Silver Nanoparticles Synthesized from Lawsonia inermis against Uropathogenic Escherichia coli: Antimicrobial, Antibiofilm, and Molecular Docking Investigations

1. Introduction

It is well known that urinary tract infections (UTIs) are among the most common infections worldwide. Uropathogenic Escherichia coli (UPEC) is responsible for a large proportion of UTIs [1]. UPEC is particularly problematic because it can express different virulence factors that help adhesion of bacteria to the epithelial cells of urinary tract tissues, invasion of host epithelial cells, and colonization on the surface for a long time [2]. A significant factor contributing to the persistence of bacterial infection is biofilm production. Bacterial cells are embedded within a self-produced extracellular polymeric matrix. This biofilm lifestyle enhances bacterial tolerance to antibiotics and immune defenses, making infections difficult to cure and leading to chronic disease [3].

There is a continued increase in the number of multidrug-resistant (MDR) E. coli strains, leading to an increase in the global burden associated with UTIs [4]. Conventional antibiotics are becoming ineffective, and there is therefore an immediate requirement for new antimicrobial methods. Nanotechnology has recently emerged as a viable new area for developing novel antimicrobial agents. Of these types of nanomaterials, silver nanoparticles (AgNPs) are gaining attention for their strong, broad-spectrum antibacterial properties, since they damage cell membranes, generate oxidative stress in microorganisms, and disrupt major types of biomolecules necessary for microbial survival [5].

The green synthesis of nanoparticles from plant extracts represents a more sustainable and environmentally friendly method of producing nanoparticles compared to traditional chemical and physical synthesis methods. This method utilizes naturally occurring phytochemicals found in plants for the reduction and stabilization of nanoparticles, thereby eliminating the need for toxic reagents and often increasing the biological activity of the resulting nanoparticles [6]. Lawsonia inermis (henna) is a well-established medicinal plant that has demonstrated antimicrobial, antioxidant, and anti-inflammatory activities. The diverse range of phytochemicals found in henna, such as lawsone, flavonoids, tannins, and phenolic compounds, enables the biosynthesis of AgNPs that are effective antimicrobial agents [7].

Previously published literature reported that zinc oxide nanoparticles (ZnO NPs) were bio-synthesized using L. inermis leaf extract, and the antibacterial and antibiofilm activities of these nanoparticles were evaluated against different isolates of UPEC [8]. An earlier study evaluated the antibiofilm and antibacterial activities of biosynthesized AgNPs by checking the efficacy of this nanoparticle in disrupting bacterial biofilm [9]. Virtual molecular docking studies were conducted in prior work to evaluate potential interactions between major plant-derived compounds and key bacterial proteins central to bacterial virulence and biofilm formation. By combining experimental and computational approaches, this work provides deeper insight into the antimicrobial mechanisms of biosynthesized AgNPs. The objective of the current study is to address the above gaps related to the bactericidal, antibiofilm, and mechanistic aspects of biosynthesis-derived AgNPs.

2. Materials and Methods

2.1. Bio-AgNP Synthesis

The leaves of the plant (L. inermis) were obtained from the Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq. A 10% (w/v) aqueous extract was prepared by heating dried leaf powder in distilled water at 80°C for 30 min. The mixture was filtered to obtain a clear extract. Ten milliliters of the clear plant extract was added to 90 mL of 1 mM silver nitrate (AgNO₃; Sigma-Aldrich, USA). The mixture was mixed using a magnetic stirrer at 60°C for 4 h in the dark. Nanoparticle formation was indicated by a visible color change. The mixture was centrifuged at 12,000 rpm for 15 min, and the pellet was washed three times with sterile double-distilled water. The purified AgNPs were stored at 4°C.

Bio-AgNP formation was confirmed by UV-Vis spectroscopy (300–700 nm). Morphology and size were determined by scanning electron microscope (SEM) (MIRA3 TE Scan, China). The prepared AgNPs were characterized using atomic force microscopy (AFM; Innova® AFM; Bruker, Santa Barbara, USA). For AFM analysis, a thin film of the nanoparticles was deposited on a silica glass plate.

2.2. Bacterial Strains and Identification

Clinical isolates of UPEC were collected from patients at Baghdad Teaching Hospital (2024–2025). The bacterial isolates were cultured on selective and differential media, including MacConkey agar and eosin methylene blue agar. Additionally, specific biochemical tests were performed. Bacterial cell morphology after Gram staining was assessed to identify bacterial species. The identification of bacterial species was confirmed using the VITEK® 2 Gram-Negative identification card (GN) (bioMérieux, France).

