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. 2025 Nov 25;44:102324. doi: 10.1016/j.bbrep.2025.102324

Enhanced photodynamic disruption of multidrug-resistant Acinetobacter baumannii biofilm using silver and zinc oxide nanostructures

Reyhaneh Fadishehei a, Zahra Fekrirad a,b,⁎, Abazar Hajnorouzi c, Esmaeil Darabpour d, Seyed Latif Mosavi Gargari a,b,⁎⁎
PMCID: PMC12686648  PMID: 41378099

Abstract

Acinetobacter baumannii is a multidrug-resistant bacterium that frequently causes severe infections in hospital intensive care units. Considering the growing resistance of this pathogen, developing innovative strategies to combat it is essential. This study evaluated the efficacy of antimicrobial photodynamic inactivation (APDI) using Erythrosin B (EB) dye against this bacterium. The role of nanostructures in enhancing the effectiveness of this method was also investigated. The minimum inhibitory concentration of silver and zinc oxide nanostructures was determined against A. baumannii ATCC 19606 and A. baumannii 58ST strains. Then, APDI using EB and nanostructures was performed in the planktonic form. The effect of APDI on biofilm formation and eradication was investigated by crystal violet and colony counting methods. The impact of nanostructures on the uptake of EB by bacterial cells and their toxicity on L-929 cells was also evaluated. The results showed that EB uptake increased by 2.5–3 times in both strains in the presence of both nanostructures. APDI with EB alone did not affect the number of bacteria; however, a significant reduction in the number of bacteria was observed when EB was combined with nanostructures. This combination also showed significant inhibitory effects on biofilm formation and eradication. Nanostructures alone had a lower efficacy in inhibiting biofilm formation, but their addition to EB increased the lethal effect on biofilm. The possible mechanism of this effectiveness is the increase in the permeability of the bacterial cell membrane due to the presence of nanostructures, leading to more photosensitizer uptake and reactive oxygen species production.

Keywords: Antimicrobial photodynamic inactivation, Silver nanostructures, Zinc oxide nanostructures, Erythrosin B

Graphical abstract

Image 1

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Highlights

  • •

    Silver and zinc oxide nanostructures increased bacterial Erythrosin B uptake.

  • •

    A significant reduction of bacterial count by Erythrosin B combination with nanostructures.

  • •

    Erythrosin B combination with nanostructures, inhibits biofilm formation and enhances eradication.

1. Introduction

Acinetobacter baumannii is an opportunistic and resistant nosocomial bacterial pathogen that causes Gram-negative infections in therapeutic settings, especially in intensive care units (ICUs) and critically ill patients with dangerous and widespread epidemics. It has a remarkable ability to resist antibiotics and is recognized by the American Society of Infectious Diseases as one of the most important multidrug-resistant microorganisms (MDR) [1]. These characteristics have made treating infections caused by this organism one of the serious challenges in the field of medicine, and its clinical importance has doubled. This bacterium can grow in a wide range of temperatures and pH. The formation of biofilm not only increases its resistance to drugs and antimicrobial agents but also allows the rapid spread of the disease in dry and humid hospital environments [2,3].

Considering the significant increase in resistance to A. baumannii antibiotics in recent years, the need to research and develop new and effective treatment methods to deal with infections caused by this bacterium is felt more than ever. One promising method for dealing with multidrug-resistant infections is Antimicrobial photodynamic inactivation (APDI) [4].

Photodynamic inactivation is a process in which a non-toxic or low-toxic photosensitizer (PS) is used in the presence of oxygen with an appropriate wavelength of visible light. This process stimulates the photosensitizer and produces reactive oxygen species (ROS), including superoxide anion, singlet oxygen, and hydroxyl radicals. These active species oxidize biological molecules such as nucleic acids, proteins, and lipids and eventually lead to cell death [5]. It is also effective in fighting biofilm infections, and by inhibiting the production of factors that contribute to bacterial adhesion and biofilm matrix formation, it can lead to biofilm degradation and thus infection control [6].

