Fluconazole-Decorated Gold Nanoparticles as a Novel Therapeutic Strategy Against Carbapenem-Resistant Klebsiella pneumoniae

Introduction

The escalating crisis of antimicrobial resistance is profoundly exacerbated by opportunistic pathogens such as Klebsiella pneumoniae, an organism distinguished by its alarming capacity to cause a diverse spectrum of systemic infections.¹ These range from pneumonia and urinary tract infections to bacteremia and invasive liver abscesses, underscoring its clinical versatility and the urgency of the threat it poses.² Due to its extensive resistance to most clinical antimicrobial agents, K. pneumoniae has become a major contributor to the antibiotic resistance crisis. Figure 1 illustrates the common mechanisms by which bacteria develop drug resistance. This pathogen is estimated to cause more than over one hundred thousand deaths annually.³ The World Health Organization (WHO) has designated Carbapenem-resistant K. pneumoniae (CRKP) as a critical-priority pathogen, a status driven by the escalating global challenge of healthcare-associated infections caused by multidrug-resistant, extended-spectrum β-lactamases, and carbapenemase-producing strains. Over the past decade, these infections have solidified into a grave and persistent challenge for both clinical management and public health infrastructure worldwide.^4–6

Figure 1 Diagrammatic representation of bacterial resistance mechanisms.

To tackle CRKP, new antimicrobial medicines are desperately needed. The development of novel therapeutic strategies targeting CRKP faces challenges such as lengthy cycles, high costs, and low success rates, highlighting the urgent need for alternative strategies such as drug repurposing or structural modification of existing compounds. In addition to these obstacles, current clinical strategies for managing CRKP infections each has limitations: Although combination regimens represented by ceftazidime-avibactam have been clinically applied, and literature reports that combinations such as tigecycline with rifampicin enhance antibacterial activity against CRKP, such therapies still face inherent limitations. While combination therapy can enhance efficacy in the short term, it is often constrained by issues such as differences in drug solubility and cross-resistance; Furthermore, studies have shown that various antimicrobial peptides, including Cecropin-4 and Osmin, exhibit antibacterial activity against CRKP. However, antimicrobial peptides are difficult to widely apply due to their susceptibility to protease degradation and poor in vivo stability; Phage therapy, meanwhile, has relatively limited practical use because of its highly specific host targeting and tendency to trigger bacterial immune evasion.^7–13 These limitations compel researchers to explore antimicrobial approaches with entirely new mechanisms of action.

Nanotechnology, which involves creating materials with dimensions of 1–100 nm, is advancing at a remarkable pace.¹⁴ The small size enable their effective dispersion within tissues.¹⁵ They often exhibit unique physicochemical properties, along with biological activities such as antibacterial and antiviral effects,¹⁶ demonstrating significant application potential. This technology has been widely applied in areas such as environmental protection, health, medicine, agriculture, and food science.¹⁷ Among various nanomaterials, gold and silver nanoparticles have been extensively investigated for biomedical applications.¹⁸ A prominent research focus lies in their green synthesis, which capitalizes on biological sources such as plants and microorganisms for production.^19–23 According to existing literature, silver and gold nanoparticles synthesized via green methods using fruit waste exhibit significant antibacterial activity against foodborne pathogens.²⁴ Moreover, green-synthesized gold nanoparticles not only demonstrate antibacterial effects but also show notable anti-biofilm activity.^25,26 Previous studies have reported that nanomaterials are capable of combating acquired antibiotic resistance and biofilm-associated bacterial infections.²⁷ However, existing research on nanoparticles for the treatment of CRKP has several limitations: insufficient characterization of bacterial resistance mechanisms, inadequate evaluation of biocompatibility, and a systematic evaluation of the antimicrobial potential of drug-functionalized gold nanoparticles remains limited.

