Bioconjugated Thymol-Zinc Oxide Nanocomposite as a Selective and Biocompatible Antibacterial Agent against Staphylococcus Species

Abstract

Owing to the rapid spread of antibiotic resistance among Staphylococcus species, effective and low-risk alternatives to antibiotics are being actively searched. Thymol (THO), the most abundant component of the oil extracted from thyme, can be considered as a natural antibacterial alternative. However, the low antibacterial activity and non-selectivity of THO limit its usage as a universal anti-Staphylococcus agent. Herein, we report the bioconjugation of THO with ZnO nanoparticle (ZO), which resulted in the TZ nanocomposite (NC), as a potent and selective antibacterial agent against Staphylococcus species, particularly S. epidermidis. The cell-free supernatant (CFS) of ATCC 25923 cultures was employed for the production of TZ NC. Successful production of TZ NC was confirmed via X-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectroscopy, and ultraviolet-visible (UV-Vis) studies. TZ NC had selective efficacy against Staphylococcus species, with MIC values 2-32-fold lower than THO. The antibacterial mechanisms of TZ NC are proposed to involve membrane rupture, suppression of biofilm formation, and modulation of new cell wall and protein-synthesis-associated cellular pathways. Its biocompatibility against HCT116 cells was also checked. Our findings suggest that the TZ nanocomposite could improve the selectivity and bactericidal activity of THO against target species.

Keywords: Staphylococcus; antimicrobial; biocompatibility; selectivity; thymol.

Figure 1

X-ray diffraction (XRD) patterns of the ZO and TZ NC samples.

Figure 2

(a) UV–vis spectrum of THO, ZO, and TZ NC samples. Inset shows the enlarged spectrum (as marked) of TZ NC sample. (b) FT-IR spectrum of THO and TZ NC samples.

Figure 3

Growth curve analysis. (i) Growth of S. epidermidis strain (ATCC 14990) at an absorbance of 600 nm (OD₆₀₀) following treatment with different concentrations of (a) ZO, (b) THO, or (c) TZ NC for 20 h. The growth curve data were plotted as average values with standard deviations of n = 3 using SigmaPlot (ver. 12.5) (Systat Software Inc., San Jose, CA, USA). (d) Cell viability. (ii). Growth of MDR S. epidermidis strain (ATCC 12228) at OD₆₀₀ following treatment with different concentrations of (a) ZO, (b) THO, or (c) TZ NC for 20 h. The growth curve data were plotted as the average values with standard deviations of n = 3 using SigmaPlot (ver. 12.5) (Systat Software Inc., San Jose, CA, USA). (d) Cell viability. A fraction of cells from the end point of the growth curves were spotted on LB agar plates and incubated at 37 °C for 24 h. The plate images were captured using ChemiDoc^TM MP (Bio-Rad, Hercules, CA, USA) and ImageLab^TM Software (ver.5.2.1, Bio-Rad, Hercules, CA, USA). One of the representatives from n = 3 is presented.

Figure 4

Biofilm formation assays. Relative biofilm formation (OD₅₉₅/OD₆₀₀) was determined for cells of (a) MDR S. epidermidis (ATCC 12228) and (b) type S. aureus (ATCC 25923) strains under the presence or absence of ZO, THO, and TZ NC. The values shown in the graphs represent average values with standard deviation of n = 10 experiments (p < 0.05). The data were analyzed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA).

Figure 5

Cell morphology analysis. Scanning electron microscopy (SEM) images of MDR S. epidermidis cells of ATCC 12228 without (a–d) treatment with either 1/4 MIC (e–h) or 1/2 MIC (i–l) of TZ NC, respectively were shown. No membrane disruption was shown in 1/4 MIC of TZ NC treated cells (e–h) compared to non-treated cells. The whole membrane was aggregated with TZ NC in cells treated with 1/2 MIC of TZ NC (i–l), inducing partial membrane rupture.

Figure 6

Biocompatibility assays. WST assays were performed to determine the viability of HCT116 cells in the presence of various concentrations of ZO, THO, and TZ NC after 24 h. The values shown in the graphs are averages of n = 3 with standard deviation (p < 0.05). Data were analyzed using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA).