmorillonite on Escherichia coli K88 and Salmonella choleraesuis. Veterinary Microbiology. 2005;v. 105:113–122. [PubMed]
- Turkheim HJ. In vitro experiments on the bactericidal effect of zinc oxide eugenol cement on bacteria-containing dentin. Journal of Dental Research. 1955;v. 34:295–301. [PubMed]
- Ukaonu C, Hill DA, Christensen F. Hypokalemic myopathy in pregnancy caused by clay ingestion. Obstetrics & Gynecology. 2003;v. 5:1169–1171. [PubMed]
- van der Werf TS, van der Graaf WT, Tappero JW, Asiedu K. Mycobacterium ulcerans infection. Lancet. 1999;v. 354:1013–1018. [PubMed]
- Voroney RP. The Soil Habitat in Soil Microbiology, Ecology, and Biochemistry. 2006
- Vermeer DE, Ferrell RE., Jr Nigerian geophagical clay: A traditional antidiarrheal pharmaceutical. Science. 1985;v. 227:634–636. [PubMed]
- Wackett LP, Dodge AG, Ellis LB. Microbial genomics and the periodic table. Applied and Environmental Microbiology. 2004;v. 70:647–655. [PMC free article] [PubMed]
- Wallace WE, Headley LC, Weber KC. Dipalmitoyl lecithin surfactant adsorption by kaolin dust in vitro. Journal of Colloid and Interface Science. 1975;v. 51:535–537.
- Warren LA, Ferris FG. Continuum between sorption and precipitation of Fe(III) on microbial surfaces. Environmental Science & Technology. 1998;v. 32:2331–2337.
- Weir E. Buruli ulcer: the third most common mycobacterial infection. Canadian Medical Association Journal. 2002;v. 166:1691. [PMC free article] [PubMed]
- Williams LB, Holland M, Eberl DD, Brunet T, Courrsou LBd. Killer Clays! Natural antibacterial clay minerals. Mineralogical Society Bulletin. 2004;v. 139:3–8.
- Williams LB, Hervig RL. Crystal-size dependence of illite-smectite isotopic equilibration with changing fluids. Clays and Clay Minerals. 2006;54:531–540.
- Williams LB, Haydel SE, Giese RF, Eberl DD. Chemical and mineralogical characteristics of French green clays used for healing Clays and Clay Minerals. in press. [PMC free article] [PubMed]
- Wilson MJ. Clay mineralogical and related characteristics of geophagic materials. Journal of Chemical Ecology. 2003;v. 29:1525–1547. [PubMed]
- World Health Organization. World Health Organization targets untreatable ulcer: report from the first international conference on Buruli ulcer control and research; Report Number; Yamousoukro (Côte d'Ivoire): WHO; 1998.
- World Health Organization. Provisional guidance on the role of specific antibiotics in the management of Mycobacterium ulcerans disease (Buruli ulcer) Geneva: World Health Organization; 2004.
- World Health Organization. Buruli ulcer disease (Mycobacterium ulcerans infection) WHO Fact sheet No. 199. 2007.
- Yamamoto O, Sawai J, Sasamoto T. Change in antibacterial characteristics with doping amount of ZnO in Mg-O-ZnO solid solution. Int. J. Inorganic Materials. 2000;v. 2:451–454.
- Yamamoto O. Influence of particle size on the antibacterial activity of zinc oxide. Int. J. of Inorganic Materials. 2001;v. 3:646–646.
- Zetola N, Francis JS, Nuermberger EL, Bishai WR. Community-acquired methicillin-resistant Staphylococcus aureus: an emerging threat. Lancet Infectious Diseases. 2005;v. 5:275–286.[PubMed]
- Zhou N-l, Liu Y, Li L, Meng N, Huang Y-x, Zhang J, Wei S-h, Shen J. A new nanocomposite biomedical material of polymer/Clay-Cts-Ag nanocomposites. Current Applied Physics. 2007;v. 7S1:e58–e62.