One of the factors that helps to increase the efficiency of APDI is the use of nanoparticles and nanostructures. Nanoparticles help to improve the absorption, solubility, and permeability of PS in microbial cells, and also increase the accumulation of photosensitizers at the site of infection [7].In APDI, nanomaterials have traditionally served as nano-sensitizers or carriers for sensitizers. However, they now play a more advanced role by enabling precise control over the release timing and rate of sensitizers, which helps maximize therapeutic outcomes and boosts the programmability and multifunctional capabilities of these agents [8]. Nanoparticles and nanostructures have broad antimicrobial effects against Gram-positive and Gram-negative bacteria. These nanostructures can induce their antimicrobial effects through various mechanisms such as disruption of the bacterial membrane, production of free radicals, and interaction with proteins within the cell [9]. Silver nanoparticles have shown promising potential when combined with light-based techniques to develop innovative treatments for bacterial and fungal infections, as well as cancer, both in vitro and in vivo. Phototherapy methods such as APDI and photothermal therapy utilizing AgNPs have been employed against both Gram-positive and Gram-negative bacteria, particularly targeting strains like Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Furthermore, some studies have paired AgNPs with photosensitizers, including porphyrins (Pps), methylene blue (MB), and phthalocyanines, to investigate their effectiveness in APDI applications [10]. ZnO nanoparticles and nanoflowers were used in APDI for the management of bacterial biofilms [11]. Among the various antibacterial strategies attributed to silver and ZnO nanoparticles, their direct damage to the cell membrane remains one of the most widely recognized and effective mechanisms [12].

Therefore, in this study, EB with silver nanostructures or zinc oxide was used against A. baumannii cells in planktonic and biofilm forms, and the effects of these compounds were investigated along with APDI.

2. Materials and methods

2.1. Strains, cells, and culture conditions

The standard strain Acinetobacter baumannii ATCC 19606 and a clinical isolate designated as A. baumannii 58ST were obtained for this study. Both strains were provided by the microbial collection of Shahed University and cultured routinely in Luria-Bertani (LB) medium at 37 °C. Standard biochemical and molecular methods were used for the identification of the isolate. Our previous study showed that both bacterial strains have antibiotic resistance to streptomycin, tetracycline, ciprofloxacin, oxacillin, cotrimoxazole, cefoxitin, ampicillin, and clindamycin. Strains were susceptible to fosfomycin and chloramphenicol [13]. Mouse fibroblast cell line L-929 was acquired from the Iranian Biological Research Center (IBRC). Cells were cultured using Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10 % heat-inactivated fetal bovine serum (FBS), and incubated under a controlled atmosphere of 5 % CO2 at 37 °C until optimal density was achieved.

2.2. Chemical agents and light source

The photosensitizer erythrosine B (EB, Sigma) was prepared at 1 mM concentration in 0.9 % saline and sterilized by filtration through a 0.2 μm polycarbonate membrane. Illumination was provided by a diode laser emitting green light at a wavelength of 530 nm with an output power of 45 mW, a power density of 0.075 W/cm2, and an irradiance of 4.5 J/min.cm2. The nanomaterials used in this research were obtained from FAPAN Co., Ltd, and were evaluated by applying Field Emission Scanning Electron Microscopy (FESEM) (TESCAN mira3). Silver nanostructures are almost spherical, with an average diameter of about 22 nm. However, the zinc oxide nanostructures used in this experiment are almost spherical with an average diameter of 40 nm, and rod-shaped with a cross-sectional diameter of approximately 80 nm.

2.3. Determination of minimum inhibitory concentration (MIC)

MIC determination was carried out in a 96-well microplate format. Mueller-Hinton broth was inoculated with bacterial suspensions at a concentration of 1 × 108 CFU/ml. Two-fold serial dilutions of Ag (initial concentration: 10 mg/ml in sterile 0.9 % saline) and ZnO (initial concentration: 16 mg/ml in sterile 0.9 % saline) were prepared. After incubation at 37 °C for 24 h, bacterial growth inhibition was evaluated. Minimum bactericidal concentration (MBC) was identified by culturing aliquots from wells without visible growth onto LB agar and assessing colony formation after further incubation [14].

