Bactericidal Properties of Rod-, Peanut-, and Star-Shaped Gold Nanoparticles Coated with Ceragenin CSA-131 against Multidrug-Resistant Bacterial Strains

Abstract

Background: The ever-growing number of infections caused by multidrug-resistant (MDR) bacterial strains requires an increased effort to develop new antibiotics. Herein, we demonstrate that a new class of gold nanoparticles (Au NPs), defined by shape and conjugated with ceragenin CSA-131 (cationic steroid antimicrobial), display strong bactericidal activity against intractable superbugs.

Methods: For the purpose of research, we developed nanosystems with rod- (AuR NPs@CSA-131), peanut-(AuP NPs@CSA-131) and star-shaped (AuS NPs@CSA-131) metal cores. Those nanosystems were evaluated against bacterial strains representing various groups of MDR (multidrug-resistant) Gram-positive (MRSA, MRSE, and MLS_b) and Gram-negative (ESBL, AmpC, and CR) pathogens. Assessment of MICs (minimum inhibitory concentrations)/MBCs (minimum bactericidal concentrations) and killing assays were performed as a measure of their antibacterial activity. In addition to a comprehensive analysis of bacterial responses involving the generation of ROS (reactive oxygen species), plasma membrane permeabilization and depolarization, as well as the release of protein content, were performed to investigate the molecular mechanisms of action of the nanosystems. Finally, their hemocompatibility was assessed by a hemolysis assay.

Results: All of the tested nanosystems exerted potent bactericidal activity in a manner resulting in the generation of ROS, followed by damage of the bacterial membranes and the leakage of intracellular content. Notably, the killing action occurred with all of the bacterial strains evaluated, including those known to be drug resistant, and at concentrations that did not impact the growth of host cells.

Conclusions: Conjugation of CSA-131 with Au NPs by covalent bond between the COOH group from MHDA and NH₃ from CSA-131 potentiates the antimicrobial activity of this ceragenin if compared to its action alone. Results validate the development of AuR NPs@CSA-131, AuP NPs@CSA-131, and AuS NPs@CSA-131 as potential novel nanoantibiotics that might effectively eradicate MDR bacteria.

Keywords: Au NPs; CSA-131; MDR (multidrug-resistant); ceragenins; nanosystems.

Figure 1

Overview STEM image of the obtained AuR NPs (a,a1), AuP NPs (b,b1), and AuS NPs (c,c1); Selected area diffraction (SEAD) patterns of the AuR NPs (a2), AuP NPs (b2), AuS NPs (c2); size distribution of the AuR NPs (a3), AuP NPs (b3), AuS NPs (c3); and unenhanced Raman spectra of the MHDA (black spectra)-functionalized nanoparticles by MHDA (blue spectra) and immobilized CSA-131 in the NPs surface (red spectra) for the AuR NPs (a4), AuP NPs (b4), and AuS NPs (c4).

Figure 2

Bactericidal activity of AuR NP@CSA-131, AuP NP@CSA-131, AuS NP@CSA-131, and CSA-131 against Staphylococcus aureus Xen 30 (A), Staphylococcus epidermidis 175 (B), Klebsiella pneumoniae ATCC 700603 (C), Klebsiella oxytoca 329 (D), Pseudomonas aeruginosa LESB58 (E), and Pseudomonas aeruginosa 510 (F). Bactericidal activity of the nanosystems at concentrations of 0.1–10 µg/mL was determined using a standard colony counting assay. Results show the mean ± SD from six measurements. * indicates statistical significance at p ≤ 0.05.

