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Journal of Antimicrobial Chemotherapy (2008) 61, 353 – 361doi:10.1093/jac/dkm468Advance Access publication 10 December 2007 Broad-spectrum in vitro antibacterial activities of clay minerals against antibiotic-susceptible and antibiotic-resistant bacterial Shelley E. Haydel1,2*, Christine M. Remenih1 and Lynda B. Williams3 1Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ, USA; 2School of Life Sciences, Arizona State University, Tempe, AZ, USA; 3School of Earth and Space Exploration, Arizona State University, Tempe, AZ, USA Received 10 September 2007; returned 15 October 2007; revised 5 November 2007; accepted 12 November 2007 Objectives: The capacity to properly address the worldwide incidence of infectious diseases lies in theability to detect, prevent and effectively treat these infections. Therefore, identifying and analysinginhibitory agents are worthwhile endeavours in an era when few new classes of effective antimicrobialshave been developed. The use of geological nanomaterials to heal skin infections has been evidentsince the earliest recorded history, and specific clay minerals may prove valuable in the treatment ofbacterial diseases, including infections for which there are no effective antibiotics, such as Buruliulcer and multidrug-resistant infections.
Methods: We have subjected two iron-rich clay minerals, which have previously been used to treatBuruli ulcer patients, to broth culture testing of antibiotic-susceptible and antibiotic-resistant patho-genic bacteria to assess the feasibility of using clay minerals as therapeutic agents.
Results: One specific mineral, CsAg02, demonstrated bactericidal activity against pathogenic Escherichiacoli, extended-spectrum b-lactamase (ESBL) E. coli, Salmonella enterica serovar Typhimurium, Pseudo-monas aeruginosa and Mycobacterium marinum, and a combined bacteriostatic/bactericidal effectagainst Staphylococcus aureus, penicillin-resistant S. aureus, methicillin-resistant S. aureus (MRSA) andMycobacterium smegmatis, whereas another mineral with similar structure and bulk crystal chemistry,CsAr02, had no effect on or enhanced bacterial growth. The <0.2 mm fraction of CsAg02 and CsAg02heated to 200 or 5508C retained bactericidal activity, whereas cation-exchanged CsAg02 and CsAg02heated to 9008C no longer killed E. coli.
Conclusions: Our results indicate that specific mineral products have intrinsic, heat-stable antibacterialproperties, which could provide an inexpensive treatment against numerous human bacterialinfections.
Keywords: infections, nanominerals, therapeutics, natural, bactericidal, bacteriostatic aiding digestive processes and cleansing and protecting the skin.4,5Due to the small particle size (,2.0 mm), these natural geological Medicinal and therapeutic use of mineral products has impacted products have a vast surface area (hundreds of m2/g of clay) with human health for thousands of years, and pure clay minerals, such high concentrations of ions and compounds located on the surfaces.
as smectite and illite, are nanomaterials of geological origin. The Despite the clear, beneficial effects on human health related to intentional consumption of earth materials, such as clays, by ridding the body of foreign substances, few studies have investi- humans and animals is known as geophagy, a complex behaviour, gated the antibacterial properties of clay minerals.6 – 10 Moreover, largely attributed to religious beliefs, cultural practices, psychologi- to our knowledge, there have been no published scientific reports cal disorders, cosmetics, dietary/nutritional needs and medicinal that examine the antibacterial activities of clay minerals on a benefits.1 –3 Early research focused on the extraordinary adsorptive broad-spectrum panel of bacterial pathogens, including antibiotic- properties of clay minerals and the health benefits recognized in resistant strains, that infect and cause disease in humans.
*Corresponding author. Tel: þ1-480-727-7234; Fax: þ1-480-727-0599; E-mail: [email protected] # The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected] Haydel et al.
Documented use of the two clay minerals described in this Salmonella enterica serovar Typhimurium ATCC 14028, P. aer- study as a therapeutic treatment of Buruli ulcer11 suggests that uginosa ATCC 27853, S. aureus ATCC 29213, Mycobacterium these natural nanomaterials have significant effects on infectious smegmatis ATCC 19420 and M. marinum ATCC 927. With the bacteria and wound healing. In 2001, a French humanitarian exception of the mycobacterial strains, the CLSI (formerly working in the Ivory Coast of Africa began treating children with NCCLS) recommends these bacteria as quality control strains Buruli ulcer with the two clay minerals described herein. Within for laboratory testing of antimicrobials.15 Upon receipt, all bac- days of initiating treatment with clay poultices, the therapeutic terial cultures were grown in the appropriate liquid medium properties of the clay minerals were demonstrated with the (described below) and stored at – 708C prior to use. ESBL E.