2.4. Photosensitizer uptake evaluation

An overnight bacterial culture was harvested by centrifugation (6000 rpm, 10 min), and the absorbance was adjusted to 1.0 at a wavelength of 600 nm, to approximately 109 CFU/ml. Bacteria were incubated for 20 min in the dark at room temperature with EB (100 μM), alone or combined with Ag or ZnO nanostructures at sub-inhibitory concentrations (1/8 MIC). Cells were centrifuged, washed, and lysed using 1 ml of 2 % sodium dodecyl sulfate (SDS) solution overnight. Absorption of EB was quantified spectrophotometrically at 530 nm using an ELISA reader [15].

2.5. Effect of antimicrobial photodynamic inactivation (APDI) on planktonic cells

Fresh bacterial suspensions (1 × 108 CFU/ml) were treated with EB (100 μM) either alone or in conjunction with Ag or ZnO nanostructures (1/8 MIC) at room temperature in the dark for 20 min. During the light exposure process, which was conducted in a laminar flow environment, the plate cover was removed. Additionally, to prevent oxygen depletion during the process, a distance was maintained between the laser tip and the liquid surface in the wells. Treated and control samples were irradiated with the diode laser for 20 min. Samples were then serially diluted, plated onto nutrient agar, incubated at 37 °C for 24 h, and the CFU/ml was determined [16].

2.6. Effect of APDI on biofilm formation

Bacteria (1 × 108 CFU/ml) were exposed to EB (100 μM) alone or in combination with nanostructure at 1/8 MIC for 20 min in the dark. Samples were subsequently irradiated using the diode laser for 20 min. Then, 100 μl of Tryptic Soy Broth (TSB) medium containing 0.2 % glucose was added to each well and incubated for 24 h at 37 °C. Biofilm formation was assessed by staining with 0.4 % crystal violet solution, washing, and measuring optical density at 490 nm following dye extraction using 30 % acetic acid [17].

2.7. Effect of APDI on biofilm eradication

Bacterial biofilms formed in 96-well plates after 24 h of incubation at 37 °C were exposed to EB alone or in combination with nanostructure (Ag or ZnO, 1/8 MIC) for 20 min in the dark. Biofilms were then irradiated for 20 min. Cells were mechanically detached using scraping and vigorous pipetting of the wells, serially diluted, plated on nutrient agar, and incubated at 37 °C for colony-forming unit enumeration (CFU/ml) [18].

2.8. Cytotoxicity evaluation

MTT assay was employed to evaluate the cytotoxicity of EB with Ag or ZnO nanostructure on L-929 cells. Cells at 90 % confluency were incubated for 20 min at 37 °C with EB-nanostructure combinations, followed by exposure to laser irradiation or darkness. After overnight incubation, cells were treated with MTT solution (0.5 mg/ml) for 4 h. Optical density at 540 nm was measured post-dissolution with dimethyl sulfoxide (DMSO) [19].

2.9. Statistical analysis

All experiments were performed in triplicate. Data were expressed as mean ± standard error (SE). Statistical significance was evaluated using the Post Hoc Tukey test (p < 0.05) via GraphPad Prism software version 8.

3. Results

3.1. Nanoparticle minimum inhibitory concentration

The minimum inhibitory concentration of both bacterial strains was determined for Ag and ZnO nanostructures. The results are listed in Table 1. Both nanostructures' sub-MIC (1/8 MIC) concentration was used in all experiments.

Table 1.

The minimum inhibitory concentration of Ag and ZnO nanostructures against A. baumannii strains.

Acinetobacter baumannii ZnO nanoparticle
 Ag nanoparticle
MBC (mg/ml) MIC (mg/ml) MBC (mg/ml) MIC (mg/ml)
ATCC 19606 8 8 5 1.6
58ST 8 8 5 1.6
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3.2. Effect of nanostructures on PS uptake

As shown in Fig. 1A, the amount of EB uptake by A. baumannii ATCC 19606 increased significantly after treatment with a combination of Ag or ZnO nanostructures. The percentage of EB uptake after using both nanostructures increased by about 2.5 times (P < 0.05). Fig. 1B showed that the amount of EB uptake by A. baumannii 58ST increased significantly by Ag or ZnO nanostructures. The percentage of EB uptake after using both nanostructures increased by about 3 times (P < 0.05).

Fig. 1.

