Novel dual-targeting of biofilm formation and pyocyanin production in clinical Pseudomonas aeruginosa isolates using glutamine-modified thiosemicarbazone-conjugated ZnO nanoparticles

Abstract

Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen frequently associated with chronic and hard-to-treat infections, particularly in burn units and immunocompromised patients. Its intrinsic and acquired resistance to multiple antibiotics poses a major therapeutic challenge. While ZnO nanoparticles conjugated with thiosemicarbazone (TSC) have shown promise in general antimicrobial applications, their potential for simultaneously inhibiting biofilm formation and pyocyanin production—key virulence factors—in clinical P. aeruginosa strains remains unexplored. In this study, ZnO nanoparticles were synthesized via a hydrothermal route and conjugated with a glutamine-modified TSC ligand (ZnO@Glu-TSC) to enhance their antimicrobial efficacy. The nanoconjugate was comprehensively characterized using UV–Vis spectroscopy, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). Functional evaluations were conducted against clinical isolates of P. aeruginosa, including minimum inhibitory concentration (MIC), fractional inhibitory concentration (FIC) index, biofilm inhibition, and pyocyanin suppression assays. ZnO@Glu-TSC nanoparticles exhibited a sharp UV–Vis absorption peak at 380 nm with a band gap of 3.26 eV, and XRD confirmed a hexagonal wurtzite structure with an average crystallite size of ~ 19.8 nm. The nanoconjugate demonstrated significantly enhanced antibacterial activity with MIC values ranging from 128 to 512 µg/mL and synergistic effects in 70% of clinical isolates (FIC ≤ 0.5, p < 0.01). Biofilm inhibition assays revealed an 80% reduction in biomass (OD values approaching those of the negative control), while pyocyanin production decreased by more than 75% at 512 µg/mL (p < 0.001). These results represent the first demonstration of ZnO@Glu-TSC's dual antivirulence action against clinical P. aeruginosa strains, underscoring its therapeutic promise as a potent, multi-targeted nanoantimicrobial candidate and warranting further development for translational nanomedicine applications in combating persistent infections.

