Naturally occurring layered-mineral magnesium as a bactericidal against Escherichia coli

Abstract

Escherichia coli (E. coli) is a versatile organism that can colonize and adapt in the environment, and an example of antibiotic resistant bacteria, persistent throughout the world because they have evolved to express various defense mechanisms to cope with antibiotics and the immune system, therein the need to find good strategies for their treatment, such as the application of industrial minerals. Located in the outer section of the external membrane of E. coli, gram-negative bacteria, is lipid A, the lipid component of an endotoxin that consists of lipopolysaccharides, which is responsible for its toxicity of the bacteria. Arguably, the antibacterial activity of naturally occurring minerals is mediated by surface interactions with lipid A. Here, we study the antibacterial activity of layered mineral Mg against E. coli using in vitro and in vivo tests, and report on: (i) in-vitro E. coli (ATCC 25922) growth behavior (bacteriostatic or bactericidal activity); and (ii) in-vitro RAW264.7 cells growth. Furthermore, the oxidative stress, oxidative degradation of lipids in cell membrane resulting in cell damage via lipid peroxidation (LP), was quantified in vivo using the Thiobarbituric Acid Reactive Substances (TBARS) assay. Finally, since inflammation is the first reaction of an organism to defend itself from outside attack by microorganisms or toxic compounds, clinical tests for inflammation using two independent methods, namely to quantify the inhibition of edema [12-O-tetradecanoylphorbol-13-acetate (TPA) method] and the migration of neutrophils [Myeloperoxidate (MPO) method] were in turn assessed. Growth curves (log phase) for E. coli showed that adding brucite or talc in concentrations as low as 20μgmL−1 cause death. Absorbance values at λ=600nm were found to be significantly lower in magnitude relative to positive controls, with no evidence for the presence of Colony Forming Units (CFUs) in agar plates at 24 or 48h. Critical to E. coli growth, Mg acts as a cofactor of alkaline-phosphatase metaloenzyme(s); however, as a layered mineral, Mg exerts irreversible negative effects. Meanwhile, results showed a poor bactericidal activity of sepiolite, owing a higher microporosity and number of surface reaction sites, which were then attributed to gelification at the surface vicinity, provoked by dislocations at inversion sites of the tetrahedral sheet. Taken together these results showed that the bactericidal activity of layered mineral Mg was restricted by physical transport over chemical transfer processes, instigated by specific structural properties, namely stacking over bioavailability Mg and by existing surface interactions with lipid A. On the other hand, in vitro viability tests for RAW264.7 cells showed significant decreases if replacing the source of mineral Mg, talc for brucite which, however, was restored after treatment with EDTA. In vivo tests showed that brucite induced LP. The opposite found to be true for talc, with brucite inducing the quantitative production of TBARS by 9.4±1.2nmolTBARSmgprot−1. Brucite surfaces are uncharged and facilitate the stabilization of hydroxyl radical species (HO), a neutral radical species, consequently contributing to lower the overall activation energy for LP. Finally, clinical tests on the effect of mineral Mg on inflammation, as the first immune reaction were conducted. Layered-Mg minerals effectively inhibited edema (edema forms due to increases in the liquid level within the stroma, which changes vascular permeability, especially for capillaries). Exposing inflamed tissue to brucite, talc, and sepiolite inhibited inflammation by 8.3±2.4, 30.2±7.8, and 61.4±8.6%, respectively. Besides, as evidenced by the MPO model, short-time exposure to mineral Mg inhibited the migration of neutrophils. In all, the anti-inflammatory activity by mineral Mg decreased according to: sepiolite≫talc˃brucite. Competing mechanisms thus prevailed, with structural dislocations favoring an abatement of inflammation in tissue while notably deterring its bactericidal properties.