Bactericidal Properties of Rod-, Peanut-, and Star-Shaped Gold Nanoparticles Coated with Ceragenin CSA-131 against Multidrug-Resistant Bacterial Strains
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Media, and Growth Conditions
2.2. Antibacterial Compounds
2.2.1. Ceragenin (CSA-131)
2.2.2. Gold Nanoparticles (Au NPs) Functionalized by CSA-131
2.2.3. Physicochemical Properties of AuR NPs@CSA-131, AuP NPs@CSA-131 and AuS NPs@CSA-131 Nanoparticles
2.3. Estimation of Antibacterial Activity of the Nanosystems
2.3.1. Antimicrobial Susceptibility Testing
2.3.2. Killing Assay
2.4. Analysis of the Bacterial Response to Tested Nanosystems at Molecular Level
2.5. Haemolytic Activity of CSA-131 Nanosystems
2.6. Statistical Analysis
3. Results
3.1. Physicochemical Nature of Rod-, Peanut-, and Star-Shaped Au NPs
3.2. Antimicrobial Activity of the Rod-, Peanut-, and Star- Shaped Au NPs
3.3. Bactericidal Mechanism of the Nanosystems Involves the Induction of Oxidative Stress as well as the Destruction and Depolarization of Bacterial Membranes and Protein Leakage
3.4. The Nanosystems Exert High Biocompatibility at Bactericidal Doses
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strain | Mechanism of Resistance | Type of Strain/Source |
---|---|---|
S. aureus Xen 30 | - MRSA, - constitutive MLSb | - Reference strain/purchased from Caliper Life Sciences (Hopkinton, MA) |
S. epidermidis 175 | - MRSE, - constitutive MLSb | - Clinical strain/isolated from tracheobronchial secretions |
K. pneumoniae ATCC 700603 | - ESBL caused by SHV-18 - impermeability to cephamycin as a mechanism associated with the alteration of porins | - Reference strain/purchased from American Type Culture Collection (ATCC, USA) |
K. oxytoca 329 | - ESBL - acquired plasmid-mediated cephalosporinase AmpC | - Clinical strain/collected from the urine |
P. aeruginosa LESB58 | - highly resistant to antibiotics with production of chromosomally encoded inducible AmpC-β-lactamase | - Hypervirulent and an epidemic cystic fibrosis strain isolated from sputum |
P. aeruginosa 510 | - ESBL related to the active efflux pump - alteration of the outer membrane permeability resulting in OprD2 deficiency and finally resistance to carbapenems | - Clinical strain/collected from tracheobronchial secretions |
Antibiotic/Chemotherapeutic | Staphylococcus aureus Xen 30 | Staphylococcus epidermidis 175 |
---|---|---|
Interpretation | Interpretation | |
Screening test with cefoxitin | Positive | Positive |
Oxacillin | R | R |
Erythromycin | R | R |
Clindamycin | R | R |
Amikacin | R | R |
Gentamicin | R | R |
Ciprofloxacin | R | R |
Levofloxacin | R | R |
Linezolid | S | S |
Daptomycin | S | S |
Teicoplanin | S | S |
Vancomycin | S | S |
Antibiotic/Chemotherapeutic | Klebsiella pneumoniae ATCC 700603 | Klebsiella oxytoca 329 |
---|---|---|
Interpretation | Interpretation | |
Ampicillin | R | R |
Amoxicillin/Clavulanic acid | I * | R |
Piperacillin/Tazobactam | I * | R |
Cefuroxime | R | R |
Cefuroxime axetil | R | R |
Cefotaxime | R | R |
Ceftazidime | R | R |
Ertapenem | S | S |
Meropenem | S | S |
Amikacin | S | S |
Gentamicin | R | R |
Ciprofloxacin | S | R |
Norfloxacin | S | R |
Antibiotic/Chemotherapeutic | Pseudomonas aeruginosa LESB58 | Pseudomonas aeruginosa 510 |
---|---|---|
Interpretation | Interpretation | |
Piperacillin | R | R |
Piperacillin/Tazobactam | R | R |
Ticarcillin/Clavulanic acid | R | R |
Ceftazidime | R | R |
Cefepime | R | R |
Imipenem | S | R |
Meropenem | S | R |