Introduction

Pseudomonas aeruginosa is a very adaptable opportunistic pathogen that is often linked to chronic and hard-to-treat infections, especially in burn units and immunodeficient patients. This bacterium is among the most prominent contributors to global morbidity and mortality due to its role as one of the most common causes of nosocomial infections in vulnerable patients, including individuals in intensive care units, or with cystic fibrosis, and burdens healthcare systems and the society as a whole [1]. This is an important model organism used in microbiological and infectious disease studies of bacterial pathogenesis, antibiotic resistance, and host–pathogen interactions. The complexity of its virulence mechanisms and resistance allows a good understanding of how multidrug-resistant bacteria evolved, as well as new therapeutic approaches [2]. Even with the current development of antimicrobial treatment, P. aeruginosa is an extremely difficult-to-treat microorganism because of its capacity to form biofilms and its capacity to produce virulence factors including pyocyanin. These processes allow the bacterium to survive in adverse conditions and avoid host immune system and traditional therapies that result in chronic infection with high treatment failure rates [3]. Biofilms composed of self-assembled extracellular matrices of DNA, polysaccharides, and proteins form protective barriers and account for approximately 80% of nosocomial infections and contribute to the global crisis in antimicrobial resistance [4]. Pyocyanin is a blue-green, two-component regulated phenazine pigment, which causes oxidative stress, interferes with immune responses, facilitates biofilm formation, and regulates virulence genes, which makes the management of infections especially challenging [5]. It is known that P. aeruginosa uses quorum sensing (QS)-mediated mechanisms, i.e. lasI/lasR and rhlI/rhlR, to control biofilm formation and pyocyanin synthesis, and researchers have demonstrated that the interference with such mechanisms can reduce pathogenicity [6, 7]. Moreover, the contribution of efflux pumps and membrane impermeability to development of multidrug resistance has also been discussed, which is why new treatment methods should be developed [8]. The emergence of several drug-resistant bacteria has increased the demand for novel and potent antimicrobial drugs, prompting the development of new strategies such as CRISPR-Cas-antimicrobials, phage therapy, nanobiotics, and probiotics to combat resistant pathogens [9, 10]. There is, however, an important gap in knowledge to develop multi-targeted agents that simultaneously prevent biofilm formation, pyocyanin production and bacterial growth in clinical multidrug-resistant strains of P. aeruginosa. These interlinked virulence mechanisms are usually resistant to traditional antibiotics, which is why new approaches such as functionalized nanomaterials are needed [11]. This study produced zinc oxide nanoparticles (ZnO NPs) through a hydrothermal process and functionalized them to produce ZnO@Glu-TSC, adding a glutamine-modified thiosemicarbazone ligand. Comprehensive characterization was conducted using UV–Vis spectroscopy, XRD, FTIR, SEM, and EDX. It also involved functional tests against clinical isolates of P. aeruginosa MIC, determination of FIC index, biofilm inhibition by crystal violet staining and pyocyanin by spectrophotometry and statistical evaluation using ANOVA (p < 0.05). Similar effects are highlighted by recent articles on the use of metal-based nanoparticles (ZnO NPs) which have been attributed to such mechanisms as release of Zn²⁺ ions, the formation of ROS, and membrane disruption [12]. They have become biocompatible, efficient with the advancement in green synthesis; numerous examples of ZnO NP synthesized by mycosynthesis under the influence of Pleurotus sajor-caju extract demonstrated good antibacterial and anticancer activity, being in the form of a sphere and approximately 10 nm diameter which has shown the significance of fungal metabolites in nanoparticle stabilization [13]. Similarly, the ZnO NPs mediated by Pterolobium hexapetalum were identified to possess antimicrobial effect on P. aeruginosa, antioxidant effect and low toxicity thereby facilitating the synthesis of nanoparticles that are environmentally-friendly [14]. Additionally, AgNPs of the same plant had a broad-spectrum antimicrobial effect, which can imply the possibility of hybrid nanomaterials [15]. The following studies form a basis of our conjugation approach, where the benefits of green-synthesized NPs are noted in minimizing the environmental footprint and increasing bioactivity with multidrug-resistant pathogens [13, 15]. Nanoparticles exhibit anti-bacterial properties, and nanotechnology involves nanoparticles of 1-100nm in diameter, where silver-based nanoparticles (AgNPs) have attracted a significant amount of interest due to their antimicrobial and disinfectant properties. Green synthesis, which is applied in this research, stands on using natural biological systems to achieve a sustainable solution with naturally sourced materials and low-energy reactions and can provide a valid alternative to the conventional methods [10]. This study specifically aimed to evaluate the synergistic efficacy of thiosemicarbazone-conjugated ZnO nanoparticles in inhibiting biofilm formation and pyocyanin production in clinical isolates of P. aeruginosa, with the goal of developing a novel multi-targeted nanoantimicrobial agent against multidrug-resistant infections.

Materials and methods

Synthesis of zinc oxide nanoparticles

Zinc oxide nanoparticles (ZnO NPs) were synthesized using a modified version of the method described by Nejabatdoust et al. [16]. Initially, 20 mL of an aqueous solution of Zn (ClO₄)₂·6H₂O with a concentration of 0.75 M was prepared. While stirring continuously at 400 rpm, NaOH solution (1.5–2 M) was added dropwise until the pH reached 12. The mixture was then heated at 100 °C for 3 h, leading to the formation of a white ZnO precipitate. After cooling to room temperature, the precipitate was centrifuged at 800 rpm and washed three times with deionized water and 96% ethanol. Finally, the sample was dried in an oven at 100 °C for 8 h.

Synthesis of thiosemicarbazone

Thiosemicarbazone was synthesized via a Schiff-base condensation reaction, according to the method described by Habibi et al. [17]. In brief, thiosemicarbazide (0.91 g) and 2-pyridinecarboxaldehyde (0.95–1 mL) were mixed in 100 mL of ethanol in a 250 mL round-bottom flask and stirred at 80 °C. A reflux condenser was employed to prevent solvent loss during the reaction. After two hours, two drops of glacial acetic acid were added to the reaction mixture, which was then refluxed for an additional 4–6 h. To concentrate the reaction, the condenser was removed, and the mixture was heated for another hour. The resulting pale yellowish-white precipitate of thiosemicarbazone was separated by centrifugation and washed twice with 5–10 mL of diethyl ether.

