Biogenic Silver Nanoparticles Strategically Combined With Origanum vulgare Derivatives: Antibacterial Mechanism of Action and Effect on Multidrug-Resistant Strains

Abstract

Multidrug-resistant bacteria have become a public health problem worldwide, reducing treatment options against several pathogens. If we do not act against this problem, it is estimated that by 2050 superbugs will kill more people than the current COVID-19 pandemic. Among solutions to combat antibacterial resistance, there is increasing demand for new antimicrobials. The antibacterial activity of binary combinations containing bioAgNP (biogenically synthesized silver nanoparticles using Fusarium oxysporum), oregano essential oil (OEO), carvacrol (Car), and thymol (Thy) was evaluated: OEO plus bioAgNP, Car plus bioAgNP, Thy plus bioAgNP, and Car plus Thy. This study shows that the mechanism of action of Thy, bioAgNP, and Thy plus bioAgNP involves damaging the membrane and cell wall (surface blebbing and disruption seen with an electron microscope), causing cytoplasmic molecule leakage (ATP, DNA, RNA, and total proteins) and oxidative stress by enhancing intracellular reactive oxygen species and lipid peroxidation; a similar mechanism happens for OEO and Car, except for oxidative stress. The combination containing bioAgNP and oregano derivatives, especially thymol, shows strategic antibacterial mechanism; thymol disturbs the selective permeability of the cell membrane and consequently facilitates access of the nanoparticles to bacterial cytoplasm. BioAgNP-treated Escherichia coli developed resistance to nanosilver after 12 days of daily exposition. The combination of Thy and bioAgNP prevented the emergence of resistance to both antimicrobials; therefore, mixture of antimicrobials is a strategy to extend their life. For antimicrobials alone, minimal bactericidal concentration ranges were 0.3-2.38 mg/ml (OEO), 0.31-1.22 mg/ml (Car), 0.25-1 mg/ml (Thy), and 15.75-31.5 μg/ml (bioAgNP). The time-kill assays showed that the oregano derivatives acted very fast (at least 10 s), while the bioAgNP took at least 30 min to kill Gram-negative bacteria and 7 h to kill methicillin-resistant Staphylococcus aureus (MRSA). All the combinations resulted in additive antibacterial effect, reducing significantly minimal inhibitory concentration and acting faster than the bioAgNP alone; they also showed no cytotoxicity. This study describes for the first time the effect of Car and Thy combined with bioAgNP (produced with F. oxysporum components) against bacteria for which efficient antimicrobials are urgently needed, such as carbapenem-resistant strains (E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) and MRSA.

Keywords: ESKAPEE pathogens; Fusarium oxysporum; MRSA; carbapenem resistance; carvacrol; green nanotechnology; oregano oil; thymol.

FIGURE 1

Characterization of the biogenically synthesized silver nanoparticles (bioAgNP) regarding their plasmonic band and morphology. (A) UV-Vis spectra of the bioAgNP and fungal-free solution. (B) Scanning electron microscopy (SEM) micrograph of the bioAgNP.

FIGURE 2

Time-kill curves of Gram-negative and Gram-positive bacterial strains exposed to oregano essential oil (OEO), carvacrol (Car), thymol (Thy), and the bioAgNP individually at minimal bactericidal concentration (MBC), and in the combination containing Thy (0.5× MIC) and bioAgNP (0.5× minimal inhibitory concentration, MIC). Control indicates bacterial growth with no antimicrobial. (A) Escherichia coli ATCC 25922. (B) KPC-producing Klebsiella pneumoniae. (C) Carbapenem-resistant Acinetobacter baumannii. (D) Methicillin-resistant Staphylococcus aureus (MRSA) N315. Values of log10 colony-forming units (CFU)/ml are the mean ± standard deviation.

FIGURE 3

Measurement of oxidative stress over time in E. coli ATCC 25922 exposed to antimicrobials at subinhibitory concentrations (oregano-derived antimicrobials and the bioAgNP individually and in combination) by quantification of intracellular reactive oxygen species (ROS) generation and lipid peroxidation levels. Controls indicate bacterial ROS and malondialdehyde (MDA) generation by cells without the antimicrobials. (A) ROS production levels expressed as relative light units (RLU) whose values are the mean ± standard deviation. (B) Lipid peroxidation levels measured by malondialdehyde (MDA) production after 1, 2, and 3 h of treatment. Values of MDA are the mean ± standard deviation. ^a–d Indicate statistically significant difference (p < 0.05, Kruskal–Wallis test) among treatments and control at the same time in term of oxidative stress; different letters indicate difference, and same letters indicate absence of difference.

