We have identified a natural clay mixture that exhibits in vitro antibacterial activity against a broad spectrum of bacterial pathogens. We collected four samples from the same source and demonstrated through antibacterial susceptibility testing that these clay mixtures have markedly different antibacterial activity against Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Here, we used X-ray diffraction (XRD) and inductively coupled plasma – optical emission spectroscopy (ICP-OES) and – mass spectrometry (ICP-MS) to characterize the mineralogical and chemical features of the four clay mixture samples. XRD analyses of the clay mixtures revealed minor mineralogical differences between the four samples. However, ICP analyses demonstrated that the concentrations of many elements, Fe, Co, Cu, Ni, and Zn, in particular, vary greatly across the four clay mixture leachates. Supplementation of a non-antibacterial leachate containing lower concentrations of Fe, Co, Ni, Cu, and Zn to final ion concentrations and a pH equivalent to that of the antibacterial leachate generated antibacterial activity against E. coli and MRSA, confirming the role of these ions in the antibacterial clay mixture leachates. Speciation modeling revealed increased concentrations of soluble Cu 2+ and Fe 2+ in the antibacterial leachates, compared to the non-antibacterial leachates, suggesting these ionic species specifically are modulating the antibacterial activity of the leachates. Finally, linear regression analyses comparing the log 10 reduction in bacterial viability to the concentration of individual ion species revealed positive correlations with Zn 2+ and Cu 2+ and antibacterial activity, a negative correlation with Fe 3+ , and no correlation with pH. Together, these analyses further indicate that the ion concentration of specific species (Fe 2+ , Cu 2+ , and Zn 2+ ) are responsible for antibacterial activity and that killing activity is not solely attributed to pH.

Introduction

Recent epidemiological studies have demonstrated a steady increase in infections due to antibiotic-resistant bacteria [1], [2]. These trends of increasing antibiotic resistance demonstrate an ongoing need to develop novel therapeutic treatments for bacterial infections. Clays have been used for medicinal applications throughout recorded history. The ancient tablets of Nippur, written approximately 5,000 years ago, listed clays as medicament for healing wounds and stopping “fluxes from the body”. The Ebers Papyrus, the world’s oldest medical text, dated approximately 1600 BC, lists clay as a mineral remedy for ailments such as diarrhea, dysentery, tapeworm, hookworm, wounds, and abscesses [3]. During the late 19th century, clays were used as topical treatments for surgical wounds with demonstrated beneficial effects on pain management, inflammation, putrefaction, and healing processes [4]. Reinbacher [5] describes German physician Dr. Julius Stumpf’s treatment strategy in 1898 of a patient who had long been suffering from a deep and suppurating ulcer of the tibia. This patient refused amputation, so the physician began treatment with a thick layer of fine clay powder. The wound immediately stopped producing a malodorous discharge and after four days of repeated clay application and bandaging, the ulcer healed. More recently, clays have been applied in a similar manner for the treatment of bacterial infections caused by Mycobacterium ulcerans, the causative agent of Buruli ulcer, which is a difficult-to-treat necrotic skin disease. A French humanitarian working in the Ivory Coast of Africa applied thick clay poultices daily, alternating between two types of clay, to individuals afflicted with Buruli ulcer. After several months of treatment, the infections often healed with some scarring and a resumption of normal motor function [6]–[8].

The healing property of clay has been attributed to both the physical and chemical properties of the minerals [8]–[10]. Clay minerals have a negative surface charge that allows the free exchange of particles from the environment such as bacteria, viruses, proteins, nucleic acids, and cations [11]. In particular, kaolinite, talc, and smectite clay minerals are highly absorptive and capable of adhering to the skin, thus offering mechanical protection against external physical or chemical agents and serving as dermatological protectors [11].

While clay minerals have been used with favorable outcomes for centuries in pharmaceutical applications, technology, and dermopharmacy as excipients and as active substances, their use in medical applications does not come without possible side effects. Several important factors that contribute to mineral toxicity include (i) site of ingress to the body (inhalation, ingestion, absorption), (ii) duration of exposure to the particles, and (iii) particle size [9]. The predominant pathogenicity of clay minerals is due to inhalation of the minerals, potentially leading to lung cancer, pneumoconiosis, or silicosis [12]. Moreover, due to their net negative charge, clays bind toxic metals to their surface. When in a hydrated environment, the ionic species adsorbed to clay surfaces can be exchanged into the surrounding medium in a manner that depends on the ionic strength of the aqueous medium and cation selectivity of the clay [13]. Mascolo et al. [14] fed mice one of three different clay samples and detected increased levels of As, Ni, and Se in urine samples, thus indicating that desorptive processes occur after clay ingestion. In addition, Tateo et al. [15] showed that ions, specifically Li, Sr, B, I, Rb, Br, Ba, Na, Cl, Se, and Ca, bound to pelotherapy clays were released from the mineral surface and penetrated human skin. While several of these elements are essential, caution must be taken to ensure that the transferred ion concentrations do not reach hazardous levels.

We have identified a natural clay mixture, designated CB, which has antibacterial activity against a broad spectrum of bacterial pathogens. Previously, we prepared aqueous clay mixture extracts (leachates) and demonstrated that the in vitro antibacterial activity of the natural clay sample is associated with the generation of low pH environment and the chemical desorption of ions from the surface of the clay particles [10]. However, as described in this report, we have collected four samples from the same source and demonstrated that the in vitro antibacterial activity of these minerals depends on the chemical properties of the clay mixtures. This discrepancy in antibacterial activity must be controlled if these clay mixtures or developed clay minerals are to be used therapeutically against topical bacterial infections. In order to define the properties that are associated with varied antibacterial efficacy, we characterized the mineralogical and geochemical composition of the four clay mineral samples. Furthermore, while metals ions have been shown to be toxic, the total ion concentration does not always correlate directly to toxicity. Rather, ion toxicity is directly linked to ion speciation changes influenced by the pH, redox state, ion solubility, osmotic strength, and temperature during experimental conditions [22]. We evaluated the interplay between total ion concentration, ion speciation, and leachate antibacterial activity by performing antibacterial susceptibility testing of leachates supplemented with additional ions and subjected to pH adjustments. Finally, we used Visual MINTEQ to model the speciation and solubility changes of ions present in the leachates to predict which species specifically contribute to the toxicity of the leachates.