Functionalization of ZnO nanoparticles with thiosemicarbazone

According to the method described by Mokhtari et al. [18], ZnO nanoparticles were functionalized via a condensation reaction mediated by glutamic acid (Glu). Initially, to synthesize ZnO@Glu nanoparticles, a mixture containing 20 mL of 0.1 M ammonia solution, 0.5 M Zn(ClO₄)₂·6H₂O solution, and glutamic acid in a 1:2 molar ratio was prepared and heated at 100 °C for 60 min. The resulting ZnO nanoparticles were separated, washed thoroughly with deionized water and ethanol, and then dried at 80 °C for 8 h. For conjugation of thiosemicarbazone (TSC) with ZnO@Glu, 500 mg of ZnO@Glu and 200 mg of TSC were dispersed in 100 mL of ethanol and subjected to sonication for 30 min. The mixture was then stirred overnight at 40 °C. The ZnO@Glu-TSC nanocomposite was collected by centrifugation, washed with deionized water and ethanol, and dried at 60 °C for 6 h (Fig. 1).

Fig. 1.

Schematic Flow Diagram of Thiosemicarbazone Synthesis and Functionalization of ZnO Nanoparticles into ZnO@Glu-TSC Nanocomposites

The synthesized compounds were subsequently submitted to certified laboratories for characterization using FTIR, SEM, EDS, UV–Vis spectroscopy, XRD, and zeta potential analysis.

Identification tests

In this study, twenty clinical isolates of P. aeruginosa were obtained from wound samples of hospitalized patients in hospitals across Tehran Province. Samples were collected using sterile swabs from various clinical wounds, including burn wounds, surgical wounds, and chronic ulcers.

The identification of the isolates was performed based on standard biochemical and microbiological tests, including catalase, oxidase, citrate utilization, methyl red–Voges-Proskauer (MR-VP), indole production, and growth at 42 °C in oxidation-fermentation (OF) medium. These tests were conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The results revealed characteristic features of P. aeruginosa, including positive oxidase activity and the ability to grow at 42 °C. The reference strain P. aeruginosa ATCC 27853 was used as a positive control in all biochemical assays.

Minimum inhibitory concentration (MIC) assay

To determine the minimum inhibitory concentration (MIC), the broth microdilution method was employed as described by Valadbeigi et al. [19]. Initially, 50 μL of Mueller–Hinton Broth (MHB) was dispensed into each well of a 96-well microtiter plate. Subsequently, various concentrations (128, 256, 512, and 1024 μg/mL) of ZnO nanoparticles, TSC, and ZnO@Glu-TSC—prepared in double-distilled water—were added to the wells. Then, 50 μL of a standardized bacterial suspension (1 × 10⁴ CFU/mL), adjusted to a 0.5 McFarland turbidity standard, was added to each well. The plates were incubated at 37 °C for 24 h. Following incubation, the MIC values were recorded as the lowest concentration of each synthesized compound that inhibited visible bacterial growth. P. aeruginosa ATCC 25213 was used as the positive control strain in this assay.

Biofilm detection

Biofilm formation was assessed using the crystal violet microtiter plate assay as described by O’Toole [20]. Briefly, bacterial isolates were cultured in Tryptic Soy Broth (TSB) supplemented with 1% glucose in 96-well microtiter plates and incubated at 37 °C for 24 to 48 h. After incubation, the supernatant was carefully discarded, and the wells were washed with phosphate-buffered saline (PBS) to remove planktonic cells. The adherent biofilms were then stained with 0.1% crystal violet solution for 15 min at room temperature. Excess stain was washed off, and the bound dye was solubilized using 95% ethanol. The absorbance was measured at 590 nm using a spectrophotometer to quantify biofilm biomass.

All assays were performed in triplicate. P. aeruginosa ATCC 25213 was used as the positive control, and sterile culture medium served as the negative control.

Anti-biofilm assay

The biofilm inhibition assay was performed according to the method described by Rafiq et al. [21], with minor modifications. Briefly, biofilm-forming P. aeruginosa strains were cultivated in 96-well microtiter plates. Each well was initially filled with 100 μL of nutrient broth (Oxoid, UK), followed by the addition of 100 μL of synthesized compounds, including ZnO nanoparticles, thiosemicarbazone (TSC), and TSC-functionalized ZnO nanoparticles, all prepared in 10 mg/mL DMSO. Subsequently, 20 μL of bacterial suspension was inoculated into each well. Ciprofloxacin was used as a positive control. The plates were sealed with adhesive lids and incubated at 37 °C for 24 h under aerobic conditions. After incubation, non-adherent planktonic cells were removed by pipetting, and the wells were washed twice with phosphate-buffered saline (PBS, pH 7.1). The plates were air-dried at room temperature for 15 min, and the adherent biofilms were fixed by heating at 60 °C for 60 min. Then, 200 μL of 0.1% crystal violet solution was added to each well and left undisturbed for 20 min. Excess stain was removed, and the wells were washed with 200 μL of PBS to eliminate unbound dye. To solubilize the bound crystal violet, 200 μL of 95% ethanol was added to each well, and the plate was incubated at 4 °C for 30 min. Absorbance was measured at 590 nm using a microplate reader (BIORAD 680), and the percentage of biofilm inhibition was calculated.