Fluconazole (FCZ), a triazole antifungal agent, inhibits fungal cell membrane synthesis and is primarily used to treat Candida albicans infections.^28–30 However, its relatively large molecular size, hydrophobicity, and low solubility pose significant challenges for topical application. To address these limitations, researchers have loaded FCZ onto nanomaterial surfaces to enhance drug delivery and improve skin penetration.³¹ The antifungal drug FCZ has a well-established safety profile in clinical applications. According to previous studies, the co-release of nitric oxide and FCZ from the same biomaterial surface can effectively combat both Candida albicans and Escherichia coli.³² The triazole ring is a molecular structure within fluconazole. The nitrogen atoms in the triazole ring contain lone pair electrons, which exhibit high affinity for the surface of gold nanoparticles, enabling stable and firm binding to form a stable nanoconjugate. The fluconazole molecule possesses certain amphiphilic properties, and its structure may help nanoparticles anchor to or disrupt the bacterial cell membrane.^33,34 Therefore, immobilizing FCZ on biomaterial surfaces may not only improve its delivery efficiency but also potentially enhance its antimicrobial efficacy. Nevertheless, the application of FCZ-decorated gold nanoparticles (FCZ_Au NPs) for combating CRKP has not yet been explored. In this study, FCZ was conjugated to gold nanoparticle surfaces via a one-pot synthesis method, and the antibacterial efficacy of the resulting conjugate against CRKP was evaluated. This work provides a comprehensive evaluation of the anti-CRKP efficacy of FCZ_Au NPs. Furthermore, we elucidated the underlying antibacterial mechanisms, revealing the multifaceted nature of their action. We anticipate that this work will provide valuable mechanistic insights and present a viable nanotherapeutic strategy, thereby contributing to the future development of antimicrobial agents against CRKP.

Materials and Methods

Bacteria

The bacterial used in this study were eight clinical CRKP isolates and the quality control strain K. pneumoniae ATCC 700603. All clinical isolates were sourced from the First Affiliated Hospital of Wenzhou Medical University, with species identification confirmed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS; bioMérieux, Lyon, France). The clinically isolated strains used in this experiment were part of the routine hospital laboratory procedure. Prior to each experiment, strains were subcultured from these frozen stocks.

Preparation of FCZ_Au NPs and Au NPs

Gold nanoparticles (Au NPs) and FCZ_Au NPs were created using a one-pot green synthesis process based on previously published techniques.^35,36 Briefly, FCZ (0.05 mmol), Tween 80 (30 mg), and triethylamine (50 μL) were dissolved in 10 mL of double-distilled water (ddH₂O). Subsequently, the solution was stirred at 45°C and 500 μL of HAuCl₄ (0.05 mmol) was introduced for a 1.5 h under continuous stirring. The distinctive violet-black color of FCZ_Au NPs demonstrated the effective synthesis and confirmed through comprehensive characterization. In addition, the Au NPs without any decoration were synthesized via a sodium borohydride reduction method. Undecorated Au NPs were produced in an ice-water bath by swirling Tween 80 (30 mg) and HAuCl₄ (500 μL, 0.05 mmol) in 10 mL of cold water. An ice-cold aqueous solution of NaBH4 was then added dropwise, which immediately turned the solution brown. Stirring was continued for 1 h. Nanoparticles were dialyzed against ddH₂O for 24 h before being filtered through a 0.22 μm membrane for sterilization.

Characterization of FCZ_Au NPs

The ultraviolet-visible (UV-Vis) absorption spectrum was acquired with a BioTek Synergy NE02 microplate reader (USA). The hydrodynamic diameter and zeta potential were determined by dynamic light scattering (DLS; Malvern Zetasizer Pro, UK), while the nanoparticle morphology was visualized by transmission electron microscopy (TEM; FEl Talos F200S, USA). Furthermore, the surface chemistry was probed using Fourier-transform infrared spectroscopy (FTIR; Thermo Fisher Scientific Nicolet 6700, USA).

Antimicrobial Susceptibility Testing

We determined the minimum inhibitory concentration (MIC) by the microbroth dilution method in accordance with CLSI (M100, 35th ed., 2025) and EUCAST guidelines.³⁷ Briefly, antimicrobial agents, prepared at 4 times the final test concentration, were added to the last column and serially diluted two-fold across the plate. Subsequently, added 100 μL bacterial suspension. The plate was then incubated at 37°C. After 16–18 h, the MIC was defined as the lowest concentration of the agent that completely inhibited visible bacterial growth.

