THE CELL-CLAY SEPARATION AND ELEMENTAL COMPOSITION ANALYSIS APPLIED TO ANTIBACTERIAL CLAY RESEARCH

Antibacterial clays (ABC) are capable of inhibiting bacterial growth of human pathogens. ABC research requires interdisciplinary and novel methods to map the deposits, assess ABCs effectiveness, and determine their mode of action. Testing ABCs is challenging when the aqueous leachate is not antibacterial; this is because clinical microbiology methods are based on spectrophotometry, colorimeter assays, or dilution of antibiotics. The presence of minerals often interferes with the results, rendering many of these tests unfeasible. AMZ is a natural ABC which leachate is not antibacterial. In order to gain insight into its mode of action, we have applied a cell-clay separation protocol that allowed us to observe and quantify chemical changes in Escherichia coliand in AMZ clay after treatment. These two methods provided information about the mechanism of action.

Briefly, 400µL of E. coli (10E8CFU/ml) were incubated with 100mg of clay at 37˚C for 24hrs. To separate the cells from the sediments by density, a protocol modified from Amalfitano et al. (2008) was applied. Then, the elemental composition of the recovered fractions was analyzed by ICP-MS. Controls for E. coli, clay without treatment, and growth medium were included.

Results suggest that AMZ induces membrane damage and metal toxicity. Treated E. coli shows an excess of Al, Fe, and Ti. The Al concentration is 9,027ppm vs. 28.06ppm in the control. The minimum inhibitory concentration of Al is 53ppm. At the aqueous conditions buffered by the clay (pH 4.1, Eh 360mV), the dominant Al specie is Al³⁺. Al⁺³ competes with Ca⁺² and Mg⁺² for available ionic sites in the membrane (Borrok et al 2005), threatening the membrane stability and permeability.

In summary, we propose that the mode of action AMZ clay involves Al toxicity, which attacks the cell membrane and interferes with efflux/influx processes. Thereby, trace elements from the clay invade the cell and cause viability loss and bacterial death.