Pyocyanin inhibition assay

Quantitative evaluation of pyocyanin pigment inhibition was performed according to the method described by Jiang et al. [22], with slight modifications. Briefly, clinical P. aeruginosa isolates were initially cultured overnight in LB broth at 37 °C with shaking at 200 rpm. Subsequently, 0.5% (v/v) of the overnight culture was inoculated into Pseudomonas Agar Medium for Detection of Pyocyanin (PDP) and incubated for 24 h under the same shaking conditions. The bacterial cultures were treated with MIC concentrations (256 and 512 μg/mL) of the synthesized compounds. After incubation, 2 mL of the culture was collected and centrifuged. The supernatant was transferred to a 10 mL centrifuge tube containing 3 mL of chloroform and vortexed thoroughly. After centrifugation at 10,000 rpm for 5 min, the chloroform layer was carefully transferred to a new tube. The chloroform phase containing extracted pyocyanin was then mixed with 2 mL of 0.2 M hydrochloric acid, vortexed, and centrifuged again. The upper pink-colored aqueous layer was collected, and the absorbance was measured at 520 nm using a spectrophotometer to quantify the pyocyanin content. The untreated P. aeruginosa ATCC 25213 strain was used as the positive control.

Results

Characterization of ZnO@Glu-TSC nanoparticles

UV–visible spectroscopy

To assess the optical properties of ZnO@Glu-TSC nanoparticles, we conducted UV–Vis spectroscopy across a 200–800 nm range. A pronounced absorption peak at 380 nm (Fig. 2) confirms successful ZnO@Glu-TSC nanoparticle formation. The optical band gap energy (E_g) of the ZnO nanoparticles was calculated using Planck’s equation: E_g = hc/λ = 1240/λ (nm) = 1240/380 = 3.26 eV. This 3.26 eV band gap represents a positive outcome, confirming effective synthesis. Significantly, the peak’s sharpness indicates high crystallinity, a promising attribute for antimicrobial applications.

Fig. 2.

Ultraviolet–visible (UV–Vis) absorption spectrum of ZnO@Glu-TSC nanoparticles in the wavelength range of 200–800 nm, exhibiting a characteristic absorption peak at approximately 380 nm

XRD pattern of ZnO@Glu-TSC nanoparticles

To investigate the structural properties of ZnO@Glu-TSC nanoparticles, XRD analysis was performed. The pattern displayed diffraction peaks at 2θ values of 31.8°, 34.5°, 36.3°, 47.5°, 56.6°, 62.9°, 68.0°, and 69.1° (Fig. 3), corresponding to the (100), (002), (101), (102), (110), (103), (112), and (201) planes. These peaks confirm a hexagonal wurtzite ZnO structure, matching the standard JCPDS card No. 36–1451 and indicating phase purity—a positive outcome. Notably, the high intensity of the (101) peak at 36.3° reflects excellent crystallinity, a promising feature for antimicrobial applications. The crystallite size was calculated using the Scherrer equation D = kλ/βcosθ, where k = 0.9, λ = 0.15406 nm, and β ≈ 0.42° at 36.3°, yielding approximately 19.8 nm. Lattice parameters (a = b ≈ 3.249 Å, c ≈ 5.206 Å) align closely with standard values for hexagonal ZnO.

Fig. 3.

X-ray diffraction (XRD) pattern of ZnO@Glu-TSC nanoparticles. The observed diffraction peaks at specific 2θ angles correspond to the standard JCPDS pattern for the wurtzite structure of zinc oxide, confirming the formation of a hexagonal crystalline structure

FT-IR spectrum

To verify chemical conjugation in ZnO@Glu-TSC nanoparticles, FT-IR spectroscopy was performed. The spectrum displayed key absorption bands (Fig. 4): a broad band at 3276 cm⁻¹ for O–H stretching from surface hydroxyl groups [23], peaks at 1419 cm⁻¹ and 1236 cm⁻¹ for C–H bending and C–N stretching in the thiosemicarbazone moiety [24], and a sharp peak at 1088 cm⁻¹ for C = S stretching [24]. These bands confirm successful ZnO-thiosemicarbazone conjugation, a positive outcome. The distinct C = S peak at 1088 cm⁻¹ highlights strong side-chain bonding, promising for antimicrobial stability. Peak in the 600 cm⁻¹ indicates Zn–O and Zn–S vibrations, further validating conjugation at the C = S group [25].