Growth Curve

The dynamic antimicrobial efficiency of FCZ_Au NPs was evaluated using a growth curve assay.³⁸ Briefly, the experiment included four groups: Phosphate-Buffered Saline (PBS), FCZ group, Au NPs group, and FCZ_Au NPs group. All agents were standardized to concentrations based on the MIC of FCZ_Au NPs, mixed with a bacterial suspension (1.5 × 10⁶ CFU/mL), and incubated at 37°C. A full-wavelength microplate reader (Shandong Jingdao Optoelectronic Technology Co., Ltd. JD-QMBY, China) was used to measure the optical density at 600 nm at 0, 2, 4, 6, 12, and 24 hours.

Crystal Violet Staining Assay

The experiment included four groups, as described previously.³⁹ 100 µL of bacterial suspension mixed with 100 µL of the corresponding agents, the concentration of agents comparable to 0.5 × MIC of FCZ_Au NPs. After 24 h, the supernatant was discarded. The adhered biofilms were then gently rinsed and air-dried. Subsequently, the biofilms were stained by adding 0.1% crystal violet solution for 15 min, then rinsed off excess dye and air-dried again. The bound dye was eluted with 200 µL of a destaining solution (95% ethanol and 5% acetic acid), and the absorbance was measured at 595 nm.

The effectiveness of FCZ_Au NPs in eradicating preformed mature biofilms was assessed using a similar four-group. Mature biofilms were established by inoculating a bacterial suspension of 1.5×10⁶ CFU/mL in LB broth and cultures at 37°C for 24 h. After careful remove the supernatant, 200 µL agents were added. Each at a concentration equal to the MIC of FCZ_Au NPs for the corresponding strain. Subsequent washing, staining, and elution steps were identical to the aforementioned procedure.

Reactive Oxygen Species Detection (ROS)

ROS levels were measured using the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), in accordance with previously described methods.⁴⁰ Bacterial suspensions were loaded with 10 μmol/L DCFH-DA and incubated. Then centrifugated and washed three times to thoroughly remove unincorporated probe. Subsequently, the stained bacterial pellets were resuspended and treated with the respective agents for a specified duration. Finally, the fluorescence intensity was measured at excitation/emission wavelengths of 488/525 nm using a microplate reader.

Quantitative Real-Time PCR

Total RNA from each bacterial strain was extracted using the Trizol method.⁴¹ Briefly, Trizol reagent, containing phenol and guanidine thiocyanate, was added to bacterial cells to lyse them and inhibit RNase activity. To separate the phases, the homogenate was mixed with chloroform, agitated briskly, then centrifuged. For precipitation, the RNA-containing aqueous phase was moved and combined with an equivalent amount of isopropanol. Following centrifugation and incubation, the RNA pellet was rinsed with 75% ethanol, allowed to air dry, and then dissolved in DEPC-treated water. A NanoDrop 2000/2000c spectrophotometer (Thermo Scientific, Waltham, USA) was used to measure the concentration and purity of RNA. Samples having A260/A280 ratios between 1.8 and 2.0 were used for further analysis. The PrimeScript RT Reagent Kit (Takara, Kusatsu, Japan) was used to create cDNA. Quantitative real-time PCR was then performed on a Thermo Fisher QuantStudio 5 system using SYBR Green chemistry (Takara, Kusatsu, Japan), with 16S rRNA as the endogenous control. Gene expression levels were calculated using the 2^−ΔΔCt method.⁴² All primer sequences used in this study are listed in Table 1.

