Assessing the interactions of a natural antibacterial clay with model Gram-positive and Gram-negative human pathogens

The emergence of antibiotic resistant bacteria and increasing accumulations of antibiotics in reclaimed water, drive the quest for new natural antimicrobials. We are studying the antibacterial mechanism(s) of clays that have shown an ability to destroy bacteria or significantly inhibit their growth. One possible mode of action is from soluble transition metal species, particularly reduced Fe, capable of generating deleterious oxygen radical species. Yet another possibility is related to membrane damage as a consequence of physical or electrostatic interaction between clay and bacteria. Both mechanisms could combine to produce cell death. This study addresses a natural antibacterial clay from the NW Amazon basin, South America (AMZ clay). Clay mineralogy is composed of disordered kaolinite (28.9%), halloysite (17.8%) illite (12%) and smectite (16.7%). Mean particle size is 1.6μm and total and specific surface area 278.82 and 51.23 m2/g respectively. The pH of a suspension (200mg/ml) is 4.1 and its Eh is 361mV after 24h of equilibration. The ionic strength of the water in equilibrium with the clay after 24 h. is 6 x10-4M. These conditions, affect the element solubility, speciation, and interactions between clay and bacteria. Standard microbiological methods were used to assess the viability of two model bacteria (Escherichia coli and Bacillus subtilis) after incubation with clay at 37 degC for 24 hrs. A threefold reduction in bacterial viability was observed upon treatment with AMZ clay. We separated the cells from the clay using Nycodenz gradient media and observed the mounts under the TEM and SEM. Results showed several membrane anomalies and structural changes that were not observed in the control cells. Additionally, clay minerals appeared in some places attached to cell walls. Experiments showed that exchanging AMZ clay with KCl caused loss of antibacterial property. Among the exchangeable -and potentially toxic- ions we measured Al+3, Cu+2, Zn+2, Ba+2 and Co+2. Besides being toxic at high concentrations, these species affect the electrophoretic interactions between clay and bacteria surfaces. Additionally, the cation exchange neutralizes the clay surface charge thus modifying further the behavior of particles in suspension. Therefore, we evaluated the clay and bacteria zeta potential (ζ) as an index for possible electrostatic forces and modeled the total interactions using DLVO theory. We suspended the particles in water equilibrated with clay (leachate). Results show that at pH 4, the ζ of clays is -14 mV while it is -3mV for bacteria. The divalent ions and trivalent Aluminum, present in the AMZ leachate, compress the thickness of the double layer (hydration shell) thus decreasing electrostatic repulsion and allowing particles to come closer. The proximity of particles increases the probability of attractive forces to bind clays and cells. In summary, results indicate that a process other than simple chemical transfer from clay to bacteria is operating. The electrostatic attraction and physical proximity may enhance the toxic action of metals and interfere with the membrane properties or processes.