Metal Ions, Not Metal-Catalyzed Oxidative Stress, Cause Clay Leachate Antibacterial Activity

Abstract

Aqueous leachates prepared from natural antibacterial clays, arbitrarily designated CB-L, release metal ions into suspension, have a low pH (3.4–5), generate reactive oxygen species (ROS) and H2O2, and have a high oxidation-reduction potential. To isolate the role of pH in the antibacterial activity of CB clay mixtures, we exposed three different strains of Escherichia coli O157:H7 to 10% clay suspensions. The clay suspension completely killed acid-sensitive and acid-tolerant E. coli O157:H7 strains, whereas incubation in a low-pH buffer resulted in a minimal decrease in viability, demonstrating that low pH alone does not mediate antibacterial activity. 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 the antibacterial leachate. Further, supplementation with numerous ROS scavengers eliminated lipid peroxidation, but did not rescue the cells from CB-L-mediated killing. In contrast, supplementing CB-L with EDTA, a broad-spectrum metal chelator, reduced killing. Finally, CB-L was equally lethal to cells in an anoxic environment as compared to the aerobic environment. Thus, ROS were not required for lethal activity and did not contribute to toxicity of CB-L. We conclude that clay-mediated killing was not due to oxidative damage, but rather, was due to toxicity associated directly with released metal ions.

Natural and ion-exchanged illite clays reduce bacterial burden and inflammation in cutaneous meticillin-resistant Staphylococcus aureus infections in mice

“…All experiments were completed in triplicate. The antibacterial activity of the leachates was previously determined and published elsewhere (Otto & Haydel, 2013;Otto et al, 2014).…”

“…Clays subjected to ion exchange were purchased from commercial suppliers (kaolinite from SigmaAldrich; EC12 illite and DV12 montmorillonite from Argiletz) and were autoclaved for 1 h at 121 uC before experimental use. (Otto et al, 2014) and mixing at 300 r.p.m. at room temperature for 20-24 h. The clay suspensions were subsequently centrifuged (31 000 g) for 3 h at 4 uC to separate insoluble and soluble fractions.…”

“…The SMM16 solution alone was used as a control and exhibited a 2-log 10 decrease in viability of MRSA after 24 h ( Fig. 1) (Otto et al, 2014). Natural kaolinite did not display antibacterial activity, exhibiting an average 0.4-log 10 unit decrease in viability after 24 h, a lesser decrease than the H 2 O-only control (Fig.…”

“…After ion-exchanging the three different clay samples with an antibacterial mixture of metal ion salts (SMM16) (Otto et al, 2014), we assessed the in vitro antibacterial activity of the IE clays and the natural clays (non-IE clays). The SMM16 solution alone was used as a control and exhibited a 2-log 10 decrease in viability of MRSA after 24 h ( Fig.…”

“…These five exchangeable ions were subsequently shown to be responsible for in vitro antibacterial activity of natural clays (Otto & Haydel, 2013). Synthetic microbicidal mixtures (SMMs), which are aqueous solutions that contain low concentrations of Fe, Co, Cu and Zn metal chloride salts, exhibit in vitro antibacterial activity (Otto et al, 2014). We used SMMs to ion-exchange natural, nonantibacterial clays and determined that the ion-exchanged clays were imparted with in vitro antibacterial activity.…”