Table 1 Oligonucleotides Used in This Study

Membrane Permeability Assays

The integrity of the bacterial membrane following FCZ_Au NPs treatment was assessed by evaluating membrane permeability. This was done using the fluorescent probes N-phenyl-1-naphthylamine (NPN) and propidium iodide (PI), following established protocols. NPN fluoresces enter the damaged outer membrane of Gram-negative bacteria, while PI enters cells with compromised cytoplasmic membranes, providing a comprehensive assessment of membrane damage.^43,44 After adjusting the bacterial suspensions to an OD₆₀₀ of 0.3–0.4, they were treated with four groups (each at 0.5 × MIC of FCZ_Au NPs). After washing and centrifugation at 3500 rpm, the bacterial pellets were harvested and resuspended in a solution containing 30 μM NPN and 50 μg/mL PI. The suspension was then incubated at 37°C for 30 min, after which the fluorescence was measured using a microplate reader at excitation/emission wavelengths of 350/420 nm for NPN and 535/615 nm for PI.

In vivo Antibacterial Activity

We selected mice for the in vivo experiments. Healthy male CD-1 (ICR) mice aged 28 to 34 days were purchased from Vital River in Zhejiang, China, with body weights ranging between 24 and 30 g. All mouse experiments were approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Approval No. SYXK 2021–0017) and conducted in full compliance with the Wenzhou Laboratory Animal Welfare and Ethics Guidelines. We established mouse intraperitoneal infection model to evaluate the effect of FCZ_Au NPs on survival rate, following a previously described method.⁴⁵ The mice were randomly divided into four groups, with ten mice in each group. Each receiving an intraperitoneal injection of 200 µL FK8712 suspension (1.5 × 10⁸ CFU/mL). Two hours post-infection, the four groups received intraperitoneal injections of PBS, FCZ (5 mg/kg), Au NPs (5 mg/kg), or FCZ_Au NPs (5 mg/kg), respectively. Survival status was monitored and recorded every 12 h for 96 h after treatment. Additionally, a bacterial load assessment was performed using the same infection model. At the 24-hour endpoint following treatment, mice were anesthetized with 3% isoflurane and sacrificed by cervical dislocation. Peritoneal lavage fluid was then aseptically collected from each mouse, and the bacterial burden was determined via performing standard colony-forming unit (CFU) counts.

Biocompatibility Analysis

We evaluated the hemolytic activity of FCZ_Au NPs according to a previously described method.⁴⁶ Briefly, a 5% red blood cell (RBC) solution (from healthy mouse blood) was incubated with varying doses of FCZ_Au NPs in order to assess hemolytic activity. The negative control was PBS and positive control was 0.1% Triton X-100. Following centrifugation, the supernatant was carefully collected to avoid disturbing the pellet, then measured at a wavelength of 540 nm. The hemolysis rate was calculated [], and a value below 5% was considered safe.^47,48

Additionally, the cytotoxicity of FCZ_Au NPs was assessed.⁴⁹ RAW264.7 cells were seeded in 96-well plates and subsequently exposed to a range of FCZ_Au NP concentrations. Following the treatment incubation, 10 µL of CCK-8 reagent (Solarbio, China) was added. The plates were subsequently incubated for 2 h in the dark. Finally, the absorbance was measured at 450 nm using a microplate reader for the quantification of cell viability.

Statistical Analysis

All quantitative data presented in the graphs are expressed as the mean ± standard deviation (SD). Statistical analyses and graph generation were performed using GraphPad Prism 10. Survival data were analyzed by the Kaplan-Meier method. For comparisons across more than two groups, one-way analysis of variance (ANOVA) was employed, followed by Tukey’s post-hoc test for multiple comparisons. The following symbols were used to denote statistical significance throughout the study: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, p > 0.05.

Results

Characterization of Synthesized FCZ_Au NPs

Currently, the one-pot synthesis method represents a highly convenient and rapid approach for nanoparticle preparation.⁵⁰ UV-Vis spectroscopy analysis of the synthesized FCZ_Au NPs revealed a characteristic peak at 540 nm (Figure 2A), which is typical for spherical gold nanoparticles. This finding was corroborated by DLS measurements, which indicated an average hydrodynamic diameter of 38.97 nm and a polydispersity index (PDI) of 0.295 (Figure 2B). Together, these data collectively confirm the successful synthesis of relatively small and monodisperse nanoparticles. The average zeta potential of FCZ_Au NPs was measured to be −22.52 mV (Figure 2C), suggesting a negatively charged surface and good colloidal stability. Additionally, TEM images revealed that FCZ_Au NPs had a high degree of dispersion and an irregular shape (Figure 2D). FTIR analysis further verified the effective synthesis of FCZ_Au NPs by seeing distinctive changes in the O–H (3463 cm⁻¹), C=O (1733 cm⁻¹), and C–O (1092 cm⁻¹) absorption peaks. The fluconazole benzene and triazole rings’ C-H stretching was identified as the cause of a faint peak at 681 cm⁻¹. These results indicate that fluconazole was successfully loaded onto the surface of the gold nanoparticles via interactions such as hydrogen bonding, forming a FCZ_Au NPs complex (Figure 2E).