Fig. 1

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Evaluation of bacterial PS uptake. Control: Untreated group, EB: 100 μM, Ag and ZnO: 1/8 MIC concentration, ∗∗ significant at p < 0.01, ∗∗∗ significant at p < 0.001, ∗∗∗∗ significant at p < 0.0001.

3.3. Effect of nanostructures on APDI of planktonic form

Fig. 2A showed that EB alone in the presence of laser light caused a decrease of about 1.7 log in the number of A. baumannii ATCC 19606, which was significant compared to the control group. However, treatment with Ag or ZnO nanostructures, combined with EB in the presence of 20 min of laser light, significantly increased the effectiveness of this method, especially in the case of Ag nanostructures. This synergy of EB dye and Ag and ZnO nanostructures caused a decrease of about 7 and 3 logarithms in the number of bacteria, respectively (P < 0.05). These findings indicated that the use of Ag or ZnO nanostructures together with EB induced a lethal effect against the planktonic form of A. baumannii ATCC 19606 strain, but the use of EB dye alone had only a sub-lethal effect, which was also statistically significant.

Fig. 2.

Fig. 2

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Photodynamic inactivation of bacterial strains in planktonic form. Control: Untreated group, EB: 100 μM, Ag and ZnO: 1/8 MIC concentration, ∗ significant at p < 0.05, ∗∗ significant at p < 0.01, ∗∗∗ significant at p < 0.001, ns: non-significant.

EB alone in the presence of laser light did not significantly reduce the number of A. baumannii 58ST compared to the control group. The treatment with both nanostructures in the presence of laser light caused a lethal effect and a decrease of about 7 and 3 logs in the number of bacteria for Ag or ZnO, respectively (P < 0.05) (Fig. 2B).

3.4. Effect of nanostructures on APDI-mediated inhibition of biofilm formation

According to Fig. 3A, EB alone with laser light and Ag nanostructures alone significantly decreased biofilm formation of A. baumannii ATCC 19606 by about 40 % and 35.6 %, respectively, compared to the control group. The combination of the Ag nanostructures with EB in the presence of laser light induced a higher inhibitory effect on biofilm formation by 67.8 % (P < 0.05). The combination of ZnO nanostructures and EB also decreased the biofilm formation by about 64.5 % while the ZnO alone inhibitory effect was about 25 %, which was statistically significant for both groups (P < 0.05).

Fig. 3.

Fig. 3

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The effect of photodynamic inactivation and nanostructures on bacterial biofilm formation. Control: Untreated group, EB: 100 μM, Ag and ZnO: 1/8 MIC concentration, ∗ significant at p < 0.05, ∗∗ significant at p < 0.01, ∗∗∗ significant at p < 0.001, ∗∗∗∗ significant at p < 0.0001, ns: non-significant.

In the presence of EB alone and laser light, the biofilm formation of A. baumannii 58ST decreased by approximately 37 %, and Ag nanostructures alone also caused a reduction of about 20 % compared to the control group (P < 0.05). The mixture of Ag, EB, and light induced a significant and high reduction in the biofilm formation ability of this strain (63.3 %). ZnO alone had no inhibitory effect, while this nanoparticle combination with EB and light decreased the biofilm formation by about 48.6 % compared to the control (Fig. 3B).

3.5. Effect of nanostructures on APDI-mediated eradication of pre-formed biofilms

The results of the biofilm eradication of A. baumannii ATCC 19606 are shown in Fig. 4A, and of the A. baumannii 58ST in Fig. 4B. EB alone in the presence of laser light only caused a reduction of about a log in the number of both bacterial strains, which was significant compared to the control group. However, the use of Ag or ZnO nanostructures together with EB (for 40 min) and laser light had a greater effectiveness in both strains, which caused a decrease of about 3 logs in the number of bacteria (P < 0.05).

Fig. 4.

Fig. 4

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The effect of photodynamic inactivation on bacterial biofilm eradication. Control: Untreated group, EB: 100 μM, Ag and ZnO: 1/8 MIC concentration, ∗ significant at p < 0.05, ∗∗ significant at p < 0.01, ∗∗∗ significant at p < 0.001.

