Evaluating the impact of TiO2nanoparticles with varying capping agents on cell integrity of Myroides odoratus isolated from spoilt paint coating
Description
The impact of okro (O), basil (B) and spearmint (S) capped TiO2NPs on cell integrity of Myroides odoratus MZ442205 isolated from spoilt paint was investigated. TiO2NPs were synthesized through green routes. The antibacterial activity of the NPs at 5, 10, 20, 40 and 80 mg/ml was evaluated using diffusion technique. Damage on bacterium cell wall after NPs treatment was investigated using differential plating method, UV-Vis and SEM. The Zone of inhibition ranged from 2-5, 2-6, and1-3.5 mm; MIC was 20, 40 and 40 mg/ml while MBC was >80mg/ml for O, B and S capped TiO2NPs respectively. UV-Vis showed shifts in NPs compared to the bulk with absorptions at 211nm and 265nm respectively. Treated cells had 19-26% increase in absorbance at 260nm with increasing NPs concentration indicating leakages of nucleic acid materials into medium compared with the control without NPs. This was corroborated by reduced viable cell population in selective blood agar medium (8.15 x 104, 7.65 x 104 and 0.82 x 104CFU/mL) compared to general purpose nutrient broth (9.3 x 104, 8.7 x 104 and 0.88 x 104 CFU/mL) at 80mg/ml respectively. SEM showed spherical NPs, treated cells with cell wall pitting suggesting susceptibility to TiO2NPs via compromised cell integrity.
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Fresh leaves of Abelmoschus esculentus (okra) and Ocimum gratissimum (African basil) were obtained from the Food Boundary Market, Ajegunle, Lagos, Nigeria while those of Mentha spicata (spearmint) were collected from the Botanical Garden, University of Lagos, Nigeria. They were identified and authenticated by Dr. Nodza of the Botany department Herbarium, Faculty of Science, University of Lagos, Nigeria. A. esculentus, O. gratissimum and M. spicata leaves with herbarium numbers 8908, 8906 and 8907 respectively, were properly washed with distilled water and air-dried for about a week. Subsequently they were blended into fine powder with an electric blender. The capping agents were extracted by putting 10g each of powdered leaves in a beaker. The powdered sample was boiled in 50mL of distilled water at 90oC for about 20 min. The boiled extracts were cooled and filtered through a filter paper and was properly washed with ethanol. The obtained filtered extracts were kept for further procedure . TiO2 Nanoparticles were synthesized using the plant extracts as capping agents. Their antimicrobial activities were then tested . The MIC of the NPs against M. odoratus was determined using the broth dilution method [19]. The tubes containing 0.1mL of the inoculum from the standardized prepared culture was supplemented with 4mls each of the varying concentrations of the nanoparticles (5, 10, 20, 40 and 80mg/ml). Potassium hydroxide (KOH) served as positive controls while distilled water served as negative controls.. To determine cell wall integrity, 0.1ml of standardized inoculum was introduced into 4ml each of the NPs concentrations in tubes and incubated at 37oC for 1hr. After the incubation, the OD600nm for each concentration was determined and the culture plated out on nutrient agar and blood agar plates and subsequently incubated at 37oC for 24hr. After further incubation for 24hr, each tube was shaken/mixed very well and the content was decanted through a 0.2-mm syringe filter to remove the bacteria. If bacterial cell wall is disrupted, release of cytoplasmic constituents of the cell can be determined. The quantity of nucleic acid released from the cytoplasm was estimated by its detection at absorbance of 260 nm. Therefore, the filtrate was centrifuged at 2000 rpm for 10 minutes. The supernatant was subsequently diluted with 10mls of phosphate buffer solution (PBS), and Ultra Violet – Visible Spectrophotometry analysis at absorbance of 260 nm were determined and recorded. For SEM, samples were prepared and The samples were then dehydrated in a graded ethanol series up to 100% ultra-pure ethanol followed by substitution into hexamethyldisilazane and allowed to air dry, placed onto carbon adhesive coated aluminum stubs, sputter coated (Denton Desk II sputter coater, Denton Vacuum, LLC, Moorestown, NJ) with palladium/gold alloy (60/40), and imaged using a JEOL-7600F SEM (Japan Electron Optics Laboratory, Peabody, MA) at 13 kV for digital image capture.