ARTICLE pubs.acs.org/est
What Makes a Natural Clay Antibacterial? Lynda B. Williams,†,* David W. Metge,‡ Dennis D. Eberl,‡ Ronald W. Harvey,‡ Amanda G. Turner,† Panjai Prapaipong,† and Amisha T. Poret-Peterson† † ‡
School of Earth & Space Exploration, Arizona State University, Tempe, Arizona 85287, United States U.S. Geological Survey, 3215 Marine St., Suite E127, Boulder, Colorado 80303, United States ABSTRACT: 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, mineralogies, and chemistries of clays that exhibit antibacterial effects on a broad spectrum of human pathogens including antibiotic resistant strains. Natural antibacterial clays contain nanoscale ( 2)
13
Fe-smectite
8.3
mica (2M1)
Figure 1. Kill curve for a 10 mg/mL (1%) suspension of the OMT clay in DDI water. The top line shows that the control bacterial population was not significantly affected by the lack of nutrients over this time period.
3.3
biotite (1M) 13.6 mica (2M1) 4.1 chlorite
Non-Clays
ceramics can also be antibacterial;11 releasing soluble toxic compounds in proportion to their high specific surface area.12,13 Consequently, our objective is to document what soluble elements E. coli assimilate from the natural antibacterial OMT clay.
Non-Clays
6.0
Non-Clays
quartz
2.0 quartz
calcite
4.4 amphibole
0.5 plagioclase
microcline
3.4 pyrite
8.2 Fe-amphibole 3.8
orthoclase albite
2.9 jarosite 1.6 gypsum
0.2 pyrite 0.9 gypsum
38.3 quartz
apatite total nonclays
’ EXPERIMENTAL METHODS Aqueous leachates were prepared using sterilized clay and water (50 mg/mL). The mixture was ultrasonified (Branson Sonifier 450) then shaken 24 h to equilibrate. The suspension of minerals in water was centrifuged (20 000 rpm; 67 000g) 1 h to remove solids14 and the solution was decanted. E. coli (JM109) was grown to log phase and a cell density of ∼109 cfu/mL in Luria Broth (LB). The cell population produced ∼30 mg of cells (dry wt.) per experiment and each experiment was repeated in triplicate. E. coli was incubated in 1:1 volume ratio with clay leachate. All cultures were grown at 37 °C in a shaker-incubator for 24 h. Bacterial growth was evaluated after incubation, by standard plate counts. Bacteria (live or dead) from experiments were centrifuged at 7000 rpm for 5 min to pellet. Bacterial cells were then rinsed with DDI water (25 mL) in triplicate. Finally, one aliquot was rinsed in 25 mL of 50 mM EDTA:100 mM oxalic acid with 0.85% NaCl (pH adjusted to 6.95 with NaOH). This oxalate-EDTA solution removes metals from the exterior cell envelope15 without causing cell lysis.16 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 40 mL. 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 J. Inorg. Biochem. 1999, 76, 81–88. (32) Schmidt, D.; Jiang, Q.; MacKinnon, R. Phospholipids and the origin of cationic gating charges in voltage sensors. Nature 2006, 444, 776–779. (33) Rao, N. N.; Liu, S.; Kornberg, A. Inorganic polyphosphate in Escherichia coli: the phosphate regulon and the stringent response. J. Bacteriol. 1998, 180, 2186–2193. (34) Arosio, P.; Levi, S. Ferritin, iron homeostasis and oxidative damage. Free Radical Biol. Med. 2002, 33, 457–463. (35) Moore, D. M.; Reynolds, R. C. X-ray Diffraction and the Identification and Analysis of Clay Minerals.; Oxford University Press: New York, 1997. (36) Unden, G.; Achebach, S.; Holighaus, G.; Tran, H.; Wachwitz, B.; Zeuner, Y. Control of FNR function of Escherichia coli by O2 and reducing conditions. J. Mol. Microbiol. Biotechnol. 2002, 4, 263–268. (37) Schoonen, M. A. A.; Harrington, A. D.; Laffers, R.; Strongin, D. R. Role of hydrogen peroxide and hydroxyl radical in pyrite oxidation by molecula oxygen. Geochim. Cosmochim. Acta 2010, 74, 4971–4987. (38) Cohn, C. A.; Fisher, S. C.; Brownawell, B. J.; Schoonen, M. A. A. Adenine oxidation by pyrite generated hydroxyl radicals. Geochem. Trans. 2010, 11 (2), 8. (39) Storz, G.; Tartaglia, L. A.; Farr, S. B.; Ames, B. N. Bacterial defenses against oxidative stress. Trends Genet. 1990, 6, 363–368. (40) Park, S.; Imlay, J. A. High levels of intracellular cysteine promote oxidative DNA damage by driving the Fenton reaction. J. Bacteriol. 2003, 185, 1942–1950. (41) Imlay, J. A.; Linn, S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 1988, 240, 640–642. (42) Kohanski, M. A.; Dwyer, D. J.; Hayete, B.; Lawrence, C. A.; Collins, J. J. A common mechanism of cellular death induced by bactericidal antibiotics. Cell. 2007, 130, 797–810. (43) Tran, N.; Mir, A.; Mallik, D.; Sinha, A.; Nayar, S.; Webster, T. J. Bactericidal effect of iron oxide nanoparticles on Staphylococcus aureus. Int. J. Nanomed. 2010, 5, 277–283. (44) Repine, J. E.; Fox, R. B.; Berger, E. M. Hydrogen peroxide kills Staphylococcus aureus by reacting with Staphylococcol iron to form hydroxyl radical. J. Biol. Chem. 1981, 256, 7094–7096.
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dx.doi.org/10.1021/es1040688 |Environ. Sci. Technol. 2011, 45, 3768–3773