2.3. Antibiotic Susceptibility

The standard Kirby–Bauer disk diffusion method was used to determine the susceptibility of UPEC to the following antibiotics: Ampicillin (AMP, 10 µg), Ciprofloxacin (CIP, 5 µg), Nitrofurantoin (NIT, 300 µg), Gentamicin (GEN, 10 µg), Amikacin (AMK, 30 µg), and Imipenem (IPM, 10 µg). Antibiotic susceptibility testing was also performed after culture on Mueller–Hinton Agar (MHA, Hi-Media, India). The instructions of the Clinical and Laboratory Standards Institute (CLSI, 2023) were followed to interpret the results [10].

2.4. PCR Method

A multiplex PCR with specific gene primers was used to detect virulence genes (fimH and papC) and resistance genes (blaCTX-M, blaKPC) in UPEC. The experimental conditions were standardized before starting the experiment. Amplified products were verified by agarose gel electrophoresis and purified for subsequent Sanger sequencing to ensure the specificity and accuracy of each gene product [11].

2.5. Minimum Inhibitory and Bactericidal Concentrations

The minimum inhibitory concentrations (MICs) were determined by the broth microdilution method in cation-adjusted Mueller–Hinton broth (CAMHB) according to CLSI 2023 guidelines. One hundred microliters of serial two-fold dilutions of Bio-AgNPs ranging from 400 to 0.39 µg/mL were made in MHB (Hi-Media, India) in Nunc™ 96-Well Polystyrene Conical Bottom MicroWell™ Plates, then mixed with 5 µL of 10⁸ CFU/mL of the testing UPEC isolate. Growth inhibition was visualized after 18 hours of culture at 37°C. Minimum Bactericidal Concentration (MBC) was determined by no-visible growth in subcultures on MHB agar from growth-negative wells. Selectivity index (SI) was calculated as CC₅₀ (HEK-293) / MIC.

2.6. Biofilm Formation and Antibiofilm Activity

The standard method of Al-Mutalib & Zgair (2023) was followed to measure the biofilm formation of UPEC isolates. Biofilm formation was quantified by crystal violet (CV) staining (OD₅₉₀). Isolates were classified as non-biofilm (OD ≤ 2× control), weak, moderate, or strong producers [10]. Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC) were determined against preformed 24-hour biofilms exposed to serial dilutions of Bio-AgNP. Biofilm inhibition and disruption at MIC, 2×MIC, and 4×MIC were quantified using CV biomass and expressed as % reduction relative to untreated controls (two-way ANOVA).

2.7. Time-Kill Kinetics

Time-kill assays were performed with UPEC isolates at MIC, 2×MIC, and 4×MIC. Aliquots (100 µL) were collected at 0, 15, 30 min, and 1, 2, 4, 6, 8, 12, and 24 hours, plated in triplicate on brain heart infusion (BHI, Hi-Media, India) agar, and incubated for 24 hours at 37°C. Bactericidal activity was defined as ≥3 log₁₀ (≥99.9%) reduction in initial CFU/mL.

2.8. Molecular Docking

Protein and Ligand Preparation

Three UPEC targets were selected: FimH adhesin (PDB: 4XO8), PapC usher (PDB: 3RFZ), and DNA Gyrase B (PDB: 4DUH). Structures were prepared in AutoDockTools 1.5.7 (removal of water molecules, addition of Gasteiger charges, PDBQT format). Grid box dimensions were 25 × 25 × 25 Å (spacing 0.375 Å). Ligands tested were the Bio-AgNP Ag⁺ core and lawsone (PubChem CID: 10685), with ciprofloxacin and novobiocin as reference ligands. Docking was performed using AutoDock Vina 1.2.0 (exhaustiveness = 20). Visualization was carried out using UCSF ChimeraX 1.6 and Discovery Studio Visualizer 2021.

2.9. Statistical Analysis

Statistics were analyzed using OriginPro (OriginLab Corporation, USA). Each experiment was repeated three times, and valid means were used for the analysis. Results were expressed as mean ± standard deviation (SD). To compare the means of different experimental groups, statistical analysis was performed using one-way and/or two-way ANOVAs; where significant results were found, appropriate post-hoc tests were used to determine which experimental group means differed significantly from one another. A threshold of P < 0.05 was used to indicate statistical significance. Dose–response and time–dose response curves were created using Origin software.