initiation of rapid, non-surgical debridement of the destroyed tissue. Extended treatment with the clay minerals resulted in con- Laboratories (Tempe, AZ, USA) and confirmed by MicroScan tinued debridement of the ulcer, tissue regeneration and wound healing. After several months of daily clay applications, the Sacramento, CA, USA) testing to be resistant to 11 antibiotics Buruli ulcer wounds healed with soft, supple scarring and the (Table 1). Penicillin-resistant S. aureus (PRSA) was obtained return of normal motor function.9,11 These therapeutic observations from the ASU School of Life Sciences microbiological culture are highly relevant since antibiotic treatment is only effective for collection, subjected to MIC disc diffusion susceptibility pre-ulcerative lesions and has generally been unsuccessful with the testing by Sonora Quest Laboratories and confirmed to be ulcerative form of Buruli ulcer disease.12 Currently, the only resistant to penicillin (data not shown). MRSA was obtained accepted treatment of an advanced Mycobacterium ulcerans infec- from Sonora Quest Laboratories and confirmed by MicroScan tion, the causative agent of Buruli ulcer disease, is surgical exci- Positive MIC Panel Type 20A (Dade Behring) testing to be sion of the ulcerative lesion along with extended healthy tissue to resistant to 10 antibiotics (Table 2).
prevent persistent subcutaneous infection.13 This costly anddangerous treatment often leads to significant loss of tissue and Table 1. Antimicrobial susceptibility patterns of the ESBL E. coli possible permanent disability. To begin understanding how these ATCC 51446 strain used in the CsAg02 and CsAr02 antimicrobial two mineral products are effective at healing patients infected with assays; the susceptible concentration of the ESBL E. coli strain and M. ulcerans, we have initiated antimicrobial susceptibility testing the qualitative susceptibility interpretation for each antibiotic are of several human bacterial pathogens.
Here, we report an assessment of the broad-spectrum antibac- terial properties of two different clay minerals: CsAg02, an iron-rich smectite and illite clay mineral enriched with magnesium and potassium, and CsAr02, an iron-rich smectite and illite clay Antimicrobial agent mineral enriched with calcium.9 To assess the usefulness of thesenanominerals as antibacterial agents, we tested a range of human bacterial pathogens, including Pseudomonas aeruginosa, extended- susceptible, 8/4 spectrum b-lactamase (ESBL) Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium marinum.
M. marinum is genetically closely related to M. ulcerans and is a cutaneous pathogen that can cause nodular and ulcerated skin lesions, and can invade deeper structures including synovia, bursae and bone.14 M. marinum infection generally occurs when traumatized skin of an extremity is exposed to contaminated aquariums, salt water or marine animals.14 Our in vitro microbiological investigations indicate that the two clay minerals, although similar in major phases and bulk chemistry,9 have striking and opposite effects on bacterial popu- lations, ranging from enhanced microbial growth to complete growth inhibition. This research documents the therapeutic effect of the clay minerals previously used to treat Buruli ulcer patients,9,11 and represents initial investigations aimed at identi- fying the mechanism by which particular clay nanomaterials exhibit antibacterial behaviour. Our goal is to identify new inhibitory agents in an era when bacterial antibiotic resistance continues to challenge human health and the availability of new antimicrobial compounds is limited.
Materials and methods intermediate, 32 – 64 Bacterial strains susceptible, 2/38 The following bacterial strains, obtained from the American Type Culture Collection (ATCC), were used as target antibiotic-susceptible organisms in these studies: E. coli ATCC 25922, aThe endpoint of susceptibility interpretation is indicated for each antibiotic.
