Characterisation, antibacterial activity and mode of action of clay against food-borne Salmonella Typhimurium and Staphylococcus aureus

In halal logistics, the discovery of clay with effective antibacterial activity is needed to combat bacterial contamination on surfaces, to ensure halalan toyyiban aspects of the products. Clays have unique properties such as fine particle size (<2μm), high cation exchange capacity, mineralogical and geochemical composition, generating antibacterial properties. The abundance of clay materials from local deposits have remained largely unstudied for their antibacterial potential, focusing on the removal of food-borne pathogens. This study was undertaken to discover the antibacterial activity of locally sourced clay soils against representative food-borne bacteria, which were gram- negative Salmonella Typhimurium ATCC 14028 and gram-positive Staphylococcus aureus ATCC 13565, followed by characterisation and the study of antibacterial mode of action. A total of 13 soils were sampled from Selangor, and their physicochemical analysis such as moisture content, soil particle size, pH and cation-exchange capacity (CEC) were determined. Antibacterial screening was performed on 13 soils using disc diffusion, which revealed that Munchong and Carey soils significantly inhibit S. Typhimurium (11.17 ± 0.76 mm) and S. aureus (7.90 ± 0.17 mm). Further treatment (untreated and treated) with these 2 soils were carried out to evaluate their difference in pH, CEC, metal content and further antibacterial analysis. Treated preparation caused significant contrasting effect for Carey and Munchong in pH, and the increased in CEC values of both samples were in line with the increased clay fraction. Soluble metal analysis revealed significantly higher concentration of treated Carey in comparison to untreated for all metals tested, except Pb. For bacterial viability testing, treated Carey clay leachate and suspension were the most effective with complete killing of S. Typhimurium, while S. aureus viability is reduced (2 to 3 log) after 6 h of exposure. The minimum inhibitory concentration (MIC) and minimum bactericidal activity (MBC) were 125 mg/mL and 250 mg/mL, respectively, for leachate and suspension, for both of the tested bacteria. Time-kill experiments revealed bactericidal effects of treated Carey on S. Typhimurium within 1 h exposure to both leachate or suspension at 4x MIC. Meanwhile, S. aureus was completely killed after 8 h and 24 h of treatment with treated Carey suspension and leachate, respectively, at 4x MIC. XRD analysis confirmed the presence of pyrite and magnetite, while EDX analysis revealed the presence of Al, Fe and S, consistent with the presence of pyrite. Correlation analysis indicated that the high concentration of metals, especially Al, Cu, Fe, Ni and Zn could be responsible in contributing to antibacterial activity against S. Typhimurium, while Ag was found to have strong positive correlation with log reduction of S. aureus. Results of transmission electron microscopy (TEM) showed abnormalities, including disrupted cell walls following exposure to Carey clay, while analysis of membrane integrity also highlighted leakage of proteins, cellular constituents and changes in membrane potential in treated cells with the generation of reactive oxygen species (ROS) that likely promotes bacterial toxicity. In conclusion, treated Carey clay showed effective antibacterial activity, thus supports the potential application of treated Carey clay as surface sanitising agent in halal logistics.

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