Photodynamic Effectiveness of Copper-Iminopyridine Photosensitizers Coupled to Zinc Oxide Nanoparticles Against Klebsiella pneumoniae and the Bacterial Response to Oxidative Stress

Abstract

One of the most urgent threats to public health worldwide is the ongoing rise of multidrug-resistant (MDR) bacterial strains. Among the most critical pathogens are MDR-Klebsiella pneumoniae strains. The lack of new antibiotics has led to an increased need for non-antibiotic antimicrobial therapies. Photodynamic therapy (PDT) has become increasingly significant in treating MDR bacteria. PDT uses photosensitizer compounds (PS) that generate reactive oxygen species (ROS) when activated by light. These ROS produce localized oxidative stress, damaging the bacterial envelope. A downside of PDT is the limited bioavailability of PSs in vivo, which can be enhanced by conjugating them with carriers like nanoparticles (NPs). Zinc nanoparticles possess antibacterial properties, decreasing the adherence and viability of microorganisms on surfaces. The additive or synergistic effect of the combined NP-PS could improve phototherapeutic action. Therefore, this study evaluated the effectiveness of the copper(I)-based PS CuC1 compound in combination with Zinc Oxide NP, ZnONP, to inhibit the growth of both MDR and sensitive K. pneumoniae strains. The reduction in bacterial viability after exposure to a PS/NP mixture activated by 61.2 J/cm² of blue light photodynamic treatment was assessed. The optimal PS/NP ratio was determined at 2 µg/mL of CuC1 combined with 64 µg/mL of ZnONP as the minimum effective concentration (MEC). The bacterial gene response aligned with a mechanism of photooxidative stress induced by the treatment, which damages the bacterial cell envelope. Additionally, we found that the PS/NP mixture is not harmful to mammalian cells, such as Hep-G2 and HEK-293. In conclusion, the CuC1/ZnONP combination could effectively aid in enhancing the antimicrobial treatment of infections caused by MDR bacteria.

Keywords: Klebsiella pneumoniae; ZnO nanoparticles; copper(I) complex; multi-drug resistance; photodynamic therapy.

Figure 1

Synthesis and characterization of the photosensitizer and nanoparticles complex. Synthesis of copper(I) complexes CuC1–3 (A). Molecular structure of CuC3 obtained by XRD. Thermal ellipsoids of CuC3 are shown to have a 30% probability. Hydrogen atoms and the counter ion were omitted for clarity (B). Absorption of CuC1–3 complexes, CuC1 + ZnONP in acetone solution and ZnONP in ethanol solution (C). X-ray diffraction of the ZnO obtained from Zincite JC PDF 36–1451 and for synthesized ZnO particles (D). TEM images of ZnONP. The dimensions bar represents 200 nm length (E).

Figure 2

Characterization of ESBL-producing K. pneumoniae strains. The K. pneumoniae KPPR-1 and ST258 laboratory strains were confirmed to be susceptible and to be extended-spectrum β-lactamase producers, respectively. The Kirby–Bauer diffusion test identified the KPPR-1 K. pneumoniae as sensitive to Caz (upper left) and Cro (lower left) and susceptible to the combinations of Caz + Cla (upper right) and Cro + Cla (lower right), where the inhibition halo exceeded 5 mm in each case (A). In contrast, the ST258 strain was not sensitive to Caz or Cro (upper right and lower right, respectively) but was sensitive to the combinations of Caz + Cla (upper left) and Cro + Cla (lower left), categorizing it as an ESBL producer (B). Through PCR, the genotypes of the KPPR-1 (SHV) and ST258 (SHV and TEM) strains were analyzed and compared to the control strain (SHV, CTX-M, and TEM) (C). Both strains carried the SHV resistance gene, which is common to all K. pneumoniae; only the TS258 strain contained the TEM gene that encodes resistance to ceftazidime. A control DNA showing three bands was utilized (+), while a negative control (−) without DNA template exhibited only the primer dimer bands.

Figure 3

Determination of the photodynamic activity of copper-based photosensitizer compounds. The cooper-based photosensitizers CuC1–C3 photodynamic properties were tested in K. pneumoniae KPPR-1 (A) and ST258 (B) strains. The CuC1 MEC was determined at 0–32 µg/mL (C). Bacterial viability is expressed as log₁₀ of mean ± SD. ns = p > 0.05; * = p < 0.05 two-tailed t-test compared to control bacteria in the dark.

Figure 4

Determination of the photodynamic activity of CuC1 coupled to nanoparticles. The ZnONP MEC was determined in an interval of 0–128 µg/mL and was tested against the KPPR-1 and ST258 K. pneumoniae strains (A). The photodynamic properties of 4 µg/mL of CuC1 photosensitizer coupled with 80 µg/mL of the zinc oxide nanoparticle (B). Bacterial viability is expressed as log₁₀ of mean ± SD * = p < 0.05; *** = p < 0.001 two-tailed t-test compared to control bacteria in the dark.

Figure 5

Determination of the minimum inhibitory concentration of CuCl + ZnONP mix. The minimal effective concentration of serial dilutions of CuCl photosensitizer mixed with a fixed concentration of 80 µg/mL of ZnONP was determined in K. pneumoniae KPPR-1 and ST258 strains (A). The minimal effective concentration of serial dilutions of 0–128 µg/mL of ZnONP photosensitizer mixed with a fixed concentration of 2 µg/mL of CuCl was determined in K. pneumoniae KPPR-1 and ST258 strains (B). Bacterial viability was assessed after PDT exposure and expressed as log₁₀ of mean ± SD * = p < 0.05; ** = p < 0.01 ANOVA test compared to untreated control.

Figure 6

Detection of reactive oxygen species produced by PS/NP mix. Luminescence (RLU) of the H₂O₂ (A) and the fluorescence of the MB (C), standard curves. Luminescence (B) and fluorescence (D) of the 1:32 PS/NP mix at 2 μg/mL CuC1 activated (PDT) or without light activation (PS) and probe solely without PS/NP mix (ROS-Glo or SOSG, respectively).

Figure 7

Transcriptional response of K. pneumoniae to photooxidative stress. The transcriptional response of K. pneumoniae strains KPPR-1 and ST258 to photodynamic stress induced by the CuCl/ZnOPN mixture was determined using RT-qPCR. Changes in the expression of genes related to the oxidative response, including oxyR, sodA, rpoE and hfq (A), as well as extracytoplasmic genes involved in the restoration of the bacterial envelope, such as mrkD, acrB, magA and rmpA (B), were assessed. Variations in gene expression were evaluated by analyzing RNA abundance through relative quantification using the 2^−ΔCt method compared to the untreated controls.

Figure 8

Cytotoxicity in mammalian cells of the CuC1 + ZnONP mixture. The survival of human cell lines Hep-G2 and HEK-293 exposed to a mix of 2 µg/mL CuC1 with 64 µg/mL ZnONP, unexcited (PS/NP), and excited with 61.2 J/cm² of blue light (PDT) was compared to the untreated control cells (Ctrl) (A). Cell death was determined by the exclusion of trypan blue and expressed as a percentage of living cells of treated compared to untreated control cells (B). Cell viability was determined by colony-forming assay expressed as surviving fraction (C). ns = p > 0.05, using Student’s t-test between treated and untreated cells.