Figure 2 Synthesis and characterization of FCZ_Au NPs. (A) Ultraviolet absorption spectrum of FCZ_Au NP. (B) Particle size and dispersity of FCZ_Au NPs. (C) Zeta potential of FCZ_Au NPs. (D) TEM images of FCZ_Au NPs (E) FTIR spectra of FCZ_Au NPs, FCZ, and Au NPs.

In vitro Antibacterial Activity of FCZ_Au NPs

By using broth microdilution, the MICs of compounds against the eight CRKP strains were ascertained (Table 2). While FCZ and Au NPs alone showed poor activity (MICs of 256 and 512 μg/mL, respectively), FCZ_Au NPs exhibited significantly enhanced efficacy, with MICs ranging from 4 to 16 μg/mL.

Table 2 Antimicrobial Susceptibility of the FCZ, Au NPs, and FCZ_Au NPs Against Carbapenem-Resistant K. pneumoniae Strains

The dynamic antibacterial effects of FCZ_Au NPs in vitro were monitored via bacterial growth curves over a 24-h period (Figure 3). A key observation was the complete suppression of bacterial growth throughout the entire incubation period in the FCZ_Au NPs group. This stood in stark contrast to the robust and significant growth observed in the other groups. Together, these data collectively validate the potent and superior anti-CRKP efficacy of FCZ_Au NPs.

Figure 3 The growth curve results of eight CRKP strains after treatment in each group.

Anti-Biofilm Activity of FCZ_Au NPs

Biofilms maintain bacterial structural integrity, facilitate adhesion to surfaces, and enhance antibiotic resistance by protecting embedded cells from antimicrobial agents.^51,52 This highlights the importance of eradicating bacterial biofilms. We assessed the anti-biofilm efficacy of FCZ_Au NPs via the crystal violet staining method. The results demonstrated that FCZ_Au NPs effectively inhibited both biofilm formation (Figure 4A) and pre-existing mature biofilms (Figure 4B).

Figure 4 Anti-biofilm activity of FCZ_Au NPs. (A) Inhibition of biofilm formation of eight bacteria strains after treatment in each group. (B) Removal of mature biofilm of eight bacteria strains after treatment in each group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Investigation of the Antibacterial Mechanisms of FCZ_Au NPs

By inducing severe oxidative stress, excessive ROS leading to bacterial cell death.⁵³ We measured intracellular ROS levels following drug treatment. The FCZ_Au NPs-treated group exhibited a marked increase in ROS levels relative to all other groups, implicating oxidative stress as a key antibacterial mechanism (Figure 5). To molecularly substantiate this finding, we quantified the expression of the soxS. This gene serves as a key transcriptional regulator activated during oxidative stress and is rapidly upregulated when bacteria encounter adverse environments, making it a sensitive indicator of intracellular oxidative damage.⁵⁴ Relative to other groups, soxS expression was significantly upregulated in the FCZ_AuNPs-treated group (Figure 6). This indicates that the bacteria encountered unfavorable conditions after FCZ_AuNPs treatment, which triggered a compensatory stress response.

Figure 5 The level of ROS in eight bacteria strains after treatment in each group. ****P < 0.0001.

Figure 6 The expression level of soxS in eight bacteria strains after treatment in each group. *P < 0.05; **P < 0.01; ****P < 0.0001.

