What makes a clay antibacterial?

What makes a clay antibacterial?

What makes a clay antibacterial?


Lynda B. Williams t, *, David W. Metge tt, Dennis D. Eberl tt, Ronald W. Harvey tt,

Amanda G. Turner t, Panjai Prapaipong t, Amisha T. Poret-Peterson t


t School of Earth & Space Exploration, Arizona State University, Tempe, AZ 85287

tt U.S. Geological Survey, 3215 Marine St., Suite E127, Boulder, CO, 80303


Keywords: biogeochemistry, antibacterial clay, illite-smectite, Fenton reaction

Corresponding author:

Lynda B. Williams, School of Earth and Space Exploration, Arizona State University, Tempe, AZ 85282


Natural clays have been used in ancient and modern medicine, but the mechanism(s) that make certain clays lethal against bacterial pathogens has not been identified. We have compared the depositional environments, mineralogy’s and chemistries of clays that exhibit antibacterial effects on a broad spectrum of human pathogens including antibiotic resistant strains. Natural antibacterial clays contain nano-scale (<200nm), illite-smectite and reduced iron phases. The role of clay minerals in the bactericidal process is to buffer the aqueous pH and oxidation state to conditions that promote Fe2+ solubility.

Chemical analyses of E. coli killed by aqueous leachates of an antibacterial clay show that intracellular concentrations of Fe and P are elevated relative to controls. Phosphorus uptake by the cells supports a regulatory role of polyphosphate or phospholipids in controlling Fe2+. Fenton reaction products can degrade critical cell components, but we deduce that extracellular processes do not cause cell death. Rather, Fe2+ overwhelms outer membrane regulatory proteins and is oxidized when it enters the cell, precipitating Fe3+ and producing lethal hydroxyl radicals.


   Overuse of antibiotics in healthcare is a major concern because of the consequential proliferation of antimicrobial resistance. Our studies of natural antibacterial minerals were initiated to investigate alternative antimicrobial mechanisms. Indigenous people worldwide have used clays for healing throughout history. Recently, French green clay poultices were documented for healing Buruli ulcer (1), a necrotizing fasciitis caused by Mycobacterium ulcerans. However, only one of the French clays used for healing proved to be antibacterial (2). Other sources of French green clay increased bacteria growth relative to controls (3).

Continued testing of over 50 clays worldwide, used for healing, revealed only a few deposits that are truly antibacterial. Each deposit is mineralogically different in detail, but the depositional environments are similar. All of the deposits are from hydrothermally altered volcaniclastic environments, either altered pyroclastic material or bentonite (volcanic ash).

This study examines the geochemical characteristics of the most effective antibacterial clay that we have found to date. It is mined by Oregon Mineral Technologies (Grants Pass, Oregon, USA), hereafter referred to as Oregon blue clay. It was shown to completely eliminate Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium and antibiotic resistant extended-spectrum beta lactamase (ESBL) E. coli and methicillin resistant S. aureus (MRSA) within 24 hrs (4). The source of this material is an open pit mine in hydrothermally altered, pyroclastic material associated with volcanic activity in the Cascade Mountains (OR, USA). We present evidence for the natural antibacterial components in this clay and deduce the chemical conditions required for its antibacterial effect.

Antibacterial clay mechanisms

A variety of physical and/or chemical processes can make clays antibacterial. Physical bactericide can occur by surface attraction between clay minerals and bacteria, which can hamper passive and active uptake of essential nutrients, disrupt cell envelopes or impair efflux of metabolites (5). The natural antibacterial clays we have studied do not kill by physical associations between the clay and bacterial cells (3). The Oregon blue clay shows no zone of inhibition when applied dry to bacterial colonies growing in Petri dishes. However, the clays are antibacterial when hydrated and incubated with bacteria. Furthermore, when an aqueous suspension of Oregon blue clay (50 mg/ml water) was placed in dialysis tubing (25000 MDCO) and submerged into a beaker of log phase E. coli suspended in sterile Tryptic soy broth, the bacteria were killed over the course of 24 hrs. This indicates exchange of soluble constituents (either toxins liberated from the clay or sequestration of essential nutrients required for bacterial growth) across the membrane (6).

