Short CommunicationAntibacterial activity of reduced iron clay against pathogenic bacteria associated with wound infections
Introduction
Humans have used clay for medicinal purposes since prehistoric times, and this practice is now being considered for adoption into the biomedical compendium. A recent report described the application of a French green clay to Buruli ulcers, resulting in apparent activity against Mycobacterium ulcerans and wound re-epithelialization [1], [2]. This example and others like it have generated interest in the pharmacologic potential of certain clays for treatment of infected wounds.
Clay is a size classification of natural minerals referring to particulate diameters of < 2 µm [3]. The mineralogical and chemical compositions of individual clay deposits vary by geologic environment. Clays identified as potentially antibacterial share mineralogical and chemical compositions that provide buffering capacity to fluids in contact with them, which include reduced transition metals (most commonly Fe2+) and whose immense surface areas (hundreds of m2/g) control the water chemistry, which is key to sustained mineral viability [1], [4]. Many underlying mechanisms for the antibacterial activity of various clays have previously been investigated [5], [6], [7], [8].
A variety of clays were previously evaluated against planktonic liquid cultures incubated with equal parts of a clay suspension followed by quantitation over 24 hours [4], [9]. In these studies, one Fe2+-bearing clay from a deposit in Oregon (mined by Oregon Mineral Technologies, OMT) exhibited superior activity compared with controls when evaluated against: Escherichia coli ATCC 25922; extended-spectrum β-lactamase-producing E. coli ATCC 51446; Pseudomonas aeruginosa ATCC 27853; Salmonella enterica subspecies enterica serovar Typhimurium ATCC 14028; Staphylococcus aureus ATCC 29213; S. aureus USA300; Staphylococcus epidermidis ATCC 14990; methicillin-resistant S. epidermidis (MRSE) ATCC 35984; and a methicillin-resistant S. aureus (MRSA) isolate from Sonora Quest Laboratories, Tempe, AZ [6], [9], [10]. This natural clay is dominated by illite-smectite (a group of clay minerals containing an expandable interlayer structure) pyrite, Ca-plagioclase, and quartz [1], [9]. The smectite interlayer region acts as a reservoir from which metals, which may have antibacterial effects, are gradually released via cation exchange [10], [11]. When clay containing reduced transition metals is taken from its natural environment and mixed with oxygenated water, soluble metals from the minerals likely provide aqueous reactants that drive an antibacterial process [9]. The proposed general mechanism for the antibacterial activity of OMT Blue Clay is that hydration of the clay results in dissolution of reduced Fe2+ and Al3+ from the minerals, which together damage the bacterial membranes, allowing excess Fe2+ to cause intracellular protein damage by oxidation [9]. The soluble metals assumed to be involved in the antibacterial action are protected from rapid oxidation by adsorption into the expandable clay interlayer, potentially conferring to the clay a more sustained effect than metals in the leachate solution alone [1], [9].
The current study examined the effect of the OMT Blue Clay and its aqueous leachate on monomicrobial pathogenic bacteria in planktonic and biofilm states.
Section snippets
OMT Blue Clay and leachate preparation
Vials containing clay were autoclaved, followed by adding sterile water (BarnsteadTM NanopureTM, Thermo Fisher ScientificTM Marietta, OH) for a clay concentration of 200 mg/mL; the mixture was homogenized on a stir plate overnight. A leachate was extracted from a portion of the equilibrated suspension by ultra-centrifugation at 9000 rpm for 1 hour at room temperature; the supernatant constituted the leachate and was collected and stored at 4°C. The leachate remained at pH < 4 and Eh ≈ 400–600
Planktonic experiments
The OMT Blue Clay and leachate were bactericidal against all Gram-positive bacteria tested over 24 hours, with the exception of OMT leachate-treated S. aureus IDRL-6169 and USA300 (Fig. 1). Growth of all Gram-positive organisms was reduced to the limit of detection over 24 hours following OMT Blue Clay and leachate treatments except leachate-treated S. aureus IDRL-6169 and USA300. The OMT Blue Clay and leachate were also bactericidal against all Gram-negative bacteria tested over 24 hours,
Discussion
While clays have previously been used in a variety of medical applications, the antibacterial activity of clays is just beginning to be scientifically evaluated [1], [2], [7], [9], [13], [14]. This study investigated the activity of a specific clay against pathogenic bacteria common to wound infections in both the planktonic and biofilm states. The OMT Blue Clay that was tested has been mineralogically and chemically defined in detail [1].
The current results support the hypothesis that
Conclusions
In summary, this study demonstrated that the application of OMT Blue Clay and its aqueous leachate to monomicrobial planktonic communities and biofilms results in significant reductions in population size. These results are provocative because they demonstrate susceptibility to clay-based treatment among both Gram-positive and Gram-negative bacteria, including strains resistant to traditional antibiotics. While this line of antibacterial therapeutics requires further development, the current
Acknowledgements
We thank Oregon Mineral Technologies for permission to study their clay deposit, as well as the Antibacterial Resistance Leadership Group (ARLG) for permission to use the A. baumannii strain. The authors acknowledge Kerryl E. Greenwood-Quaintance, M.S., for her expertise and review of the present work. We thank Henry Chambers (University of California, San Francisco) for the kind gift of S. aureus USA300.
Funding
Supported by the National Science Foundation (EAR-1719325). The National Science Foundation
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