The bacterial cell membrane is a critical barrier against antimicrobial agents, and its damage increases permeability, thereby enhancing bactericidal effects.^55,56 In this study, membrane permeability was assessed using the fluorescent probes NPN and PI for outer and inner membrane integrity, respectively. The results demonstrated that treatment with FCZ_Au NPs at 0.5 × MIC increased outer membrane permeability (Figure 7A) as well as inner membrane permeability (Figure 7B) across all eight tested strains.

Figure 7 Antibacterial mechanisms of FCZ_Au NPs. (A) Assessment of bacterial outer membrane permeability using the NPN assay. (B) Assessment of bacterial inner membrane permeability using the PI assay. ***P < 0.001; ****P < 0.0001.

Evaluation of the in vivo Antibacterial Activity of FCZ_Au NPs

To establish the abdominal infection model, mice were intraperitoneally inoculated with a standardized bacterial suspension. Treatments were administered 2 h post-inoculation, using PBS as a control, and survival rates were monitored at 12-hour intervals. The results demonstrated a significant survival advantage in the FCZ_Au NPs-treated group over the other three groups at 96 h (Figure 8A). Furthermore, bacterial colony counts from peritoneal fluid revealed a highly significant reduction in bacterial counts in mice treated with FCZ_Au NPs (Figure 8B). Collectively, these findings demonstrate that FCZ_Au NPs possess a dual advantage: significant in vitro antibacterial activity against CRKP, coupled with promising in vivo therapeutic outcomes, as evidenced by improved survival rates and decreased bacterial burden in the murine intraperitoneal infection model. This underscores their potential as a viable candidate for further development against challenging CRKP infections.

Figure 8 In vivo antibacterial activity of FCZ_Au NPs. (A) 96-hour survival rates of mice in different treatment groups after FK8712 infection. (B) Bacterial load in the peritoneal cavity of mice from different treatment groups after FK8712 infection. **P < 0.01; ****P < 0.0001.

Safety Assessment of FCZ_Au NPs

The safety of the drug concentrations used in this study was assessed through a series of experiments. The RBC hemolysis assay showed that at 32 μg/mL and below, the hemolysis rate induced by FCZ_Au NPs was less than 5%, indicating that FCZ_Au NPs exhibited no significant hemolytic activity at antibacterial effective concentrations (Figure 9A). Meanwhile, results from the cytotoxicity assay demonstrated that FCZ_Au NPs exhibited no significant toxicity to RAW264.7 cells even at 64 μg/mL (Figure 9B). In summary, these preliminary experiments showed that FCZ_Au NPs exhibit a favorable safety profile at the tested concentrations.

Figure 9 The Biocompatibility of FCZ_Au NPs. (A) Hemolytic rate of red blood cells after FCZ_Au NPs-treatment with different concentrations. (B) Cytotoxicity of FCZ_Au NPs at different concentrations. ns, P > 0.05.

Discussion

Antimicrobial resistance (AMR) poses a worldwide concern driven by the dissemination of bacteria and resistance genes across human, animal, and environmental reservoirs.^57,58 Infections caused by carbapenem-resistant Enterobacteriaceae (CRE), especially CRKP, lead a formidable challenge in global infectious disease control due to limited therapeutic options.^59–61 Given this challenge, new treatment modalities are urgently needed. Au NPs have garnered significant research interest, Au NPs have tunable optical characteristics and ease of surface functionalization, which underpin their diverse applications across biomedical and other fields. Previous studies have shown that Au NPs possess inherent antibacterial potential and can enhance the efficacy of antimicrobial photodynamic therapy.^62–64

FCZ_Au NPs was successfully synthesized by conjugating the surface of Au NPs with the therapeutically utilized antifungal drug fluconazole. The nanoparticles had a PDI of 0.29 and an average hydrodynamic size of 38.97 nm. This narrow size distribution suggests high dispersion stability, which is favorable for cellular uptake. The antibacterial activity of FCZ_Au NPs was confirmed by both in vivo and in vitro tests, underscoring their promising potential to combat challenging infections. The biosafety assessments, including hemolysis and cytotoxicity assays, confirmed the preliminary safety of FCZ_Au NPs at tested concentrations. However, future efforts should focus on optimizing the formulation of FCZ-Au NPs to enhance its stability and targeting efficiency. Additionally, comparative studies with existing nanotherapeutic strategies, such as silver nanoparticles or other drug-functionalized platforms, will be essential to establish the relative advantages and limitations of this approach. Addressing these aspects in subsequent research will be critical for advancing FCZ-Au NPs toward clinical translation.

