Evaluating the Effect of Azole Antifungal Agents on the Stress Response and Nanomechanical Surface Properties of Ochrobactrum anthropi Aspcl2.2

Abstract

Azole antifungal molecules are broadly used as active ingredients in various products, such as pharmaceuticals and pesticides. This promotes their release into the natural environment. The detailed mechanism of their influence on the biotic components of natural ecosystems remains unexplored. Our research aimed to examine the response of Ochrobactrum anthropi AspCl2.2 to the presence of four azole antifungal agents (clotrimazole, fluconazole, climbazole, epoxiconazole). The experiments performed include analysis of the cell metabolic activity, cell membrane permeability, total glutathione level and activity of glutathione S-transferases. These studies allowed for the evaluation of the cells' oxidative stress response to the presence of azole antifungals. Moreover, changes in the nanomechanical surface properties, including adhesive and elastic features of the cells, were investigated using atomic force microscopy (AFM) and spectrophotometric methods. The results indicate that the azoles promote bacterial oxidative stress. The strongest differences were noted for the cells cultivated with fluconazole. The least toxic effect has been attributed to climbazole. AFM observations unraveled molecular details of bacterial cell texture, structure and surface nanomechanical properties. Antifungals promote the nanoscale modification of the bacterial cell wall. The results presented provided a significant insight into the strategies used by environmental bacterial cells to survive exposures to toxic azole antifungal agents.

Keywords: environment; hydrophobicity; oxidative stress; permeability; pharmaceuticals; roughness; xenobiotics.

Figure 1

Chemical structures of azole antifungal molecules used in the experiments. Fc—fluconazole, Ep—epoxiconazole, Cb—climbazole, Cl—clotrimazole.

Figure 2

Changes of (a) cell metabolic activity; (b) cell membrane permeability; (c) optical density of bacterial cells exposed to fluconazole (Fc), epoxiconazole (Ep), climbazole (Cb) and clotrimazole (Cl). Ctrl stands for control samples—microbial cultures without the addition of azole molecules. The metabolic activity is defined in MRU (MTT reducing unit). One MRU corresponds to the absorbance of a solution resulting from the dissolution of the formazan crystals formed by mL of cells per OD600. Cell membrane permeability is expressed as a %.

Figure 3

Microbial stress response to the presence of azole antifungal agents: (a) the level of total glutathione (oxidized glutathione, GSSG + reduced glutathione, GSH); (b) activity of glutathione-S-transferases (GSTs). Bacterial cells were exposed to fluconazole (Fc), epoxiconazole (Ep), climbazole (Cb) and clotrimazole (Cl). Ctrl stands for control samples—microbial cultures without the addition of azole molecules; ns = not significant. See Section 4.7. for the description of statistical analysis.

Figure 4

Representative atomic force microscopy (AFM) three-dimensional and height images supplemented with surface roughness parameters (mean value ± SD) showing morphological differences between (a) untreated control cells of O. anthropi AspCl2.2; cells exposed to: (b) fluconazole; (c) epoxiconazole; (d) climbazole; (e) clotrimazole. Asterisks (*) indicate a statistically significant difference (Cb vs. Ctrl: Rq p = 0.0046; R3z p = 0.0226; Ra = 0.0124). See Figure S1 for the bacterial AFM images of a larger scale.

Figure 5

Bacterial cell adhesive properties: (a) adhesion energy; (b) adhesion force; (c) cell surface hydrophobicity. In violin plots, the difference between each of the treated samples and control sample was statistically significant (p < 0.0001); ns = not significant. See Section 4.7 for the description of statistical analysis.

Figure 5

Bacterial cell adhesive properties: (a) adhesion energy; (b) adhesion force; (c) cell surface hydrophobicity. In violin plots, the difference between each of the treated samples and control sample was statistically significant (p < 0.0001); ns = not significant. See Section 4.7 for the description of statistical analysis.

Figure 6

Empirical distribution of the data of bacterial cell elastic properties measurements: (a) changes in bacterial cell wall deformation; (b) Young’s modulus; (c) stiffness. The difference between each of the treated samples and control sample was statistically significant (p < 0.0001). See Section 4.7 for the description of statistical analysis.