Antibacterial activities of clay minerals Table 2. Antimicrobial susceptibility patterns of the MRSA strain 808C) and grinding, before use. X-ray diffraction analyses used in the CsAg02 and CsAr02 antimicrobial assays; the (limits of detection ,5%) of the French clay samples indicate susceptible concentration of the MRSA strain and the qualitative that they are composed predominantly of smectite and illite min- susceptibility interpretation for each antibiotic are shown erals. The bulk CsAg02 clay is composed of 24% Fe-illite and50% Fe-smectite, whereas the bulk CsAr02 clay is composed of27% Fe-illite, 30% Fe-smectite, 3% kaolinite and 3% chlorite.9 The surface areas of CsAg02 and CsAr02 are 115.4 and Antimicrobial agent 90.5 m2/g, respectively,9 which provides a potentially large reac-tive area for ion exchange. Major element analyses of the clay sized fraction (,2.0 mm) of CsAg02 and CsAr02 showed that both are iron-rich ( 6% wt),9 while other clays obtained from the supplier of CsAg02 have lower iron content (data not susceptible, 16/8 shown). Levels of trace elements, including silver, arsenic and lead, in the clay minerals were all below MICs and will be reported elsewhere. Before use in any susceptibility testing, all mineral samples were sterilized by autoclaving at 1218C for 1 h.
susceptible, 0.5 Particle size fractionation of clay minerals Since clay samples are not pure clay minerals and can contain numerous detrital minerals, such as quartz, feldspar and carbon- ates, clay-sized particles of ,2.0 mm in diameter were collected and separated by particle size. After a series of washes and ultra- sonication of a clay suspension in distilled-deionized water to remove chloride salts, the clay sample suspension (at room temperature) was centrifuged in 50 mL conical tubes in a swing- ing arm rotor with an axial distance of 15 cm from the centre axis to the bottom of the tube. According to Stokes' law, a cen- trifuge speed of 750 rpm for 3.3 min will settle particles .2.0 mm as long as the water temperature, viscosity and settling distance are fixed.17 The suspended material, which contains ,2.0 mm particles, was centrifuged at 2000 rpm for 1.8 min to susceptible, 2/38 settle the 1.0 – 2.0 mm particles. For purification, this process was performed in triplicate. After collecting the 1.0 – 2.0 mm particles, the remaining suspension containing ,1.0 mm par- ticles was centrifuged for 29 min at 2500 rpm to settle the 0.2 – The endpoint of susceptibility interpretation is indicated for each antibiotic.
1.0 mm particles. The remaining ,0.2 mm particles, in theresulting suspension, were dried at 608C or less. All fractionated Bacterial growth media and growth conditions and collected CsAg02 clay particles were dried, crushed with a E. coli, S. enterica serovar Typhimurium and P. aeruginosa mortar and pestle, and sterilized by autoclaving at 1218C for 1 h.
were cultured using Luria – Bertani (LB) broth or agar, nutrient broth or agar and trypticase soy broth or agar, respectively, at378C. S. aureus strains were cultured using trypticase soy broth To remove the natural exchangeable cations, CsAg02 (1 g) was or agar at 378C. Bacterial cultures of M. smegmatis and M.
saturated with 1 M KCl (25 mL) and shaken for 24 h. The clay marinum were grown in supplemented Middlebrook 7H9 broth was then washed and dialysed to remove Cl2, leaving Kþ ions [Middlebrook 7H9 broth supplemented with 10% OADC (oleic in the exchangeable sites of the clay. Potassium-exchanged acid, albumin, dextrose and catalase), 0.2% glycerol and 0.05% CsAg02 was dried, crushed with a mortar and pestle, and steri- Tween 80] at 37 and 308C, respectively.16 Viable cell counting lized by autoclaving at 1218C for 1 h.
of mycobacteria was performed on supplemented Middlebrook7H10 agar (Middlebrook 7H10 supplemented with 10% OADC) Thermal treatment of clay minerals after 48 h of growth at 378C for M. smegmatis and after 5 days To gradually destroy the water and hydroxyl bonds in the of growth at 308C for M. marinum. For the assays described mineral structure, separate aliquots of CsAg02 were heated to below, all bacteria were cultured with gentle rotary mixing to 200 and 5508C for 24 h. Heating to 2008C dehydrates the inter- layer of smectite,18 whereas heating to 5508C dehydroxylates most iron-rich clays.19 Finally, heating to 9008C for 24 hdestroys the clay structure completely, leaving only oxide com- ponents of the clay. For this procedure, 1 – 2 g of clay was The CsAg02 and CsAr02 minerals used in this study were both placed in a porcelain crucible, fitted with a porcelain lid and supplied by Brunet de Courssou11 from the batches used in the heated to 200, 550 or 9008C in a muffle furnace. All thermal Buruli ulcer clinics located in western Africa. The clays were treatments were performed in air to generate a highly oxidizing subjected to factory processing, which included drying (below environment. Although microorganisms would be destroyed by

Haydel et al.
thermal treatments, the heated CsAg02 minerals were sub-sequently sterilized by autoclaving at 1218C for 1 h before per-forming antimicrobial assays.