3. Results

3.1. Characterization of Bio-AgNPs

The present study demonstrated that L. inermis extracts reduced AgNO₃ within 30–45 minutes, as indicated by a color change from pale yellow to reddish-brown. UV-Vis spectroscopy revealed a surface plasmon resonance (SPR) peak at 418 nm, confirming the formation of silver nanoparticles (AgNPs).

SEM analysis showed well-dispersed, semispherical nanoparticles, with most particle diameters <50 nm. SEM showed a particle size range of 15–65 nm, with a mean of 38 ± 11.5 nm. AFM showed that most prepared Bio-AgNPs were less than 50 nm. The study clearly showed that the procedure used for preparing biosynthesized silver nanoparticles was a highly efficient method, as the yielded particles were consistently within the nanoparticle size range.

3.2. Characteristics and Resistance Profiles of Seven Clinical Isolates

All seven isolates exhibited multidrug resistance. Resistance to ampicillin was observed in all isolates (7/7). However, resistance to fosfomycin was observed in only one isolate (1/7). The study also showed that six isolates were resistant to ciprofloxacin and four to nitrofurantoin. The blaCTX-M gene was detected in 4 of the 7 isolates, while blaKPC was found in one isolate (UE-07). The fimH gene was present in all isolates, whereas papC was detected in 5 of the 7 isolates. Notably, all papC-positive isolates demonstrated strong biofilm production (Table 1).

3.3. Bio-AgNPs Antibacterial Activity

Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and selectivity index of Bio-AgNPs were evaluated against all UPEC isolates. Bio-AgNPs exhibited significant antibacterial activity against all UPEC strains (Table 2). MIC values ranged from 3.12 µg/mL to 12.5 µg/mL, with the highest value observed for UE-07, the blaKPC-positive isolate. Similarly, MBC values ranged from 6.25 µg/mL to 25 µg/mL. The MBC/MIC ratios were ≤4 for all isolates, confirming the bactericidal mechanism of Bio-AgNPs. Cytotoxicity analysis revealed a CC₅₀ value of 49.9 µg/mL, while selectivity index (SI) values ranged from 8.0 to 16.0. ESBL-positive isolates did not exhibit significantly higher resistance to Bio-AgNP, consistent with the hypothesis that Bio-AgNPs exert antibacterial effects through a multi-target, non-antibiotic mechanism.

3.4. Biofilm Formation and Antibiofilm Activity

The biofilm formation capacity varied among the UPEC clinical isolates, with five of the seven isolates classified as strong biofilm producers (OD₅₇₀ > 0.68), while the remaining two were identified as moderate producers (Table 3). Minimum biofilm inhibitory concentration (MBIC) values ranged from 12.5 µg/mL to 50.0 µg/mL (4–8× MIC), whereas the minimum biofilm eradication concentration (MBEC) was ≥200 µg/mL for 5 of the 7 clinical isolates, highlighting the high tolerance of established UPEC biofilms.

A clear concentration-dependent inhibitory effect on biofilm formation was observed, as shown in Figure 1. The effect of varying concentrations of AgNPs on biofilm production was evaluated by measuring OD at 590 nm. As the concentration of Bio-AgNPs increased from 1/32 MIC to 1/2 MIC, optical density values progressively decreased, indicating reduced biofilm formation. By contrast, the untreated control groups consistently exhibited the highest OD₅₉₀ values, confirming robust biofilm production under normal growth conditions.

While sub-inhibitory concentrations of Bio-AgNPs (1/32 and 1/16 MIC) reduced biofilm formation compared with control groups, higher sub-MIC concentrations (1/8, 1/4, and 1/2 MIC) produced markedly greater inhibition. These findings indicate that Bio-AgNPs can significantly impair bacterial adhesion and extracellular matrix production, thereby inhibiting biofilm development even at concentrations below the MIC.

3.5. Time-Kill Kinetics

The bactericidal time course of AgNPs against six isolates of uropathogenic E. coli (UPEC1, UPEC2, UPEC3, UPEC4, UPEC5, and UPEC7) showed a concentration- and time-dependent killing effect. While the growth of untreated isolates (control groups) remained stable or increased slightly over the 30-h incubation period (6.5–8.1 log₁₀ CFU/mL), bacterial counts declined gradually across all tested isolates treated with AgNPs at MIC, 2×MIC, and 4×MIC concentrations. The decline accelerated over time, with very low levels of bacterial growth by the end of the experiment (approximately 0–1 log₁₀ CFU/mL at 25–30 h). Effective killing at MIC showed a slower rate than at higher concentrations, and exposure to 2×MIC resulted in a more rapid decrease in viable bacterial counts. Most isolates showed a sharp decline within the first 5–15 h and near-complete eradication of viable cells by approximately 20–25 h; this pattern was also observed under the 4×MIC condition. These findings demonstrate that AgNPs exhibit bactericidal activity against UPEC isolates, and that increasing the concentration from MIC to 2×MIC and 4×MIC significantly accelerates bacterial killing.