3.6. Cytotoxicity of nanostructures and APDI

The cytotoxicity test on the L-929 mouse fibroblast cell line is shown in Fig. 5. The cell viability after treatment with Ag or ZnO nanostructures was about 71 % and 69 %, respectively. The cell viability did not significantly alter after treatment with both nanostructures, EB, and light.

Fig. 5.

Fig. 5

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Cell viability of L-929 mouse fibroblast cell line following treatment with APDI and nanostructures. Control: Untreated group, EB: 100 μM, Ag and ZnO: 1/8 MIC concentration, ∗ significant at p < 0.05, ∗∗ significant at p < 0.01.

4. Discussion

A. baumannii is an important nosocomial pathogen that causes drug-resistant infections, especially in patients admitted to intensive care units. This bacterium is considered a serious public health challenge due to its ability to form resistant biofilms. In this regard, APDI has been proposed as a non-invasive and innovative method to inactivate bacteria in biofilm form. Despite its potential, broader application remains constrained by several challenges, including the tendency of the photosensitizer to aggregate, its hydrophobic nature, and limited tissue penetration. These factors collectively reduce reactive oxygen species (ROS) generation and diminish therapeutic efficacy. However, integrating nanoparticles offers a promising solution. Their small size, modifiable architecture, and extensive surface area facilitate enhanced uptake across cellular membranes and biological barriers [20].

Green light wavelengths we used in this study align well with the absorption spectrum of EB, making them highly effective for enhancing the antibacterial action of APDI. In contrast, red light is commonly used in photodynamic therapies involving porphyrins due to its superior penetration into deeper skin layers, such as the dermis. However, for treating superficial skin lesions, green light presents a compelling alternative. Its limited penetration depth not only targets surface-level infections more precisely but also significantly reduces patient discomfort during irradiation [21,22].

Antibiotics are the main approach for treating bacterial infections. However, the extended and excessive use of standard antibiotics is leading to the rapid development of antibiotic-resistant bacteria. Simultaneously, the number of new antibiotics available on the market is steadily declining. There is an urgent need to develop innovative antimicrobial materials and techniques to improve non-antibiotic strategies and enhance the performance and advantages of photodynamic antimicrobials. Antimicrobial approaches like APDI can be utilized independently or in conjunction with other treatments, such as nanomaterials or antibiotics. The latest findings on the combination of APDI and antibiotics suggest that photoinactivation makes microbes more vulnerable to commonly used antimicrobials. Besides, approaches utilizing nanomaterials are also among the most significant strategies in antimicrobial therapy. Nanomaterials serve as effective drug carriers, promoting the penetration of natural compounds, enabling controlled release, and facilitating targeted uptake by bacteria, which enhances drug stability and efficacy, resulting in potent bactericidal effects even at low doses [23].

The nanostructures help to combat resistant biofilms more effectively by improving the penetration and solubility of photosensitizers [24,25]. This study aimed to enhance the efficacy of APDI against both planktonic and biofilm forms of A. baumannii by combining silver (Ag) and zinc oxide (ZnO) nanostructures with the photosensitizer erythrosine B (EB). Both Ag and ZnO nanostructures disrupt bacterial cells by damaging the cell membrane, increasing permeability, improving solubility, and inducing oxidative stress through ROS production, which affects intracellular proteins [26]. To enhance the efficacy of APDA, the PS must be internalized by the target microorganisms rather than remaining on or within the plasma membrane. However, this uptake is notably slow in Gram-negative bacteria due to their structurally complex membranes, which pose a significant barrier to the entry of neutral or negatively charged PS molecules. As a result, efficient microbial inactivation is often hindered [7].

Our findings showed that the PS uptake rate was significantly increased by the combination of EB with nanostructures, which resulted in a more effective and greater reduction in bacterial number in the planktonic form of both A. baumannii strains, as well as increased lethality. While the use of EB alone had no significant effect on bacterial cells. In addition, the use of this compound in APDI increased the lethal effect and effective biofilm destruction in both strains, while the use of EB alone did not affect the destruction of bacterial biofilm. Similarly, Sun et al. demonstrated that silver nanoparticles enhanced photodynamic therapy (PDT) against Staphylococcus aureus and Escherichia coli biofilms, where silver nanoparticles and PDT exhibited a synergistic lethal effect on biofilm cells [24]. Darabpour et al. showed that gold nanoparticles combined with methylene blue (MB) effectively inactivated Staphylococcus aureus biofilms, achieving a significant reduction in biofilm counts, while MB alone had minimal impact [27]. Similarly, another study by Darabpour et al. with MB combined with chitosan nanoparticles demonstrated enhanced antibiofilm effects against S. aureus and P. aeruginosa [18].