Antimicrobial assays Bacterial strains were grown overnight and diluted with freshmedium to achieve an approximate density of 1  107 cfu/400 mL. To confirm the initial bacterial counts, serially dilutedbacterial cultures were plated on the appropriate agar plates andenumerated. After dilution, sterilized clay minerals (200 mg)were introduced into 400 mL of media containing the initialpathogen inoculum to achieve a consistency similar to thehydrated clay poultices used to treat Buruli ulcer patients. Thebacteria – mineral mixtures were incubated with constant rotaryagitation for the appropriate times and temperatures, asdescribed above. Positive controls for growth of bacteria in theabsence of clay minerals were included in each series of inde-pendent experiments. To ensure that the clay samples were steri-lized after autoclaving and maintained sterilization duringstorage, negative control growth experiments with clay mineralsin LB broth were performed several times throughout the courseof the study. Incubation of the bacteria – clay mixtures was per-formed as follows: E. coli, S. enterica serovar Typhimurium, P.
aeruginosa, S. aureus and MRSA were incubated for 24 h at378C, M. smegmatis was incubated for 48 h at 378C and M.
marinum was incubated for 5 days at 308C. After incubation, themixtures were subjected to successive 10-fold serial dilutions in Figure 1. Bactericidal activity of CsAg02 against susceptible and resistant the appropriate medium, mixed with a vortex shaker to ensure Gram-negative pathogens: (a) E. coli ATCC 25922; (b) ESBL E. coli ATCC dispersion and quantitatively cultured in duplicate onto agar 51446; (c) S. enterica serovar Typhimurium ATCC 14028; and (d) P.
plates to determine the number of viable bacteria. Additionally, aeruginosa ATCC 27853. The reported values represent the average and SD 100 mL of the bacteria – clay suspension was directly plated onto of at least three independent experiments. Statistical significance ( paired agar plates to assess the bacterial viability in undiluted samples.
t-test) of Cs-inoculated 24 h bacterial growth compared with standard 24 h At least three independent antimicrobial assays with specific bacterial growth: ***P ¼ 0.0006; **P ¼ 0.007.
clay minerals and specific bacterial strains were performed.
Viable cell counts are expressed as log10 cfu per 400 mL.
Statistical analysis and P. aeruginosa (Figure 1d). Most notably, CsAg02 demon-strated a bactericidal effect against ESBL E. coli (Figure 1b), Statistical analysis was performed using Prism 4 (GraphPad which was resistant to 11 of the 27 tested antibiotics (Table 1).
Software, San Diego, CA, USA) and was calculated using a two- In contrast, in the presence of the CsAr02 clay minerals, growth tailed paired Student's t-test. A P value of ,0.05 was con- of the susceptible and resistant E. coli strains was significantly sidered statistically significant.
enhanced (Figure 1a and b), whereas growth of S. entericaserovar Typhimurium and P. aeruginosa was not significantlydifferent compared with bacterial growth in media alone (Figure 1c and d). No microbial growth was evident in mineralsthat were subjected to sterilization before use in the antimicro- To assess the effect of the CsAg02 and CsAr02 minerals on the bial susceptibility assays (data not shown).
growth of clinically relevant Gram-negative bacteria, suscepti- Susceptibility testing with S. aureus ATCC 29213 was per- bility testing of E. coli ATCC 25922, ESBL E. coli ATCC formed as described above to assess the potential broad-spectrum 51446, S. enterica serovar Typhimurium ATCC 14028 and P.
inhibitory activity of CsAg02. In addition, to assess the inhibitory aeruginosa ATCC 27853 was performed in liquid cultures. To activity of CsAg02 against antibiotic-resistant S. aureus, a single achieve poultice consistencies similar to those used to treat antibiotic-resistant strain, PRSA, and a multidrug-resistant strain, Buruli ulcer patients, initial bacterial cultures (107 bacteria/ MRSA, were also subjected to susceptibility testing. The MRSA 400 mL) were mixed with 200 mg of clay minerals and incu- strain exhibited resistance to 10 of the 23 tested antibiotics, includ- bated at 378C on a rotating drum for 24 h. After incubation, the ing methicillin (oxacillin) (Table 2). Incubation with CsAg02 bacteria – clay mixtures were diluted and plated onto agar to minerals resulted in partial growth inhibition of all three S. aureus determine the number of viable bacteria (Figure 1). Based on strains while growth was unaffected in the presence of CsAr02 these susceptibility experiments performed in triplicate, incu- minerals (Figure 2). In contrast to the complete bactericidal effect bation with the CsAg02 clay minerals resulted in complete demonstrated with Gram-negative bacteria, incubation with CsAg02 killing of several antibiotic-sensitive Gram-negative bacteria: appears to have more of a bacteriostatic effect on the S. aureus E. coli (Figure 1a), S. enterica serovar Typhimurium (Figure 1c) strains. Compared with the initial S. aureus cfu, viability of the

Antibacterial activities of clay minerals Figure 3. Effects of CsAg02 and CsAr02 on mycobacterial growth. Forantimicrobial susceptibility testing, M. smegmatis ATCC 19420 (a) and M.