Fig. 4.

FTIR spectrum of ZnO@Glu-TSC nanoparticles in the 400–4000 cm⁻¹ range, confirming the presence of functional groups and successful conjugation

FE-SEM micrograph of ZnO@Glu-TSC nanoparticles

To determine surface morphology and particle size of ZnO@Glu-TSC nanoparticles, FE-SEM analysis was conducted. Micrographs revealed discrete nanoparticles and aggregates (Fig. 5). The clustered particles ranged from 19.6 to 20.2 nm, a positive result confirming nanoscale dimensions. Notably, the consistent 20 nm range aligns with literature [26], an interesting finding for antimicrobial efficacy.

Fig. 5.

Field emission scanning electron microscopy (FESEM) image of ZnO@Glu-TSC nanoparticles. The image reveals a spherical morphology of the nanoparticles with an average size of 19.6—20.2 nm nm (scale bar: 500 nm)

EDX analysis

To confirm elemental composition and conjugation in ZnO@Glu-TSC nanoparticles, EDX analysis was performed. The spectrum showed 70.98% Zn, 19.46% O, 8.50% C, and 1.06% N by weight (Fig. 6). These percentages highlight a ZnO core coated with thiosemicarbazone, a positive outcome. The O:Zn atomic ratio of 1.12:1 closely matches theoretical ZnO values, while the C:N ratio of 9.4:1 confirms ligand attachment. One limitation of this study is the absence of S peaks in the EDX spectrum, potentially due to low sulfur content or instrumental detection limits.

Fig. 6.

Energy-dispersive X-ray spectroscopy (EDX) analysis of ZnO@Glu-TSC nanoparticles. The spectrum confirms the presence of zinc (Zn), oxygen (O), carbon (C), and nitrogen (N), validating the chemical composition of the nanoparticles. The sulfur (S) peak was not detected due to its low content or instrumental limitations

Zeta potential analysis

To evaluate surface charge and stability of ZnO@Glu-TSC nanoparticles, zeta potential measurement was conducted at 25.2 °C. The average zeta potential was –20.9 mV (Fig. 7). This negative value highlights thiosemicarbazone-derived functional groups on the nanoparticle surface, a positive outcome confirming conjugation. Strikingly, the –20.9 mV zeta potential value implies adequate colloidal stability, with strong potential for biomedical applications.

Fig. 7.

Zeta potential distribution of glutamic acid-thiosemicarbazone-conjugated zinc oxide nanoparticles (ZnO@Glu-TSC) measured at pH 7. The zeta potential value of -20 ± 2 mV indicates good colloidal stability of the nanoparticles

Evaluation of antibacterial activity via MIC assay

To assess antimicrobial efficacy of ZnO@Glu-TSC nanoparticles, MIC determination was performed on clinical P. aeruginosa isolates and the ATCC 25213 strain (Fig. 8).

Fig. 8.

Assessment of P. aeruginosa growth in treated and positive control groups using a 96-well microtiter plate assay. The left plate represents the treated group, exposed to ZnO–TSC nanoparticles, whereas the right plate represents the positive control group (ATCC 25213), containing bacteria treated with the ZnO–TSC nanoparticles

The MIC values are shown in Fig. 9. ZnO@Glu-TSC exhibited lower MICs (256–512 µg/mL) than ZnO or TSC (512–1024 µg/mL) in most isolates, a positive result highlighting enhanced potency. For the ATCC strain, the conjugate's MIC of 128 µg/mL was significantly lower than 512 µg/mL for both components. One-way ANOVA confirmed overall differences (F(2,30) = 39.2, p < 0.0001), with Bonferroni post hoc tests showing ZnO@Glu-TSC superiority (p < 0.001 vs ZnO; p < 0.01 vs TSC). Intriguingly, the fourfold MIC reduction in the ATCC 25213 strain suggests strong synergistic effects, an interesting finding for clinical translation.

Fig. 9.