Other clays, e.g., allophane and imogolite, have been made antibacterial by chemical sorption of known bactericidal elements (Ag, Cu, Co, Zn) onto certain crystallographic sites of the mineral surfaces (7-9). Nanoparticulate metal oxides and ceramics can also be antibacterial;(10) releasing soluble toxic compounds in proportion to their high specific surface area (11, 12). Therefore, we evaluated what soluble elements E. coli assimilate from the naturally antibacterial Oregon blue clay.

Mineralogy of antibacterial clays

            Each antibacterial clay deposit is mineralogically different (Table 1) but they have in common the presence of expandable clay minerals (smectite) and Fe-rich phases (e.g., biotite, jarosite, pyrite, magnetite, hematite, goethite, and amphibole). The presence of pyrite in some samples may be important for bactericidal action (13), but not all of the antibacterial clays contain pyrite. The average crystal diameter of the antibacterial clays (<200nm) is an order of magnitude smaller than standard clay reference materials (www.clays.org). The nano-minerals in these deposits may enhance solubility of toxic elements (14). Additionally, the smectite may sequester toxic ions in the interlayer sites, which could be released upon rehydration in a clay poultice.

Because the Oregon blue clay is only antibacterial when hydrated, we studied the soluble elements in aqueous leachates of the clay (Supporting Information). Table 2 presents the chemistry of aqueous leachates from the Oregon blue clay compared to the French antibacterial clay (3) and a non-antibacterial clay (NAB) (15) used medicinally. The elemental concentrations are all below minimum inhibitory concentrations (MIC) published for E. coli at circum-neutral pH (16-18). However, under the pH, oxidation state and aqueous chemistry conditions created within a poultice, the MIC may be quite different due to stabilization of different soluble species.

Experimental Methods

Aqueous leachates were prepared using sterilized clay and water (50mg/ml). The mixture was ultra-sonified (Branson Sonifier 450) then shaken 24h to equilibrate. The suspension of minerals in water was centrifuged (20Krpm) to remove solids.

  1. coli (JM109) was grown to log phase and a cell density of ~109 cfu/ml in LB. The cell population produced ~30mg of cells (dry wt.) per experiment and each experiment was repeated in triplicate. E. coli was incubated in 1:1 ratio with clay leachate. All cultures were grown at 37˚C in a shaker-incubator for 24 hrs. Bacterial growth was evaluated after incubation, by standard plate counts. Bacteria (live or dead) from experiments were centrifuged at 7000rpm for 5 minutes to pellet. Bacterial cells were then rinsed with DDI water (25mls) in triplicate. Finally, one aliquot was rinsed in 25 ml of 50mM EDTA:100mM oxalic acid with 0.85% NaCl (pH 6.95). This solution removes metals from the exterior cell envelope (19). Test tubes containing bacteria were dried at room temperature, ground and accurately weighed. Twelve milliliters of 10% nitric acid (Omnitrace Ultra, EMD chemicals) was used to digest cells, and then diluted to 40mls.

Leachates, whole cells, and cells with exterior metals removed, were analyzed for elemental compositions with a Thermo Fisher Element 2 single-collector, double-focusing magnetic sector inductively coupled plasma mass spectrometer (ICP-MS) in low-, medium- and high-resolution modes, depending on spectral interferences. The samples, blanks and standards were acidified with nitric acid and spiked with 1 ppb indium solution to correct for instrumental response affected by solution matrix. Analyses of river water standard reference materials NIST 1640, NIST 1643e, and NRC SLRS4 determined analytical accuracy and precision. The measurement uncertainties were <5% (1s). Major anion concentrations were measured by ion chromatography using a Dionex DX 600 Dual IC System with IonPac AS11-HC column.