The bacterial cell membrane serves as the first barrier against external threats. Consequently, disruption of this barrier through altered permeability can induce cell lysis and death. Our results revealed that treatment with FCZ_AuNPs increased the permeability of membranes. This process may originate from two aspects: the interactions of nanoparticle surface functional groups with the bacterial outer membrane, along with the specific binding of FCZ to its components. Together, these effects may lead to membrane structural disruption, creating conditions for subsequent intracellular actions. Further studies revealed that FCZ_Au NPs induce ROS accumulation, confirming that oxidative stress represents a major mechanism underlying the antibacterial action. ROS production compromises cellular antioxidant defenses and induces mechanical damage to cell membranes.^65,66 As highly reactive oxidizing molecules, ROS can cause cell death by damaging bacterial DNA, proteins, and lipid membranes. Moreover, membrane disruption may exacerbate intracellular ROS accumulation: increased membrane permeability promotes the cellular uptake of FCZ_Au NPs, where their interaction with components such as respiratory chain proteins directly triggers ROS generation. However, this still requires further experimental investigation and validation. The soxS, a central regulator of the oxidative stress response, is upregulated under ROS-induced stress to mitigate and repair oxidative damage, once the damage exceeds the bacterial regulatory capacity, it can still cause irreversible harm to the cells.⁶⁷ In this study, FCZ_AuNPs treatment significantly elevated ROS levels, triggering the upregulation of soxS as an adaptive response to oxidative stress. However, when the damage exceeded the bacterial regulatory capacity, it resulted in irreversible bacterial death. Consequently, the upregulation of soxS also serves as corroborating evidence that ROS accumulation is one of the antibacterial mechanisms. The specific underlying mechanisms require further investigation.

In this study, we successfully constructed FCZ_Au NPs and systematically evaluated their activity against CRKP. Through a series of experiments, we confirmed the significant bactericidal effect of this nanoplatform and preliminarily revealed that it may damage bacteria by inducing oxidative stress. However, this study has certain limitations that need to be addressed in future work. First, regarding the antibacterial mechanism, the current exploration remains relatively superficial and has not yet reached the molecular level. Specifically, the interaction between FCZ_Au NPs and specific bacterial outer membrane proteins has not been clarified. Moreover, although we observed an increase in ROS levels, the decisive intracellular targets attacked by ROS that lead to bacterial death remain to be identified. Elucidating these issues requires screening differentially expressed proteins using proteomics and validating them by constructing specific gene knockout strains, thereby fully elucidating the antibacterial mechanisms at the molecular level. Second, in terms of in vivo efficacy evaluation, the current infection model is relatively singular. The pharmacodynamic data in this study are primarily based on an abdominal infection model. However, clinical epidemiological data indicate that CRKP infections are most common in the lungs of patients receiving mechanical ventilation and in the urinary tract environment with indwelling catheters. Given that different infection microenvironments may affect the activity of nanoparticles, future research should establish pneumonia and urinary tract infection models to more accurately assess their actual efficacy in primary clinical lesions. Finally, concerning clinical translation, the distribution, degradation, and excretion pathways of FCZ_Au NPs in vivo remain to be clarified. Although preliminary observations suggest a certain level of biosafety, data on their long-term toxicity and comprehensive pharmacokinetic characteristics are still lacking. Addressing these issues is crucial for advancing their clinical translation.

Conclusion

This study developed a novel nanoconjugate by decorating gold nanoparticles with fluconazole, which exhibited potent antimicrobial and anti-biofilm effects against CRKP. The underlying mechanism was attributed to a multi-target action, including simultaneous damage to the bacterial membranes and induction of intracellular ROS production. This nanoconstruct also showed excellent biocompatibility in preliminary safety assessments, thus representing a promising nanoplatform worthy of further preclinical investigation.