Minimum inhibitory concentration (MIC) of zinc oxide nanoparticles (ZnO), thiosemicarbazone (TSC), and glutamic acid-thiosemicarbazone-conjugated zinc oxide nanoparticles (ZnO@Glu-TSC) against clinical isolates of P. aeruginosa. The results demonstrate lower MIC values for ZnO@Glu-TSC (range: 256–512 µg/mL) compared to ZnO and TSC alone, confirming synergistic effects

Evaluation of the fractional inhibitory concentration (FIC) index

To assess interactions between ZnO@Glu-TSC components, FIC index calculations were performed on P. aeruginosa isolates. FIC indices, calculated as the sum of individual FICs (MIC in combination / MIC alone), are reported in Table 1. The indices revealed synergistic effects (FIC ≤ 0.5) in 70% of isolates, additive (> 0.5–1) in 20%, and indifferent (> 1–4) in 10%, a positive outcome. No antagonistic interactions (> 4) were observed, a favorable result. Synergistic responses dominated in clinical strains, while additive/indifferent cases showed milder cooperation. Remarkably, the 77% pyocyanin reduction at 512 µg/mL reveals dose-responsive inhibition, providing crucial insights into virulence modulation.

Table 1.

Fractional inhibitory concentration (FIC) index of zinc oxide nanoparticles (ZnO), thiosemicarbazone (TSC), and ZnO@Glu-TSC against clinical isolates of Pseudomonas aeruginosa, demonstrating synergistic effects in 70% of isolates, additive effects in 20%, and indifferent effects in 10%

P. aeruginosa strain	MIC of ZnO@Glu (alone)	MIC of TSC (alon)	MIC of ZnO–TSC combination	MIC of ZnO@Glu in combination (0.714 × combined MIC)	MIC of TSC in combination (0.286 × combined MIC)	FIC of ZnO@Glu	FIC of TSC	FIC Index	Type of interaction (based on FIC Index)
Sample1	512	512	512	365.18	146.83	0.713	0.287	1	Additive
Sample2	512	1024	512	365.18	146.83	0.713	0.143	0.856	Additive
Sample3	1024	1024	512	365.18	146.83	0.357	0.143	0.5	Synergistic
Sample4	256	512	512	365.18	146.83	1.426	0.287	1.713	Indifferent
Sample5	1024	1024	256	182.5	73.1	0.178	0.071	0.249	Synergistic
Sample6	1024	1024	512	365.18	146.83	0.357	0.143	0.5	Synergistic
Sample7	512	1024	256	182.5	73.1	0.356	0.071	0.427	Synergistic
Sample8	512	1024	256	182.5	73.1	0.356	0.071	0.427	Synergistic
Sample9	1024	1024	256	182.5	73.1	0.178	0.071	0.249	Synergistic
Sample10	1024	1024	256	182.5	73.1	0.178	0.071	0.249	Synergistic
ATCC:25,213	512	512	128	91.49	36.71	0.179	0.072	0.251	Synergistic

Biofilm formation assay

To evaluate biofilm inhibition by ZnO@Glu-TSC nanoparticles, optical density (OD) measurements were conducted. OD values ranged from 1.213 to 2.192 (Fig. 10), with the negative control at 0.0013 (no biofilm) and positive control at 1.809 (strong biofilm). Samples 1–3, 5–7, 9, and 10 showed significantly lower OD than the positive control (p < 0.05), a positive outcome. Samples 4 and 8 exhibited no significant difference (p > 0.05), a negative result. One-way ANOVA confirmed overall differences (F(11,24) = 505.15, p < 0.0001), with t-tests validating pairwise reactions. Notably, Sample 1's lowest OD (1.213) highlights superior inhibition, an interesting finding for clinical efficacy.

Fig. 10.

Assessment of biofilm formation by clinical isolates of P. aeruginosa using the crystal violet assay (OD₅₇₀). Mean OD values of the isolates ranged from 1.213 to 2.192, with significant differences in 8 isolates compared to the positive control (OD = 1.809, p < 0.05)

To assess biofilm inhibition by ZnO@Glu-TSC nanoparticles, OD measurements were conducted on treated P. aeruginosa isolates. OD values are shown in Fig. 11, with the positive control (untreated) at 0.296 and negative control at 0.155. ZnO@Glu-TSC (Sample 10) showed the lowest OD (0.110), a positive result. ANOVA confirmed significant treatment effects (F(3,32) = 147.8, p < 0.0001). t-tests revealed ZnO@Glu-TSC significantly outperformed ZnO and TSC (p < 0.01), a favorable reaction. No negative effects were observed, with consistent reductions across samples. Intriguingly, the reduced SD in ZnO@Glu-TSC treatments signals enhanced uniformity, a valuable insight for clinical dependability.