3.6. Molecular Docking Results

To evaluate the interactions among active components responsible for antibacterial activity, molecular docking simulations were performed. Three-dimensional binding poses of the bio-AgNP (Ag⁺ core) and its associated capping agent, lawsone, were mapped against three key bacterial targets. The key spatial orientation of the Ag⁺ core is shown in Table 4 and Figures 3 and 4.

Binding to FimH Adhesin (PDB: 4XO8)

Bacterial adhesion (FimH) is a crucial virulence factor that mediates initial bacterial adhesion to host cell surfaces. As shown in the structural outline and magnified inset of Figure 3A, the bio-AgNP core showed a highly favorable, strong binding affinity within the FimH mannose-binding lectin domain (ΔG = −9.84 kcal/mol, Ki = 68.3 nM). The visualization clearly demonstrates a multi-dentate coordination sphere around the central Ag⁺ ion, driven by robust electrostatic and hydrogen-bonding interactions with key polar residues, specifically Asp140, Asn163, and Asp162. The binding is stabilized by central hydrophobic contacts with Phe1 and Ile13, anchoring the nanoparticle core firmly within the pocket. Lawsone also exhibited significant binding in this region (ΔG = −7.21 kcal/mol, Ki = 5.40 µM), interacting predominantly with Tyr48, Gln133, and Asp140. Both the Ag⁺ core and the bio-capping agent substantially outperformed the reference antibiotic, ciprofloxacin (ΔG = −5.63 kcal/mol, Ki = 73.2 µM), at this non-classical antibacterial target, indicating a strong antiadhesive mechanism.

Binding to PapC Usher (PDB: 3RFZ)

PapC usher protein is an integral membrane channel essential for the assembly and translocation of P-fimbriae. The Bio-AgNP Ag⁺ core exhibited profound binding affinity for this target (ΔG = −10.17 kcal/mol, Ki = 36.8 nM). Figure 3B shows that the spherical silver core closely corresponds to the PapC plug domain, the region responsible for gating pilus subunit translocation. The predictive model shows direct, stabilizing interactions with an array of amino acids, including Arg651, Phe628, Thr632, Asp636, and Glu620. Furthermore, lawsone showed moderate affinity for this site (ΔG = −6.94 kcal/mol, Ki = 8.70 µM). In stark contrast, ciprofloxacin scored only −5.01 kcal/mol, confirming a distinct, non-quinolone binding mode for the synthesized nanoparticles that likely sterically blocks the PapC channel and impedes fimbriogenesis.

Binding to DNA Gyrase B (PDB: 4DUH)

The most striking virtual result was observed against DNA Gyrase B, an enzyme crucial for bacterial DNA replication and supercoiling. The bio-AgNP (Ag⁺ ion core) showed remarkably high binding affinity at the catalytic ATP-binding site (ΔG = −11.32 kcal/mol, Ki = 6.45 nM). In this study, a direct comparison of this binding pocket was visualized (Figure 4). Figure 4A shows the highly efficient spatial accommodation of the Ag⁺ core, which forms a compact network of interactions with Asp73, Asn46, Lys103, Ile78, and Gly77. These are the same critical residues engaged by the known Gyrase B inhibitor novobiocin, shown side-by-side in Figure 4B (Ki = 189 nM). The Ag⁺ ion (core) interaction shows a predicted binding affinity approximately 29-fold higher than that of novobiocin. Additionally, lawsone showed potent binding at this site (ΔG = −8.45 kcal/mol, Ki = 0.63 µM) independently. The structural and thermodynamic data suggest that the biosynthesized AgNPs act as highly potent, two-agent competitive inhibitors of DNA Gyrase B.

4. Discussion

In recent years, several studies have highlighted that resistance among pathogenic and opportunistic bacteria to a wide spectrum of antibiotics is a serious public health problem and represents a major challenge [13,14].

A recent study suggested alternative methods, either by improving antibiotics through combinations with different materials to enhance their antibacterial activity [15], or by using other antimicrobial agents such as herbal extracts [12] and polymeric or metallic nanoparticles [16]. Various studies have reported the antibacterial efficacy of AgNPs against multiple pathogenic bacterial species [17]. The mechanism by which AgNPs affect bacterial attachment and biofilm formation needs to be explained; thus, the present study aimed to highlight the antibacterial effectiveness of Bio-AgNPs against uropathogenic E. coli. Furthermore, the present study visualized the molecular docking of interactions between silver ions and two proteins important in bacterial attachment and biofilm formation, as well as another protein important for bacterial survival (DNA Gyrase B).