E.coli (JM109) was used as a model species to evaluate mass transfer from the clay to bacteria. Incubation of the antibacterial Oregon blue clay leachate 1:1 with a population of 109 cfu/ml E. coli grown in Luria Broth (LB) to log phase, showed no surviving bacteria in three independent experiments, compared to non-antibacterial (NAB) clay leachate and control (E.coli in LB without leachate). The control group produced on average 7.9±0.4 x109 cfu/ml, and the NAB clay leachate produced 9.0±0.8 x109 cfu/ml. Elements taken up by the Oregon blue clay-amended E. coli suspensions were analyzed using direct injection ICP-MS (20) (Table 3). Only elements showing significant differences in concentrations from the controls are presented. Whole cells were rinsed in triplicate with filter-sterilized, distilled-deionized (DDI) water before elemental measurements. Intracellular elemental abundances were determined on EDTA-oxalic acid treated samples (19); which removes extracellular metals, particularly iron.

High-resolution scanning electron microscope (SEM) images of the Oregon blue clay (Fig. 1) show a) the nano-scale crystals; b) the clay matrix encompassing sub-micron spherical Fe-S particles; and c) a lath-shaped mineral, rich in Ca, Al, S and O but no Si. This sulfate crystal might be jarosite, identified by XRD, or gypsum with a metal oxide coating. Co-existence of sulfate and pyrite in the sample, places the chemical stability of the clay on the equal activity line for S-SO4 (21).

          Important energy-requiring processes, for nutrient uptake and metabolism, motility, and cell division are localized at the cell membrane. Transmission electron microscope (TEM) imaging of E.coli treated with the Oregon blue leachate (Fig. 4) shows initial development of “hairy” vesicles on the cell membrane in response to acidic conditions (22). These dark, electron-opaque particles are initially evenly distributed on the cell walls. However, after 6 hrs of incubation, the black particles are concentrated at polar ends of the cells, which indicates that the cells are metabolically active, as bacteria regulate uptake of metals through polar attractions (23). The greatest cell damage is observed after 24 hrs when the black particles appear on the interior of the E. coli cells, near large voids or vacuoles in the cytoplasm.


Significantly higher concentrations of Al, P, V, Fe Cu and Pb are associated with the whole bacteria killed by the Oregon blue leachate (Table 3), than in the live bacteria (controls). In contrast, the concentrations of Mg and Ca are much lower in the Oregon blue clay leachate-treated whole bacteria. This is consistent with results of Borrok et al. (24) who showed that protons irreversibly exchange with Mg and Ca on the surface of bacteria exposed to acidic solutions and increase adsorption of other metals. Nonetheless, the interior of the Oregon blue clay treated bacteria shows Mg and Ca concentrations similar to the NAB sample that did not kill E. coli, indicating that limitation of these nutrients was not the cause for bacterial cell death.

On the cell interior of the Oregon blue clay killed bacteria, Al, P, Fe and Cu have elevated concentrations relative to controls. Whereas Cu can be toxic to bacteria, its abundance is not statistically different than the control or NAB samples. However, concentrations of P, Fe and Al in the cell interior of Oregon blue clay killed bacteria are significantly higher than controls.

The high concentration of Al in the Oregon blue clay leachate is of interest because the antibacterial clay leachates have extreme pH (≤4 or ≥10; Table 2). Aluminum is soluble in extreme pH conditions, but precipitates in circum-neutral fluids (25). Notably, whole-cell concentrations of Al in Oregon blue clay leachate-treated samples are four times higher than those in the controls, even though the cytoplasmic Al contents in treated and control samples were similar. Apparently, Al has limited transport through cell membranes, as tri- and tetra-valent metals are often precluded by cell pore diameters (26). Aluminum precipitated on the cell envelope might inhibit influx of nutrients or efflux of waste. However, the elevated intracellular P and Fe suggest that Al did not block their influx channels.

The P-content of the E. coli killed by Oregon blue clay leachate is also four times greater than the control, and the interior cell concentration is twice as high. This indicates significant precipitation of P on the cell wall that does not preclude uptake into the cell. Phosphorus is an important part of ATP, DNA, polyphosphates and phospholipids in cells, and phosphate anions have been found essential for the regulation of cation transport across the cell membrane (27). The negatively charged phosphate group in the lipid molecules of the cell membrane counterbalance positively charged arginine in a voltage-controlled gate across pore channels. The Oregon blue clay leachate treated E. coli accumulated P, possibly in response to chemical stresses (28), whereas it was not required in excess for the normal cell function of the controls. The enhancement of P in the E. coli cell interior may represent an attempt by the bacteria to control the influx of Fe2+ across the cell membrane, or to remove the metal.