marinum ATCC 927 (b) were grown in the presence of CsAg02 or CsAr02for 48 h at 378C and 5 days at 308C, respectively. The reported valuesrepresent the average and SD of at least three independent experiments.
Statistical significance ( paired t-test) of Cs-inoculated bacterial growthcompared with standard bacterial growth: *P , 0.05.
ulcerans, and significantly reduce the growth of the non-pathogenic M. smegmatis, whereas CsAr02 clay partially inhib-ited M. marinum growth, but had no effect on M. smegmatisgrowth. The bactericidal effect of CsAg02 and bacteriostaticeffect of CsAr02 on M. marinum growth (Figure 3b) stronglysuggest that these minerals will have a detrimental effect on the Figure 2. Effects of CsAg02 and CsAr02 on S. aureus ATCC 29213 (a), growth of genetically similar M. ulcerans, as indicated by the PRSA (b) and MRSA (c) growth. Average values and SDs from three topical application of these two minerals to effect a cure for independent experiments are shown. Statistical significance ( paired t-test) of Buruli ulcer patients.11 These results are especially promising Cs-inoculated 24 h bacterial growth compared with standard 24 h bacterial considering that there is no therapeutic cure for the ulcerative growth: **P , 0.004; *P , 0.05.
form of Buruli ulcer disease.
To eliminate the potential that a non-clay mineral in the bulk susceptible S. aureus strain and the PRSA and MRSA strains clay sample is responsible for killing bacteria and to determine if after 24 h was decreased 10-fold. The comparable effect on the crystal structure of the minerals is important in the antibacterial antibiotic-susceptible S. aureus and MRSA indicates that the effectiveness, we separated the clay minerals by size fractions. By mechanism of CsAg02 growth inhibition is unique and, more differentially centrifuging the mineral particles, we concentrated importantly, is unrelated to the mode of action of b-lactam, most of the non-clay minerals in the coarse size fraction (1– macrolide and fluoroquinolone antibiotics. These results areof significant clinical relevance considering the serious healthimplications of MRSA infections.
To assess the effect of the CsAr02 and CsAg02 clay minerals on the growth of non-pathogenic and pathogenic mycobacterialstrains, susceptibility testing of M. smegmatis ATCC 19420 andM. marinum ATCC 927 was performed. Initial mycobacterialcultures (107 bacteria/400 mL) were incubated with 200 mg ofbulk clay at 378C on a rotating drum for 48 h (M. smegmatis) or5 days (M. marinum). After incubation, the bacteria – clay mix-tures were diluted and plated onto supplemented Middlebrook7H10 agar to determine the number of viable bacteria. Similarto the bactericidal effects demonstrated upon Gram-negativebacteria (Figure 1), incubation with CsAg02 resulted in com-plete growth inhibition of M. marinum (Figure 3b). Unlike thebactericidal effect upon M. marinum, M. smegmatis growth inthe presence of CsAg02 was reduced 1000-fold in comparisonwith cultures grown without minerals for 48 h (Figure 3a).
Figure 4. Effects of CsAg02 fractionation, Kþ-exchange and thermal Incubation with CsAr02 clay did not significantly affect M.
treatment on E. coli growth. E. coli ATCC 25922 was grown in the absence smegmatis growth, while M. marinum growth was decreased of clay minerals (white bars) for 24 h, in the presence of bulk CsAr02 (black 50-fold in the presence of CsAr02 (Figure 3). Notably, the bar) and in the presence of fractionated, Kþ-exchanged or thermally treated CsAg02 clay was able to completely kill the pathogenic myco- CsAg02 (grey bars) for 24 h. The reported values represent the average and bacterial strain, M. marinum, most closely related to M.