The physicochemical profile of the synthesized Bio-AgNPs — the SPR peak at 418 nm and a mean diameter of less than 50 nm on SEM examination — confirmed monodisperse nanoparticles consistent with lawsone- and flavonoid-mediated bioreduction and surface capping. The nanoscale size of AgNPs confers a high surface-area-to-volume ratio, maximizing Ag⁺ release flux across bacterial membranes [18]. All seven UPEC clinical isolates were MDR, with co-detection of fimH and papC virulence genes in strong biofilm producers, corroborating the well-established role of P-fimbriae in mediating surface attachment and biofilm initiation [19].

Biosynthesized AgNPs exhibited high bactericidal activity against all study isolates (MIC 3.12–12.5 µg/mL; MBC/MIC ≤4), independent of ESBL status, indicating that the mechanism of action targets membrane-disruptive pathways that bypass conventional β-lactamase-mediated resistance [20]. Selectivity indices of 8.0–16.0 indicate an acceptable therapeutic window for the intended topical urinary application [21]. The results of the present study are in line with a previous study that reported bio-AgNP activity against uropathogenic E. coli [22]. The current study showed UPEC biofilm tolerance of MBEC ≥200 µg/mL in 5/7 isolates, due to EPS matrix diffusion barriers and persistent cell populations [23]. However, sub-MICs produced concentration-dependent biofilm inhibition (Figure 1) by suppressing fim gene expression and fimbrial biogenesis, blocking surface colonization before mature biofilm architecture is built [24,25]. Time-course kill assays confirmed ≥3 log₁₀ bactericidal reductions, with faster kinetics observed at higher concentrations (4×MIC: eradication within 15–20 h), consistent with a multi-hit model that requires cytoplasmic Ag⁺ accumulation to reach a threshold for irreversible multi-target damage [26].

Figure 5 integrates these findings into a six-node mechanistic model. Ag⁺ ions released from the nanoparticle surface (1) destabilize the bacterial membrane by binding thiol groups and dissipating the proton motive force, triggering (2) Fenton-like lipid peroxidation and (3) intracellular ROS amplification that collectively inactivate DNA, enzymes, and structural proteins [26,27,28]. Computationally, (4) DNA Gyrase B inhibition is supported by the highest docking affinity recorded (ΔG = −11.32 kcal/mol, Ki = 6.45 nM at the catalytic Asp73 residue), representing 29-fold greater predicted affinity than novobiocin [29]. Anti-virulence targeting of (5) FimH (ΔG = −9.84 kcal/mol) and PapC (ΔG = −10.17 kcal/mol) provides a resistance-sparing mode of action by disarming adhesion and pilus assembly without bactericidal lethal pressure [30]. (6) Biofilm matrix disruption completes the model and is consistent with the significant antibiofilm activity observed across all tested concentrations [24].

The present study demonstrated that L. inermis-derived Bio-AgNPs exhibit potent, multi-mechanistic antibacterial and antibiofilm activity against MDR UPEC, collectively explained by the model depicted in Figure 5 and supported by molecular docking analyses of three clinically relevant virulence targets. While in vivo validation in murine UTI models is required to bridge the translational gap, these data position Bio-AgNPs as a promising candidate strategy for managing biofilm-associated MDR urinary tract infections in the post-antibiotic era [31].

5. Conclusion

The present study demonstrated the antibacterial and antibiofilm effects of biosynthesized AgNPs from L. inermis against MDR UPEC, including ESBL- and KPC-producing isolates. The MICs of Bio-AgNPs ranged from 3.12 to 12.5 µg/mL. The bactericidal MBC/MIC ratios were less than or equal to 4 across all studied isolates. Selectivity indices ranged from 8 to 16, indicating a safety margin for the AgNPs. The study showed that sub-MICs reduced biofilm formation in a concentration-dependent manner. AgNP time-killing was also concentration-dependent. Molecular docking revealed that the Ag⁺ core and lawsone strongly inhibited three critical UPEC targets (FimH, PapC, and DNA Gyrase B), outperforming ciprofloxacin and novobiocin at their respective binding sites. The findings of the current study suggest that Bio-AgNPs are a promising multi-target therapeutic candidate against drug-resistant UTI.

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