In vitro and In vivo assessment of the mechanism of action and efficacy of antibacterial clays for the treatment of cutaneous infections

The prevalence of antibiotic resistant bacterial pathogens has increased since the introduction of penicillin in the 1940s. Insufficient development of novel antibacterial agents is leaving us with a failing arsenal of therapies to combat these pathogenic organisms. We have identified a clay mineral mixture (designated CB) that exhibits in vitro antibacterial activity against a broad spectrum of bacterial pathogens, yet the antibacterial mechanism of action remains unknown. Antibacterial susceptibility testing of four different clay samples collected from the same source revealed that these natural clays had markedly different antibacterial activity. X-ray diffraction analyses of these minerals revealed minor mineralogical differences across the 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 resulted in antibacterial activity against E. coli and MRSA, confirming the role of these ions in the in vitro antibacterial clay mixture leachates. The prevailing hypothesis is that metal ions participate in redox cycling and produce ROS, leading to oxidative damage to macromolecules and resulting in cellular death. However, E. coli cells showed no increase in DNA or protein oxidative lesions and a slight increase in lipid peroxidation following exposure to CB-L. Supplementation of CB-L with ROS scavengers eliminated oxidative damage in E. coli, but did not rescue the cells from killing, indicating that in vitro killing is due to direct metal toxicity and not to indirect oxidative damage. Finally, we ion-exchanged non-antibacterial clays with Fe, Co, Cu, and Zn and established antibacterial activity in these samples. Treatment of MRSA skin infections with both natural and ion-exchanged clays significantly decreased the bacterial load after 7 days of treatment. We conclude that 1) in vitro clay-mediated killing is due to toxicity associated directly with released metal ions and not to indirect oxidative damage and 2) that in vivo killing is due to the physical properties of the clays rather than metal ion toxicity.