Small indentations, or “pits,” begin to form on the cell surface as silver nanoparticles accumulate. One key pathway of bacterial cell death is thought to involve the production of reactive free radicals by these nanoparticles. When silver nanoparticles interact with bacterial cells, they generate free radicals capable of compromising the integrity of the cell membrane. This damage increases membrane permeability and can ultimately lead to cell lysis [12].

The cytotoxicity test results demonstrated that both Ag and ZnO nanostructures produced about a 30 % reduction in cell viability. The study by Mironava et al. showed that the toxicity of gold nanoparticles to human dermal fibroblasts depends on several factors, including concentration, size, and duration of exposure of cells to nanoparticles [28]. Avalos et al. reported that smaller nanoparticles exhibited higher toxicity due to their larger surface area, which leads to increased ROS generation. The larger surface area creates a large number of active sites on the particle surface that can absorb oxygen molecules and produce superoxide radicals and other types of ROS. Therefore, not only the size of the primary nanoparticles but also the size of the aggregated (secondary) nanoparticles can be used as a measure to determine the toxicity of nanoparticles in cell culture [29]. Additionally, ZnO nanoparticle toxicity varies with size and concentration, with higher concentrations causing nanoparticle aggregation that reduces cellular uptake, while lower concentrations allow better absorption [30].

The available data regarding the health risks of NPs, their toxicokinetics in humans and the environment, variations in their distribution across different air, terrestrial, and aquatic milieus, as well as effective degradation mechanisms, remains uncertain. The toxicity of NPs is largely influenced by their size, shape, surface area, and chemical makeup. Furthermore, NPs' interactions with biological entities like proteins, enzymes, and DNA can have an impact on their toxicity. The new stage in NPs toxicity research involves creating safe and effective techniques for the synthesis of NPs, determining biomarkers indicative of NPs toxicity, and establishing protective measures for employees in sectors that utilize NPs. Furthermore, additional studies are required to comprehend the long-term impacts of exposure to NPs on human health [31].

The proposed mechanism for improving the effect of APDI was the increase in bacterial cell permeability by nanostructures, which was validated by increased PS uptake. As the Ag and ZnO nanoparticle concentrations were at 1/8 MIC, reducing the sub-MIC concentration is essential to improve cell viability and reduce cytotoxicity. Beyond improving permeability, nanostructures enhance APDI efficiency by creating pores in bacterial membranes and increasing ROS production. Ag nanostructures bind closely to the cell membrane, allowing greater interaction with Ag + ions, while ZnO nanostructures disrupt bacterial membranes [32].

Silver nanoparticles functionalized with methylene blue (MB-AgNPs) exhibit potent phototoxicity against the bacterial strains Staphylococcus aureus and Pseudomonas aeruginosa, both in their planktonic and within biofilm structures, while remaining non-toxic under dark conditions. Notably, these MB-AgNPs demonstrated the ability to penetrate the innermost layers of the biofilms formed by both species. Upon light activation, they generated significantly higher levels of ROS compared to free PS or AgNPs lacking a PS component. This enhanced phototoxic response was largely attributed to the continuous release of methylene blue and its efficient uptake by bacterial cells [33]. Das et al. found that Ag nanoparticles bind to the cell membrane and enter the cell, increasing ROS production, which reduces the stability of the plasma membrane and lowers the intracellular ATP level. As a result, the cellular respiratory chain is damaged, ultimately leading to cell lysis and death [34]. Prabhu et al. described several antibacterial mechanisms of Ag nanoparticles, including the release of ions that inactivate essential enzymes by interacting with thiol groups [35]. Another mechanism involves Ag nanoparticles denaturing bacterial proteins by binding to thiol groups [36].