SD of at least three independent experiments.
Haydel et al.
2 mm), while the smaller size fractions were progressively enriched antidiarrhoeal agents and to soothe the digestive tract.4 In in pure clay minerals. To determine if the relative surface area of addition, kaolinite and smectite clay minerals are hallmark addi- mineral crystals was an important parameter for antibacterial tives used by the cosmetic industry in topical applications to activity, various crystal size fractions of CsAg02 (,0.2, 0.2 – 1.0 serve as skin protectants and to absorb skin secretions.4 The data and 1.0 – 2.0 mm) were tested against E. coli (Figure 4). Results obtained in this study, together with biosafety studies with animal indicate that the finest size fraction (,0.2 mm), which makes up and controlled human trials, will be essential in supporting and the greatest percentage of the bulk sample, is bactericidal, whereas validating the use of specific minerals or derived products as the larger CsAg02 size fractions showed no statistically significant inexpensive antibacterial agents. Analyses of the chemical inter- effect on E. coli growth (Figure 4).
action at the mineral – bacterial interface are ongoing and will be Standard cation exchange was performed on CsAg02 where the pertinent in understanding the mechanism by which bacterial natural exchange sites ( primarily interlayer ions in the expandable growth inhibition occurs.
smectite mineral) were replaced by Kþ.17 Incubation of E. coli Buruli ulcer is recognized by the WHO as a global health with Kþ-exchanged CsAg02 resulted in complete loss of bacteri- threat, and recent observations showing that the clay minerals cidal activity (Figure 4). These results indicate that a chemical used in this study were effective at healing necrotic Buruli ulcer exchange from CsAg02 is responsible for the antibacterial activity disease11 prompted our investigations of these geological nano- and that surface properties of the clay have no direct effect on bac- materials. The use of clay minerals in the treatment and healing terial growth. Similar to CsAr02, Kþ-exchanged CsAg02 of Buruli ulcer lesions represents great promise for the develop- enhanced the growth of E. coli (Figure 4).
ment of an inexpensive cure for many skin diseases and topical Clay minerals can be heated to gradually remove or destroy infections. Our aim is to evaluate the effect of these minerals different bonds in the mineral structure.20 By progressively on M. ulcerans, but initial investigations have been directed destroying different bonds and vaporizing mineral elements in towards assessing the broad-spectrum antibacterial properties of thermal reactions, we may determine whether variable clay specific minerals against several bacterial pathogens.
structures and/or chemical characteristics of the mineral play a CsAg02 and CsAr02 are geological materials, primarily com- role in the antibacterial effectiveness of CsAg02. Heating the posed of the minerals smectite and illite, which contain variable clay to 2008C sterilizes the sample, dehydrates the minerals and elemental constituents and adsorbed ions. The CsAg02 and collapses the mineral interlayers, while mineral dehydroxylation CsAr02 minerals have been used previously to treat children and decomposition and combustion of organic matter occurs with Buruli ulcer.11 The CsAg02 mineral exhibits bactericidal after heating to 5508C.20,21 Heating the clay to 9008C destroys activity against E. coli, ESBL E. coli, S. enterica serovar its silicate structure, oxidizes the clay components and releases Typhimurium, P. aeruginosa and M. marinum, and significantly many elements that are volatile at lower temperatures.20 Since reduces growth of S. aureus, PRSA, MRSA and non-pathogenic autoclaving the CsAg02 clay to achieve sterilization does not M. smegmatis 1000-fold compared with cultures grown without affect its antibacterial activity, it was not unexpected to deter- added mineral products. We have demonstrated that the CsAg02 mine that heating the CsAg02 ,0.2 mm fraction to 2008C was inhibitory effect occurs in liquid culture medium, while a similar not detrimental to its bactericidal properties (Figure 4). Heating mineral, CsAr02, displays enhanced or no effect on bacterial the CsAg02 ,0.2 mm fraction to 5508C also resulted in reten- viability in liquid culture. To our knowledge, these experiments tion of its bactericidal activity on E. coli (Figure 4), indicating represent precedential investigations of a novel geological nano- that CsAg02 organic compounds and elements bound to the material that displays broad-spectrum antibacterial effects against hydroxyls are not associated with antibacterial activity. Upon human pathogens, including antibiotic-resistant strains.