Whole cell Fe concentrations in the Oregon blue clay leachate-treated E. coli are twenty times greater than in the controls. The intracellular Fe contents are eight times higher. Thus excess Fe was transported through the cell wall and is implicated as the primary reactant in the bactericidal process. The elevated concentrations of P and Fe in the E. coli interior imply that extracellular metals did not block transport channels through the cell envelope at least not initially.

Images depicting the progression of dying E. coli incubated with Oregon blue clay leachate (Fig. 4) are similar to images of E. coli treated with FeSO4 (14), where black particles were oxides of Fe3+. The vacuoles formed may result from a) polyphosphate granules that regulate intracellular metals, b) metabolic byproducts (e.g., NO), c) destruction of DNA or other cell components, d) leakage of cytoplasm after cell death. Bacteria require Fe for many metabolic processes, and use ferritin protein to regulate intracellular Fe levels and storage capacities. Ferritins can keep some Fe in solution, but excess Fe can form toxic precipitates (29). E. coli exposed to Oregon blue clay leachate may have accumulated P as a stress response (28) and may employ P to gate the cation flux across the cell wall (27), but eventually the bacteria were overwhelmed by the high Fe2+ concentration of the Oregon blue clay leachate.

Evaluating the antibacterial process

          The bioavailability of metals to bacteria depends on the aqueous metal speciation in the clay poultice. The process of transferring elements from a clay surface through water to a cell membrane involves numerous chemical reactions and the formation of rapidly reactive intermediates (radicals) that are affected by clay mineralogy and by surface complexation on the bacteria. The pH and oxidation state of the water added to clay to make a poultice is most influenced by the buffering capacity of the clay minerals with relative surface areas >100 m2/g (30).

Clays that buffer water to circum-neutral pH values are not antibacterial.(31) Therefore, we tested the tolerance of E. coli to low pH fluids. Adjusting the pH of sodium phosphate buffer to acidic conditions similar to that of Oregon blue clay leachate (pH 3.5) reduced the population of E. coli by two orders of magnitude (107.5 to 105.5 cfu/ml) over 2 hrs. However, the Oregon blue clay leachate (CB07-L) (4) completely killed the bacteria in less than 1hr, indicating that it is not pH alone that killed the E. coli but that metals soluble at low pH play an important antibacterial role.

The oxidation state of the clay poultice is also critical to the antibacterial process. A low oxidation state (log fO2 < –74) is inferred from the presence of sulfide and sulfate in the Oregon blue clay (Fig 2) where Cu+ or Fe2+ are dominant soluble cations (21). Experiments with Oregon blue leachates show loss of antibacterial capacity after oxidation. It is common for clay leachate to precipitate iron oxide coincident with the loss of bactericidal capacity. However, when small amounts (50mg/ml) of clay are suspended in the aqueous solution, it is stabilized and remains antibacterial. A rapid decrease of dissolved oxygen (D.O.), ORP and pH occurs when the Oregon blue clay is added to DDI water (Fig. 5). The pH stabilizes within the first hour, but the D.O. declines over 6 hrs, coincident with E.coli death. It is unlikely that decline in D.O. was responsible for the E. coli death, because this bacterium is fully capable of anaerobic respiration(32). However, E. coli may take up Fe2+ under the anaerobic conditions induced by the clay. The Oregon blue clay contains up to 8% pyrite, which is known to be bactericidal. In the presence of water, pyrite produces reactive oxygen species (ROS) such as hydrogen peroxide and hydroxyl radicals that degrade nucleic acids in RNA and DNA via the Fenton reaction (33). Chelation of Fe2+ in solution by EDTA, or hydroxyl radicals by catalase or other hydroxyl scavengers can prevent their destruction of DNA (34). Therefore, EDTA, thiourea, and bi-pyridal were used to chelate Fe and ROS from Oregon blue clay aqueous leachates (4). The bactericidal effect on E. coli was reduced (compared to controls), but was not eliminated. Bacteria have developed mechanisms for tolerating external oxidative stress, (35) therefore ROS formed in the extracellular leachate are not the key antibacterial components. The Fe-chelation experiments may have failed due to excess supply Fe2+ from minerals in suspension.

