Antibacterial Activity of Aluminum in Clay from the Colombian Amazon

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The Antibacterial Activity of Aluminum in Clay from the Colombian Amazon Sandra Carolina Londono, Hilairy E. Hartnett, and Lynda B. Williams Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04670 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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Environmental Science & Technology

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The Antibacterial Activity of Aluminum in Clay from the Colombian Amazon

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S. Carolina LondonoA*, Hilairy E. Hartnett A,B, and Lynda B. WilliamsA

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A. School of Earth & Space Exploration, Arizona State University, Tempe, Arizona

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B. School of Molecular Sciences, Arizona State University, Tempe, Arizona

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Corresponding Author

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Physical Sciences, PSF-686, 550 East Tyler Mall, Arizona State University

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Tempe, AZ 85287-1404

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* E-mail: [email protected]; phone (480)276-1188

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Abstract The problems of antibiotic overuse compel us to seek alternative antibacterial agents.

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Some clays have been shown to kill antibiotic-resistant human pathogens and may provide an

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alternative to known antibiotics. Here we show that Al-toxicity plays a central role in the

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antibacterial action of a kaolin-rich clay from the Colombian Amazon (AMZ). Antibacterial

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susceptibility testing shows minimum inhibitory concentrations of 80 mg/ml against a model E.

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coli (ATCC 25922). The clay buffered the media pH to ~4.6, and Eh values to +360mV.

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Chemical analysis of AMZ and bacteria showed that Al, P, and transition metals (Fe, Cu, Mn,

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Zn) were exchanged during incubation at 37˚C. Only Al derived from the clay exceeded the

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minimum inhibitory concentrations for E. coli under acidic conditions. Ion imaging showed

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elevated Al in the bacterial membrane, and high intracellular Fe, relative to untreated controls.

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Phosphorous depletion in E. coli after reaction with AMZ, together with evidence for membrane

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permeabilization suggests that Al reacts with membrane phospholipids, enhancing intracellular

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transport of metals. These results highlight the importance of dissolved Al for amplifying the

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toxicity of transition metals to human pathogens.

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Introduction The misuse and overuse of antibiotics threatens both environmental and human health by

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polluting water and soils and stimulating the proliferation of antibiotic-resistant bacteria.1,2,3

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Antibacterial clays may provide a more environmentally-benign alternative for treating certain

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health problems. Furthermore, the availability of these common minerals in developing

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countries, with limited health services, could make antibacterial clays a valuable treatment for

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certain skin infections. However, to use antibacterial clays safely and effectively, we need to

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understand how they affect pathogenic bacteria, and evaluate potential side effects.

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The antibacterial activity of clay is linked to the particular chemistry and to the mineral

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properties that control the pH and the oxidation state (Eh) of the hydrating water used to make a

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clay poultice.4,5 The presence of transition metal ions in the clay, (e.g., Fe2+) is an essential

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component of some antibacterial clay.5,6,7 Aqueous Fe2+ produces reactive oxygen species (ROS)

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that can have deleterious effects on cell components such as membranes and DNA.8 In the

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presence of Al, Fe toxicity may be enhanced because Al can rearrange the membrane structure in

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ways that favor membrane oxidation.9,10 Morrison et al. (2016) recently showed that a clay

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derived from volcanogenic hydrothermal alteration destroys bacteria through the synergistic

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actions of Fe and Al.

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Our study investigates an antibacterial clay of fluvial origin, found in the Colombian

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Amazon (AMZ), mainly composed of kaolin and smectite. Generations ago, the Uitoto people of

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the Colombian Amazon recognized and employed the curative properties of the AMZ clay. They

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ingest the clay to alleviate digestive discomfort and diarrhea. AMZ was shown to be antibacterial

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using standard antimicrobial susceptibility testing.11,12

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Most of the known antibacterial clays are of hydrothermal origin and contain illite-

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smectite (expandable clay) and reduced-Fe phases.6 Aqueous leachates of many antibacterial

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clays are also antibacterial.4,13,14 However, AMZ contains primarily kaolin-group minerals

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(kaolins) and lacks reduced Fe-bearing minerals.11 Furthermore, the aqueous leachate of AMZ is

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not antibacterial,11 indicating that exchangeable elements are not responsible for the antibacterial

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action. Yet, the AMZ clay releases Aluminum. Acid rain studies have shown an effect of Al on

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living organisms15,16,17 This paper shows (via NanoSIMS measurements) that elevated Al is

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associated with bacterial membranes, and that Al is the only element that exceeds MIC in

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solution. This differs from previous research5 showing a more important role of redox sensitive

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metals in the antibacterial process. Therefore, AMZ is an atypical antibacterial clay that appears

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to work by a different mechanism than previously reported.

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The aims of this study were to determine if 1) the observed bactericidal activity is linked

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to metal toxicity as indicated for other antibacterial clays, or 2) if nutrient (e.g., P, Mg) depletion

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via clay adsorption (e.g., Mg, Ca, P) contributes to the antibacterial activity. The results of this

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study expand our knowledge of various antibacterial mechanisms of clays.

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Experimental The AMZ clay is used as a healing clay by members of the Uitoto tribe, who granted

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permission for this research and kindly donated the samples. The clay deposit was sampled in

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northwest Amazon (0°36'48"S, 72°22'26") near Araracuara, within the basin of the Caqueta

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river, an Amazon tributary. An attempt was made to determine the sample age by palynology

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(Pers. Com. Carina Hoorn, 2016), but no pollen was observed. The fluvial deposit may have

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been transported from earlier lacustrine/swamp sediments common to the region.18 Samples were

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placed in air-tight bags for transport, dried and homogenized before separating clay size

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fractions.

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The clay size fraction (