exposure to 9008C-heated CsAg02, E. coli growth was unaf- Clay minerals are layered substances comprised of sheets of fected and was similar to control cultures lacking clay minerals silicate tetrahedra (SiO4) and octahedra (containing Al, Mg and (Figure 4). Since CsAg02 no longer kills after heating to 9008C, Fe). Smectite and illite, the primary minerals in CsAg02 and the remaining oxides, non-volatile elements in their oxidized CsAr02, are similar in structure (2:1 layer clays), but the silicate forms and increased surface area are not associated with the layers of the smectite are separated by an expandable interlayer region containing water, cations and molecules. If the interlayerregion is collapsed due to a high attraction between the silicatesheets with cations ( primarily Kþ) balancing the charge, then the mineral is illite. The interlayer surfaces of smectite are nega-tively charged due to substitution of Al3þ for Si4þ in some of Worldwide, the number of antibiotic-resistant pathogenic bac- the tetrahedral sites, and Mg2þ for Al3þ (for example) in octa- teria has substantially increased within the past 50 years.22 hedral sites. Therefore, cations are most commonly attracted to These alarming trends, reduced drug discovery and development the interlayer, with Kþ, Naþ, Ca2þ and NHþ productivity,23 and the emergence and increasing incidence of and forming weak surface bonds. The edges of the crystals can antibiotic-resistant infections, such as S. aureus, Streptococcus have a positive charge and attract anions such as hydroxyls, pneumoniae, Enterococcus faecalis and Mycobacterium tubercu- phosphates or sulphates.29 Water can enter the interlayer and losis,24 – 28 indicate a progressive need to identify and analyse expand the clay structure to accommodate additional com- new antibacterial agents.
pounds, including neutral and negatively charged species. Illite Numerous mineral products are currently used for therapeutic has the same structure as smectite, but has a higher layer charge, purposes in the pharmaceutical and cosmetic industries. For which attracts and holds interlayer cations ( particularly Kþ) example, smectite minerals adhere to the gastrointestinal mucosa tightly. Such interlayer cations are not easily exchanged with to adsorb and rid the body of dissolved toxins, bacteria and solutions, but are ‘fixed-cations'.17 Because of the special viruses, whereas kaolinite and palygorskite are primarily used as quality of smectite for incorporating various ions, the surface Antibacterial activities of clay minerals can be either hydrophilic or hydrophobic depending on the element components, exchanged ions and surface properties of charge and available solutes. A hydrophobic surface is inher- the clays are in progress. However, there is not a single com- ently organophyllic and could harbour an organic substance that ponent of the CsAg02 clay (e.g. transition metals) that stands is lethal to bacteria. If the mineral surface is hydrophilic, it might out as an obvious antibacterial agent, so it may be a fortuitous compete with bacteria for cations that are essential nutrients for combination of factors (multiple components) responsible for metabolic function or release a toxic inorganic substance that the inhibitory property. Further work is in progress to identify either inhibits a particular metabolic function or precipitates on additional antibacterial nanominerals, to isolate chemicals the cell wall.
released from the antimicrobial clays and to simulate the effect Enhanced growth of bacteria upon incubation with clay min- using synthetic materials.
erals, as demonstrated with CsAr02, is not an uncommon occur- Clay minerals can affect bacterial metabolism indirectly by rence, as microorganisms inherently require numerous trace altering the physicochemical properties of a specific environ- elements to facilitate growth. Montmorillonite, a variety of 2:1 ment or directly through surface interactions.40 Although we layer smectite and kaolinite, a 1:1 layer clay composed of one demonstrate that the nanoparticle-sized fraction (,200 nm) of tetrahedral layer and one octahedral layer, can stimulate bacterial CsAg02 retains bactericidal activities against E. coli, cation- growth and promote biofilm formation to serve as important exchanged CsAg02 completely loses its antibacterial effectiveness.