Fig. 11.

Evaluation of biofilm formation inhibition by zinc oxide nanoparticles (ZnO), thiosemicarbazone (TSC), and ZnO@Glu-TSC in five biofilm-producing clinical isolates of P. aeruginosa using the crystal violet assay (OD₅₇₀). Samples treated with ZnO@Glu-TSC exhibited significant reduction in OD₅₇₀ compared to the positive control (p < 0.05)

Pyocyanin inhibition results

To evaluate ZnO@Glu-TSC's impact on pyocyanin production in P. aeruginosa, absorbance at 520 nm was measured. Mean pyocyanin levels were 1.57 µg/mL (512 µg/mL ZnO@Glu-TSC) and 2.49 µg/mL (256 µg/mL) versus 6.70 µg/mL in controls (Fig. 12). These reductions highlight significant inhibition, a positive outcome. One-way ANOVA confirmed group differences (p < 0.001), with Tukey's test showing ZnO@Glu-TSC superiority over ZnO and TSC alone (p < 0.01). No adverse effects were noted, with consistent reductions across concentrations. Interestingly, the 77% reduction at 512 µg/mL demonstrates dose-dependent inhibition, with implications for targeting bacterial virulence.

Fig. 12.

Comparison of pyocyanin concentration (µg/mL) in clinical isolates of P. aeruginosa treated with zinc oxide nanoparticles (ZnO), thiosemicarbazone (TSC), and ZnO@Glu-TSC at concentrations of 256 and 512 µg/mL. The results indicate a significant reduction in pyocyanin concentration with ZnO@Glu-TSC compared to the control (p < 0.05)