When ZnO is exposed to photons with energy exceeding 3.3 eV, electrons are excited from the valence band to the conduction band, initiating a cascade of photochemical reactions. The resulting electrons and positive holes (h+) interact with water molecules, generating hydroxyl radicals and protons (H+). Simultaneously, the electrons react with molecular oxygen to form superoxide anions, which subsequently combine with protons to produce hydroperoxyl radicals. These radicals can further react with electrons and protons to generate hydrogen peroxide. Notably, the yield of H2O2 is closely linked to both the concentration and particle size of ZnO [37].

Xie et al. demonstrated that ZnO nanoparticles significantly upregulated genes related to oxidative stress, membrane leakage, and morphological changes in C. jejuni [38]. The bactericidal activity of ZnO-NPs depends on various factors such as their size, shape, and surface coverage, with smaller nanoparticles exhibiting higher antibacterial activity due to greater surface area. ZnO nanoparticles induce oxidative stress and regulate bacterial virulence genes, reducing pathogenicity [39].

The Core Mechanisms of the anti-biofilm effect of EB-mediated APDI are ROS generation upon light activation, biofilm matrix disruption, and cell membrane permeabilization [40]. The enhancement of this antibiofilm effect by Silver/Zinc Oxide Nanoparticles originates from the metal ions released, which interfere with bacterial enzymes and DNA replication, and penetrate biofilms. Additionally, ZnO and Ag nanoparticles downregulate quorum-sensing genes, thereby reducing the production of virulence factors and the formation of biofilms. Nanoparticles physically disrupt biofilm layers, enhancing photosensitizers’ access to deeper bacterial cells. ZnO and Ag nanoparticles can catalyze additional ROS production under light exposure, amplifying the oxidative damage initiated by erythrosine-mediated APDI [41,42].

The combined effect of Ag and ZnO nanostructures with EB enhanced the antibacterial activity against both planktonic and biofilm forms of A. baumannii strains by increasing membrane permeability, thereby allowing for more photosensitizer uptake. Additionally, the use of these nanostructures improved the eradication of pre-formed biofilms, indicating better penetration of the photosensitizer into the biofilm structure. Further studies are needed to explore the mechanisms behind nanoparticle-enhanced ROS production during the APDI process, and animal model tests could validate the safety and efficacy of this approach for clinical use.

5. Conclusions

The use of APDI combined with silver and zinc oxide nanostructures effectively eliminated and reduced the viability of A. baumannii bacteria in both planktonic and biofilm forms. This combination not only helps to eliminate free bacterial cells but also has a significant bactericidal effect on the resistant structure of the biofilm. The proposed mechanism is the improvement of the permeability of bacterial cells by nanostructures interacting with the cell membrane, weakening the membrane structure, and increasing the absorption of the photosensitizer. As a result, increases the production of ROS, leading to severe cell damage and bacterial death. These results demonstrate the high potential of using metal nanostructures in combination with APDI to combat drug-resistant infections of A. baumannii. The use of nanostructures in enhancing the efficiency of APDI is promising for eliminating bacteria and controlling biofilms, particularly for antibiotic-resistant strains. This combination not only improves penetration and destruction of biofilms but also provides an effective strategy against difficult-to-treat infections.

Funding information

This work was supported by the Iranian National Science Foundation (INSF, Grant number 4023535).

CRediT authorship contribution statement

Reyhaneh Fadishehei: Investigation, Writing – original draft. Zahra Fekrirad: Conceptualization, Funding acquisition, Methodology, Validation, Writing – review & editing. Abazar Hajnorouzi: Resources. Esmaeil Darabpour: Resources. Seyed Latif Mosavi Gargari: Conceptualization, Supervision, Validation, Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zahra Fekrirad reports financial support was provided by Iran National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors would like to thank the Deputy of Research at Shahed University.

Contributor Information

Zahra Fekrirad, Email: fekrirad@shahed.ac.ir.

Seyed Latif Mosavi Gargari, Email: slmousavi@shahed.ac.ir.

Data availability

The data utilized in this study are available upon request by the corresponding author.

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Associated Data

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Data Availability Statement

The data utilized in this study are available upon request by the corresponding author.

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