Park and Imlay (36) showed that significant damage to cellular DNA occurs when hydrogen peroxide reacts with Fe2+ to form hydroxyl radicals in vivo. Exogenous hydrogen peroxide is normally chelated in the cell envelope (37), but penetration of Fe2+ into the cell will catalyze the Fenton reaction. The rapid influx of reduced Fe2+ and possibly Cu+ produced by the Oregon blue clay may overwhelm the metal resistance mechanisms of pathogenic bacteria. Oxidation of Fe2+ occurs within the bacteria and produces destructive hydroxyl radical reactions precipitating Fe3+ near the intracellular voids (Fig. 3d). Hydroxyl radical production may be enhanced through cellular biochemical reduction of the Fe3+ by cysteine; reported as the rate-limiting step for oxidative DNA damage (36). Kohanski et al. (38) showed that production of intracellular hydroxyl radicals is a common underlying mechanism for cellular death by synthetic antibiotics. Evidence presented here suggests that antibacterial clays can provide an external stimulant of the same bactericidal reaction.

Development of protective biochemical mechanisms by bacteria (17) may not be possible on the time scale of a clay poultice application. Because of this, the external application of antibacterial clays may remain more effective for wound care than systemic antibiotics. The growth of human tissue, coincident with the antibacterial action of clays (1) remains unexplained. However, nanoparticles of Fe-oxide (magnetite) were recently found to be bactericidal against S. aureus, while increasing growth of human bone cells (39). The differential effect of Fe on eukaryotic versus prokaryotic cells is consistent with a role of host defense mechanisms that target bacterial Fe utilization, (40) and may be key to understanding the observed clay healing process(1).



            We thank Oregon Mineral Technologies, Inc. for access to their mineral deposit. T Cunningham, and D. Lowry provided TEM images; S. Haydel assisted with microbiology pilot studies. NIH grant R21 AT003618 partially supported this research. We appreciate support from the ASU School of Life Sciences, Center for Solid State Science, and NASA Astrobiology Institute. The author(s) declare that they have no competing interests.


Brief : Certain volcanic environments produce antibacterial clays with soluble reduced metals in concentrations that overwhelm bacterial defense mechanisms, causing lethal intracellular hydroxyl radical attack of biomolecules.




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Table Captions

Table 1. Mineralogical comparison of antibacterial and non-antibacterial clays discussed, determined by quantitative X-ray diffraction (41) using CuKa radiation.

Table 2. Chemical analyses of aqueous clay leachates, A) ICP-MS analyses of antibacterial French clay (ARG), Oregon blue clay and a non-antibacterial (NAB) clay leachate (23), B) Ion chromatography analysis of anions in the Oregon blue clay leachate.

Table 3. Metals assimilated by whole bacterial cells compared to intracellular concentrations.

Figure Captions

 Fig, 1. SEM of the Oregon blue clay using 30kV, 2.1nA current, A) Clay particles on graphite (dark substrate). EDS (right) shows elements over a rastered area, B) Close up of Fe-S spherules in aluminosilicate matrix. EDS spectrum is rastered over spherules, C) Lath shaped particle with high Ca, S and O (black dot) interpreted as gypsum.

Fig. 2. Time series TEM images of pressure frozen, fixed (glutaraldehyde and Os vapor), resin embedded, sectioned E. coli after incubation with Oregon blue clay leachate. A) initial incubation shows uniform black precipitate of electron opaque metal, B) after 1 h metal migrates toward cell poles, C) metals are concentrated at cell poles after 6h, D) after 24 h metal has penetrated the cell interior and voids form accompanying cell death (images by T. Cunningham).

Fig. 3. Plot showing changes in pH, ORP and dissolved O2 content of DDI water over 24 h after addition of Oregon blue clay (50mg/ml).


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