bioremediation substances.30 For example, Shewanella oneiden- Therefore, we hypothesize that the physicochemical properties sis respires structural ferric iron bound by smectite and uses it as of hydrated CsAg02 indirectly kill bacteria by generating an the sole electron acceptor for bacterial growth.31 Additionally, unfavourable environment. Since the physicochemical properties clay minerals may serve to protect environmental bacteria from of iron-rich smectite can be greatly affected by the structural Fe UV irradiation or toxic substances.32 By providing structural oxidation state,41 determining the oxidation state of Fe in the support and organic or inorganic nutrient acquisition via its high CsAg02 nanoparticle-sized fraction, which contains primarily cationic exchange capacity, clay minerals, particularly montmor- iron-rich illite and smectite minerals,9 is important. Thermal inac- illonite, may protect bacteria and serve as a minimal nutritional tivation of the antibacterial properties when CsAg02 is heated to sphere for bacterial proliferation.33 9008C could indicate that the change in oxidation state of inherent Since cation exchange eliminated the antibacterial activity elements, including Fe which is highly oxidized at 9008C, or the of CsAg02, we have analysed the exchange solution by induc- loss of vaporized elements removes the critical components of the tively coupled plasma mass spectrometry (ICP-MS) to assess the abundance of elements in the exchange solution relative to the Reactive oxygen species, including oxygen ions, free radicals total amount in the clay (L. B. Williams and S. E. Haydel, and peroxides, are by-products of aerobic bacterial metabolism unpublished results). The most significantly abundant elements with demonstrated toxic effects on bacteria.42 Mediated by the removed by cation exchange are silicon (30% of total), barium Fenton reaction, hydroxyl radicals are formed by the reduction (35% of total) and strontium (45% of total) (L. B. Williams and of hydrogen peroxide and ferrous iron,42,43 and cause oxidative S. E. Haydel, unpublished results). Since a silicon concentration damage to bacterial DNA, proteins and lipids.44 – 48 Furthermore, of .5000 mg/L (178 mM) is toxic for E. coli,34 the levels of numerous transition metals, which are inherent in clay minerals, silicon in the CsAg02 exchange solution (96 mg/L; 3.42 mM) can also participate in Fenton-like reactions to produce hydroxyl are not likely to be important for bactericidal activity. High radicals.49 The combination of elevated levels of reduced iron in levels of barium (144 mg/L; 1.05 mM) and strontium (154 mg/ CsAg02 and excessive free radical production in the presence of L; 1.76 mM) in the CsAg02 exchange solution could be of inter- oxygen could cause oxidative stress and damage to bacterial est since these elements potentially could be acquired by cation cells, resulting in death.50,51 Most importantly, Kohanski et al.52 uptake systems, inhibit transport mechanisms or substitute as recently demonstrated that three major classes of bactericidal enzymatic cofactors in bacterial cells to interfere with normal antibiotics stimulate production of hydroxyl radicals via Fenton physiological processes.35 – 39 Although the CsAr02 exchange chemistry to contribute to bacterial cell death. However, regard- solution contains slightly lower levels of barium (119 mg/L) ing CsAg02, additional experiments are necessary to assess the (L. B. Williams and S. E. Haydel, unpublished results), E. coli effect that varying redox environments and chemical speciation cultures incubated in the presence of high levels of barium changes have on microbial viability.
(144 mg/L) grew normally (data not shown). Thus far, no single During the past 25 years, 70% of newly discovered drugs element identified in the CsAg02 cation exchange solution was introduced in the USA have been derived from natural pro- greatly different than the concentration identified in the CsAr02 ducts.23 Our discovery that natural geological minerals harbour exchange solution or was determined to be solely responsible for antibacterial properties should provide impetus for exploring ter- E. coli toxicity (L. B. Williams and S. E. Haydel, unpublished restrial sources for the presence of novel therapeutic compounds.
results). Therefore, the possibility exists that the antibacterial Combining the availability of natural bioactive resources with activity could be attributed to a combination of elements and/or powerful combinatorial chemistry optimization methodologies chemical compounds that work in concert to mediate toxicity.
could result in the development of new antibacterial agents to The uniqueness of the French clay minerals is evident consid- fight existing antibiotic-resistant infections and diseases for ering that it has previously been used to treat children afflicted which there are no known therapeutic agents, such as advanced with Buruli ulcer,9,11 and shown to kill or significantly decrease M. ulcerans infections.
the growth of both antibiotic-susceptible and antibiotic-resistanthuman bacterial pathogens. Moreover, of the six independentclay samples collected from the French supplier and testedagainst various bacteria (data not shown), only CsAg02 dis-played antibacterial effects. Analyses of the various trace Haydel et al.
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porting travel to France for sample collection and geologic field investigations, Sonora Quest Laboratories (Tempe, AZ, USA) Performance Standards for Antimicrobial Susceptibility Testing— for providing quality control bacterial strains and for performing Fourteenth Edition: Approved Standard M100-S15. NCCLS, Wayne, the antimicrobial susceptibility panels and Dr Thierry Ferrand PA, USA, 2002.
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