Figure 3

Induction of reactive oxygen species (ROS) generation by Staphylococcus aureus Xen 30 (A), Staphylococcus epidermidis 175 (B), Klebsiella pneumoniae ATCC 700603 (C), Klebsiella oxytoca 329 (D), Pseudomonas aeruginosa LESB58 (E), and Pseudomonas aeruginosa 510 (F) was evaluated by DFCH-DA (2′,7′-dichlorofluorescin diacetate) fluorimetric assay. Formation of ROS by microorganisms subjected to AuR NP@CSA-131, AuP NP@CSA-131, AuS NP@CSA-131, and CSA-131 ranging from 1 to 10 µg/mL was presented. Results show the mean ± SD, n = 3; * indicates statistical significance at p ≤ 0.05, ** ≤0.01, and *** ≤0.001.

Figure 4

The increase of the outer membrane permeability of Klebsiella pneumoniae ATCC 700603 (A), Klebsiella oxytoca 329 (B), Pseudomonas aeruginosa LESB58 (C), and Pseudomonas aeruginosa 510 (D) subjected to the tested nanoparticles. Uptake of NPN by microorganisms upon treatment with AuR NP@CSA-131, AuP NP@CSA-131, AuS NP@CSA-131, and CSA-131 at a concentration of 1–10 µg/mL was investigated using the fluorimetric method. Results show the mean ± SD, n = 3; * indicates statistical significance at p ≤ 0.05, ** ≤0.01, and *** ≤0.001.

Figure 5

Depolarization of the bacterial membrane of Staphylococcus aureus Xen 30 (A), Staphylococcus epidermidis 175 (B), Klebsiella pneumoniae ATCC 700603 (C), Klebsiella oxytoca 329 (D), Pseudomonas aeruginosa LESB58 (E), and Pseudomonas aeruginosa 510 (F) was assessed using a diSC₍₃₎ assay. The evaluation of the degree of cell membrane depolarization in the presence of AuR NP@CSA-131, AuP NP@CSA-131, AuS NP@CSA-131, and CSA-131 ranging from 1 to 10 µg/mL was monitored by the enhancement of fluorescence intensity. Results show the mean ± SD, n = 3; * indicates statistical significance at p ≤ 0.05, ** ≤0.01 and *** ≤0.001.

Figure 6

Reduced survival and increase of PI-positive cells upon exposure of the representative strain of Pseudomonas aeruginosa 510 to AuR NP@CSA-131 for 1 h was evaluated using a fluorescence microscope. Panel (A) demonstrates the untreated control. On the other hand, the viability of the bacteria cells subjected to treatment with AuR NP@CSA-131 at doses of 5 µg/mL (panel B) and 10 µg/mL (panel C) was compromised. The results from one representative experiment are shown.

Figure 7

The release of cytoplasmic proteins from Staphylococcus aureus Xen 30 (A), Staphylococcus epidermidis 175 (B), Klebsiella pneumoniae ATCC 700603 (C), Klebsiella oxytoca 329 (D), Pseudomonas aeruginosa LESB58 (E), and Pseudomonas aeruginosa 510 (F) upon treatment with AuR NP@CSA-131, AuP NP@CSA-131, AuS NP@CSA-131, and CSA-131 at concentrations of 1–10 µg/mL was evaluated by Bradford protein assay. Results show the mean ± SD, n = 3; * indicates statistical significance at p ≤ 0.05, ** ≤0.01 and *** ≤0.001.

Figure 8

Hemoglobin release from human red blood cells (RBCs) incubated in the presence of AuR NPs@CSA-131, AuP NP@CSA-131, AuS NP@CSA-131, and CSA-131 (A–D) at doses of 1–50 µg/mL after 1 h (A), 6 h (B), 12 h (C), and 24 h (D). Results show the mean ± SD, n=3; * indicates statistical significance at p ≤ 0.05, ** ≤0.01 and *** ≤0.001.

Figure 9

Characteristics of gold nanoparticles functionalized by CSA-131. Proposed antimicrobial mechanism of the developed nanosystems which comprises the generation of ROS (reactive oxygen species) associated with the destruction of the bacterial membranes as well as the leakage of the intracellular content and consequently cell death. The figure was prepared using BioRender.