Amikacin | R | R |
Gentamicin | R | R |
Tobramycin | R | R |
Ciprofloxacin | R | R |
Levofloxacin | R | R |
Colistin | S | S |
Assay | Indicator Reagent | Final Bacterial Inoculum | Final Concentration of the CSA-131 | Test Conditions | Results Recording |
---|---|---|---|---|---|
ROS generation | 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Sigma-Aldrich, USA) | OD600 = 0.1 in PBS | 1–10 µg/mL | 60 min incubation at 37 °C with 20 µM DCFH-DA in PBS (in 96-well black plates) | fluorescence emission at wavelengths of 488/535 nm |
Outer membrane permeabilization * | 1-N-phenylnapthylamine (NPN, Sigma-Aldrich, USA) | OD600 = 0.1 in PBS | 1–10 µg/mL | 5 min incubation at 37 °C with 0.5 mM NPN | fluorescence intensity λex = 348 nm/λem = 408 nm |
diSC(3) | 3,3′-dipropylthiadicarbocyanine iodide (diSC(3), Sigma Aldrich, USA) | OD600–0.05 | 1–10 µg/mL | 60 min incubation at room temperature with 0.4 µM diSC(3), followed by 5 min incubation with 100 mM KCl | fluorescence emission at wavelengths of 622/670 nm |
SYTO9/PI-dual staining ** | LIVE/DEAD BacLight Bacterial Viability Kit (CA, USA) | OD600–0.5 | 5 µg/mL and 10 µg/mL | 60 min incubation at 37 °C in PBS, followed by staining using SYTO9 dye and propidium iodide (PI) for 15 min | fluorescence microscopy (Zeiss AxioObserver.A1 Fluorescence Version Inverted Optical Microscope, JPK Instruments, German). |
Protein leakage | Coomassie Brilliant Blue G-250 (Bradford reagent, Sigma-Aldrich, USA) | OD600–0.1 | 1–10 µg/mL | 60 min incubation at 37 °C, followed by 10 min centrifugation (5000 rpm at 4 °C) and incubation of supernatant with Bradford reagent (1:1 ratio) for 10 min in dark | absorbance level at 595 nm |
Ceragenins/Nanosystems/ Microorganisms | S. aureus Xen 30 MIC/MBC (µg/mL) | S. epidermidis 175 MIC/MBC (µg/mL) | K. pneumoniae ATCC 700603 MIC/MBC (µg/mL) | K. oxytoca 329 MIC/MBC (µg/mL) | P. aeruginosa LESB58 MIC/MBC (µg/mL) | P. aeruginosa 510 MIC/MBC (µg/mL) |
---|---|---|---|---|---|---|
CSA-131 | 0.5/1 | 0.5/1 | 2/2 | 1/4 | 4/8 | 2/8 |
AuR NPs@CSA-131 | 0.2/0.4 | 0.2/0.2 | 0.8/1.6 | 0.8/3.2 | 0.8/1.6 | 1.6/3.2 |
AuP NPs@CSA-131 | 0.4/1.6 | 0.4/0.8 | 0.8/3.2 | 0.8/6.4 | 0.8/0.8 | 1.6/3.2 |
AuS NPs@CSA-131 | 0.2/0.8 | 0.2/0.4 | 0.8/1.6 | 0.4/0.8 | 1.6/1.6 | 1.6/3.2 |
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Chmielewska, S.J.; Skłodowski, K.; Depciuch, J.; Deptuła, P.; Piktel, E.; Fiedoruk, K.; Kot, P.; Paprocka, P.; Fortunka, K.; Wollny, T.; et al. Bactericidal Properties of Rod-, Peanut-, and Star-Shaped Gold Nanoparticles Coated with Ceragenin CSA-131 against Multidrug-Resistant Bacterial Strains. Pharmaceutics 2021, 13, 425. https://doi.org/10.3390/pharmaceutics13030425
Chmielewska SJ, Skłodowski K, Depciuch J, Deptuła P, Piktel E, Fiedoruk K, Kot P, Paprocka P, Fortunka K, Wollny T, et al. Bactericidal Properties of Rod-, Peanut-, and Star-Shaped Gold Nanoparticles Coated with Ceragenin CSA-131 against Multidrug-Resistant Bacterial Strains. Pharmaceutics. 2021; 13(3):425. https://doi.org/10.3390/pharmaceutics13030425
Chicago/Turabian StyleChmielewska, Sylwia Joanna, Karol Skłodowski, Joanna Depciuch, Piotr Deptuła, Ewelina Piktel, Krzysztof Fiedoruk, Patrycja Kot, Paulina Paprocka, Kamila Fortunka, Tomasz Wollny, and et al. 2021. "Bactericidal Properties of Rod-, Peanut-, and Star-Shaped Gold Nanoparticles Coated with Ceragenin CSA-131 against Multidrug-Resistant Bacterial Strains" Pharmaceutics 13, no. 3: 425. https://doi.org/10.3390/pharmaceutics13030425