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.
Highlights
We have identified a natural clay mixture, arbitrarily termed CB, that displays broad-spectrum, in vitro bactericidal activity in hydrated suspensions [1,2,3,4]
We demonstrate that EDTA, not reactive oxygen species (ROS) scavengers or carbon supplements, disrupt antibacterial leachate killing and that bactericidal activity occurs in an anaerobic environment, negating the hypothesis that metal-catalyzed oxidative stress causes clay leachate antibacterial activity in vitro
Induction of AR2 with glutamate significantly enhanced E. coli O157:H7 ATCC 43889 survival in the low pH EG medium (Fig. 2) Compared to a non-inducing (NI) environment with 1% CB, glutamate-induction of AR2 in acid-tolerant E. coli O157:H7 delayed the time required for the CB suspension to completely kill the cells (Fig. 2); after a 2 h exposure to 1% CB, chemically-induced, acid-resistant E. coli O157:H7 was completely killed (Fig. 2)
Summary
We have identified a natural clay mixture, arbitrarily termed CB, that displays broad-spectrum, in vitro bactericidal activity in hydrated suspensions [1,2,3,4]. We prepared aqueous extracts from the natural clay mixtures, termed leachates, that are devoid of physical particles. These leachates, designated CB-L, maintain in vitro antibacterial activity, but do not cause cell lysis [2]. Killing is dependent on the abiotic chemical, not physical, properties of hydrated clay suspensions [1,2,3]. 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 Escherichia coli and methicillinresistant Staphylococcus aureus (MRSA), confirming the role of these ions in the antibacterial clay mixture leachates [1]
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