Discussion

Metal nanoparticles have emerged as potent alternatives to traditional antibiotics, leveraging their multifaceted antimicrobial properties and utility in targeted drug delivery systems [27, 28]. Recent investigations underscore the efficacy of silver, copper, and zinc oxide nanoparticles against multidrug-resistant pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae [29]. In vivo models have demonstrated ZnO nanoparticles' protective role against P. aeruginosa-induced histopathological damage in pulmonary, hepatic, and renal tissues [30]. Synergistic formulations, including thymol-conjugated ZnO nanoparticles with thiosemicarbazone, have exhibited superior antibiofilm activity against P. aeruginosa [18]. Likewise, thiosemicarbazone-functionalized copper nanoparticles display robust antibacterial effects [31]. Building upon this foundation, the current study pursued three core objectives: (1) to evaluate the synergistic antibacterial effects of ZnO and thiosemicarbazone (TSC) within the ZnO@Glu-TSC nanoconjugate against clinical P. aeruginosa isolates; (2) to assess its capacity to inhibit biofilm formation; and (3) to examine its suppression of pyocyanin production, a pivotal virulence determinant. Physicochemical and structural characterizations unequivocally validated the targeted synthesis of ZnO@Glu-TSC nanoparticles. UV–Vis spectroscopy detected a prominent absorption peak at 380 nm, yielding a band gap of 3.26 eV, characteristic of ZnO's semiconducting behavior and optical efficacy [32, 33]. X-ray diffraction (XRD) confirmed a hexagonal wurtzite structure with an average crystallite size of approximately 20 nm, indicative of high phase purity and crystallinity [34]. Fourier-transform infrared (FTIR) spectroscopy revealed TSC-specific functional groups alongside Zn–O and Zn–S vibrational bands, affirming successful surface conjugation [25]. Scanning electron microscopy (SEM) disclosed spherical, uniform morphologies with particle sizes below 22 nm, corroborating XRD data and nanoscale dispersion [35]. Energy-dispersive X-ray (EDX) analysis verified the integration of Zn, O, and TSC-derived organic elements [26, 34]. The zeta potential of –20.9 mV signified moderate colloidal stability, attributable to TSC-induced electrostatic repulsion [34, 36, 37]. Pertaining to the first objective, minimum inhibitory concentration (MIC) assays revealed that ZnO@Glu-TSC curtailed bacterial growth at reduced concentrations relative to individual components in most isolates, with pronounced efficacy in the ATCC 25213 strain. Fractional inhibitory concentration (FIC) indices indicated synergy (≤ 0.5) in 70% of strains, additivity (0.5–1.0) in 20%, and indifference in 10%, devoid of antagonism. Notably, the nanoparticles' consistent sub-25 nm size and spherical morphology likely facilitate enhanced bacterial membrane penetration and intracellular accumulation, amplifying bioactivity [38, 39]. The FIC index of 0.251 in the ATCC strain exemplifies robust synergy, underscoring therapeutic consistency. For the second objective, untreated P. aeruginosa isolates exhibited variable biofilm formation, with isolates 4 and 8 surpassing the positive control in intensity, while isolates 1, 2, and 10 were comparatively weaker. ZnO@Glu-TSC treatment diminished optical density (OD) values to near-negative control levels, surpassing ZnO or TSC monotherapy. The anti-biofilm reproducibility (standard deviation < 0.02) highlights reliable performance. These findings are consistent with the results of Mokhtari et al. [18]. Regarding the third objective, ZnO@Glu-TSC substantially attenuated pyocyanin biosynthesis compared to controls and standalone agents. The marked pyocyanin suppression emphasizes the nanoconjugate's interference with quorum-sensing networks, a cornerstone of P. aeruginosa virulence and persistence in chronic infections [40, 41]. Intriguingly, one clinical isolate manifested indifference (FIC = 1.713), potentially reflecting strain-specific efflux pump overexpression or adaptive resistance mechanisms, despite the universal absence of antagonism [42, 43]. These characterizations resonate with contemporary reports on ZnO nanoparticle fabrication, band gaps, and crystalline architectures [32–34, 44]. The antibacterial synergy corroborates ZnO-TSC conjugates and analogous metallic nanoparticles targeting P. aeruginosa [37, 45, 46]. Anti-biofilm outcomes echo ZnO nanocomposites disrupting P. aeruginosa extracellular matrices [18, 47, 48]. Pyocyanin inhibition supports nanoparticle-driven virulence modulation [49, 50]. While no overt contradictions emerged, the indifferent response in one isolate diverges from uniformly synergistic profiles in certain ZnO studies, possibly attributable to clinical strain heterogeneity not captured in standardized models [51, 52]. The MIC reductions and FIC synergy likely arise from ZnO's reactive oxygen species (ROS) production and membrane perturbation, synergized by TSC's enzymatic inhibition and metabolic disruption, fostering multi-target bactericidal action [45, 51, 52]. Biofilm abatement may stem from ROS-mediated exopolysaccharide degradation and TSC-enhanced permeation [53–55]. Pyocyanin curtailment could involve quorum-sensing pathway blockade, including lasR/rhlR regulators [50]. Inter-isolate biofilm variability probably reflects differential pel/psl/alg gene expression governing matrix synthesis [53, 56]. Although these in vitro data are compelling, prudence is advised in extrapolating to clinical scenarios, where host immune interactions, pharmacokinetic variables, and polymicrobial environments may modulate outcomes; rigorous in vivo corroboration is imperative [28, 57]. We posit that TSC functionalization augments ZnO's bioavailability and pathogen specificity, potentially extensible to other Gram-negative opportunists via analogous ROS-enzymatic synergies, mitigating resistance emergence [29, 58]. These observations imply ZnO@Glu-TSC's potential to minimize therapeutic dosages, attenuate toxicity, and combat resistance in P. aeruginosa-driven pathologies like cystic fibrosis or nosocomial pneumonia [30, 59]. Its antivirulence paradigm offers a resistance-sparing adjunct to antibiotics, informing nano-drug development for sustained delivery in biomedical contexts.

Conclusion

This study aimed to assess the synergistic antibacterial effects of ZnO@Glu-TSC nanoconjugates against P. aeruginosa, focusing on inhibiting bacterial growth, biofilm formation, and pyocyanin production. The nanoconjugate effectively inhibited 64% of clinical isolates, disrupted biofilms, and suppressed pyocyanin, a key virulence factor. These findings suggest ZnO@Glu-TSC as a promising tool in antimicrobial nanotechnology for combating multidrug-resistant infections. Its multifaceted approach offers a resistance-sparing strategy, potentially transformative for managing persistent infections like cystic fibrosis or nosocomial pneumonia. However, the study’s in vitro nature and limited strain diversity warrant cautious interpretation. Future research should optimize the nanoconjugate’s properties, test it against diverse resistant strains and animal models, and elucidate its molecular mechanisms to enhance its therapeutic potential.

Author contributions

A.H and Kh.I: Conceptualization, Methodology, Data analyses and Reviewing and Editing. H.M.R: Data curation, Writing- Original draft preparation. M.F.G: Visualization, Investigation and Reviewing and Editing. All authors have read and agreed to the published version of the manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

All data supporting the findings of this study are contained within the article. No additional data are available.

Declarations

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.