FIGURE 4

Extracellular ATP concentrations over the time ofE. coli ATCC 25922 exposed to the oregano-derived antimicrobials and bioAgNP individually and in combination at subinhibitory doses. Control indicates ATP leakage from untreated bacterial sample. ATP levels were measured at four time points of treatment: 0-h time point, after 15 min of treatment, after 30 min of treatment, and after 45 min of treatment. Values of ATP (nM) are the mean ± standard deviation.

FIGURE 5

Extracellular protein, DNA and RNA concentrations of E. coli ATCC 25922 exposed for 1 h to the oregano-derived antimicrobials and bioAgNP individually and in combination at subinhibitory concentrations. Control indicates extracellular biomolecule concentration of bacterial cells without the antimicrobials. (A) Extracellular total proteins (mg/ml). (B) Extracellular single-stranded DNA (ssDNA; ng/μl). (C) Extracellular double-stranded DNA (dsDNA; ng/μl). (D) Extracellular RNA (ng/μl).

FIGURE 6

Scanning electron micrographs of the antibacterial effect of the oregano-derived antimicrobials and bioAgNP individually and in combination against E. coli ATCC 25922. Bacteria were exposed for 30 min to subinhibitory concentrations of the antimicrobials. (A) Untreated control. (B) OEO-treated cells. (C) Car-treated cells. (D) Thy-treated cells. (E) BioAgNP-treated cells. (F) Bacterial cells treated with the combination of Thy plus bioAgNP. Micrographs (A–F) show bacterial cell density, size, shape, and surface morphological changes (15,000×). Inset images show in detail the morphological alterations of treated cells and typical cells of the untreated control (30,000×). Arrows: morphological changes (surface protrusions), cellular debris and size-changed cells.

FIGURE 7

Transmission electron micrographs of the antibacterial effect of the bioAgNP individually and in combination against E. coli ATCC 25922. Bacteria were exposed for 1 h to the bioAgNP at subinhibitory concentration. (A) Untreated control showing no changes in cell morphology and regular electron density. (B) BioAgNP-treated cells with morphological changes. (C,D) BioAgNP-treated cells with disruption of external ultrastructures and reduced electron density. Arrow: damaged cellular wall and cytoplasmic membrane. Arrowheads: leakage of cytoplasmic contents.

FIGURE 8

Hemolytic activity at different concentrations of the oregano-derived antimicrobials and silver-based compounds individually. (A) OEO. (B) Car. (C) Thy. (D) BioAgNP. (E) AgNO₃. (F) F. oxysporum-free solution used in bioAgNP synthesis. Maximum tested concentrations of the fungal-free solution and bioAgNP have equivalent volumes. Values of hemolysis percentage are the mean ± standard deviation. The linear model equation used to predict the CC₅₀ of each antimicrobial and its R-squared (R²) are shown.

FIGURE 9

Antibacterial mechanism of action of the bioAgNP and oregano derivatives, both alone and in combination. Oregano vulgare-derivatives, especially Thy, disturb the selective permeability of bacterial cell membrane, resulting in high leakage of cellular proteins, DNA, RNA, and ATP. Electron microscopy analysis shows they also cause physical damage in the cytoplasmic membrane and cell wall. The antibacterial mechanism of Thy also involves oxidative stress by enhancing intracellular reactive oxygen species and amount of MDA (marker of lipid peroxidation). The bioAgNP show multiple antibacterial mechanisms: ROS generation, lipid peroxidation, disruption of cytoplasmic membrane and cell wall, and loss of cytoplasmic content. The combination containing Thy and bioAgNP shows strategic antibacterial mechanism. Thy disturbs the selective permeability of the bacterial cell membrane and consequently facilitates access of nanoparticles to the bacterial cytoplasm. Thy plus bioAgNP causes loss of cytoplasmic content, physical damage in the membrane and wall cell, and oxidative stress.