Biofilm inhibition by silver nanoparticles produced from Stachys spectabilis

Abstract

Background

Green synthesis of silver nanoparticles (AgNPs) has emerged as an environmentally friendly approach for developing novel antimicrobial materials. Although several Stachys species have been investigated for their pharmacological properties, the antibacterial and antibiofilm activities of AgNPs synthesized from Stachys spectabilis have not previously been evaluated. Therefore, this study aimed to investigate the antimicrobial and antibiofilm potential of Stachys spectabilis-derived AgNPs against clinically relevant biofilm-forming bacterial pathogens.

Methods

AgNPs were fabricated through an aqueous green synthesis approach and characterized using standard analytical techniques. Antibacterial activity was assessed via liquid microdilution to determine minimum inhibitory concentrations (MICs) against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Antibiofilm activity was quantified using the crystal violet assay in 96-well microplates.

Results

Disc diffusion results revealed measurable inhibition zones only in E. coli and P. aeruginosa (6 mm), corresponding largely to the disc diameter and indicating limited nanoparticle diffusion in agar. In contrast, MIC and MBC analyses demonstrated a clear concentration-dependent antibacterial effect: S. aureus showed the highest susceptibility with a MIC of 1024 µg/mL, while the remaining strains exhibited MICs of 2048 µg/mL. Consistently higher MBC values confirmed the inhibitory-to-bactericidal threshold. AgNPs also displayed substantial antibiofilm activity, achieving biomass reductions of 59.7% in P. aeruginosa, 57.0% in E. coli, 54.9% in S. aureus, and 40.1% in K. pneumoniae. Overall, the results demonstrate that although AgNPs exhibit limited diffusion in solid media, they possess concentration-dependent antibacterial and antibiofilm activity in liquid media.

Conclusions

The synthesized silver nanoparticles demonstrated substantial antibacterial and antibiofilm efficacy despite their limited diffusion in solid media. While disc diffusion assays produced minimal inhibition zones, MIC and MBC analyses revealed concentration-dependent inhibitory and bactericidal effects, with S. aureus showing the greatest susceptibility. Additionally, significant reductions in biofilm biomass across all tested species indicate that AgNPs not only inhibit planktonic bacterial growth but also effectively disrupt established biofilms. These findings suggest that AgNPs hold considerable potential as alternative antimicrobial agents, particularly in applications where both planktonic and biofilm-associated bacterial forms must be controlled. To the best of our knowledge, this is the first study to demonstrate the antibacterial and antibiofilm activities of Stachys spectabilis-derived AgNPs, highlighting their potential as sustainable antimicrobial agents for the control of biofilm-associated bacterial infections.

Introduction

Nanotechnology has rapidly evolved into a multidisciplinary field with applications spanning biomedicine, pharmaceutics, catalysis, sensing, agriculture, textiles and energy technologies [1]. Central to this discipline are nanomaterial structures measuring between 1 and 100 nm which exhibit physicochemical characteristics distinct from their bulk counterparts [2]. Conventional physical and chemical methods for nanoparticle synthesis often rely on hazardous reagents, generate environmentally harmful by-products and demand substantial energy, making them incompatible with sustainable production goals [3]. These limitations have driven increasing interest in biological or “green” synthesis methods, in which plant-derived biomolecules act as natural reducing and stabilizing agents. Such approaches offer simplicity, lower environmental impact and improved biocompatibility compared with synthetic routes [4]. Among the various nanostructures produced through green synthesis, metal and metal oxide nanoparticles are particularly valuable due to their high surface area and broad functional versatility [5]. The efficiency of plant-mediated nanoparticle production has been supported by recent studies demonstrating strong antimicrobial and toxicological activity of green-synthesized AgNPs generated from botanical sources such as Raphanus sativus leaf extract [6]. More recent studies published in 2025 have further confirmed that green-synthesized AgNPs exhibit broad-spectrum antimicrobial activity and enhanced antibiofilm potential against clinically relevant multidrug-resistant pathogens, reinforcing their promise as sustainable alternatives to conventional antimicrobial agents. The diverse phytochemical composition of botanical extracts provides an effective platform for generating nanoparticles with tailored properties [7]. Recent reviews published in 2025 further emphasized that plant-mediated AgNP synthesis enables improved nanoparticle stability, enhanced biocompatibility and broad-spectrum antimicrobial activity through the synergistic action of phytochemical reducing and capping agents [8, 9].

The genus Stachys (Lamiaceae), comprising nearly 300 species, is well known for its ethnopharmacological importance. Stachys species are rich in flavonoids, iridoids, phenolic acids and fatty acid compounds associated with a broad range of biological activities [10]. Traditionally, these plants have been used to manage inflammatory disorders, neurological problems, gastrointestinal disturbances and respiratory conditions, and several species are valued for their anxiolytic and antioxidant properties [11]. Although the antimicrobial potential of Stachys extracts has been documented, their application in nanotechnology-based antimicrobial systems remains underexplored, particularly regarding biofilm-associated infections. Recent green synthesis studies, including those utilizing carob leaf extract to generate biologically active AgNPs [12], further highlight the potential of plant-mediated nanoparticle systems for biomedical applications.

Biofilms are highly structured microbial communities embedded in a self-produced extracellular matrix, formed following the attachment of planktonic cells to biotic or abiotic surfaces [13, 14]. The biofilm matrix facilitates nutrient retention, enhances horizontal gene transfer and provides substantial tolerance to desiccation, disinfectants and antibiotic exposure [15, 16]. Microorganisms within biofilms exhibit up to a thousand-fold higher resistance to antibiotics compared to free-floating cells [17]. These features make biofilms a major contributor to chronic wound infections, device-associated infections and persistent hospital-acquired outbreaks [18]. The limited efficacy of conventional treatments underscores the need for alternative antimicrobial strategies capable of disrupting biofilm integrity.

Recent investigations conducted in 2025 and 2026 have highlighted the growing role of nanoparticle-based approaches in overcoming biofilm-associated antimicrobial resistance, particularly through the disruption of extracellular polymeric matrices and inhibition of bacterial adhesion [19, 20].

Nanotechnology-based antimicrobial agents, particularly AgNPs, have gained significant attention for their potent activity against a wide variety of pathogens. AgNPs exhibit multiple modes of action, including membrane disruption, oxidative stress induction, interference with DNA replication and inhibition of essential enzyme functions [21]. They also prevent microbial adhesion, inhibit early biofilm formation and destabilize mature biofilms. Additionally, synergistic effects between AgNPs and conventional antibiotics may reduce the required antibiotic dose and slow the development of resistance [14]. Green-synthesized AgNPs offer further advantages, including improved biocompatibility and reduced cytotoxicity, making them strong candidates for biomedical and infection-control applications.

Recent evidence has also suggested that plant-mediated AgNPs may retain antimicrobial efficacy while exhibiting lower toxicity profiles than conventionally synthesized nanoparticles, further supporting their potential for future biomedical applications [8].

In this context, the present study evaluates the antimicrobial and antibiofilm properties of silver nanoparticles synthesized from Stachys spectabilis extract. Although the biological activities of several Stachys species have been reported, the potential of S. spectabilis to mediate nanoparticle synthesis and its influence on clinically relevant biofilm-forming bacteria has not yet been investigated. To our knowledge, this is the first study to examine the antibiofilm activity of S. spectabilis-derived AgNPs. The findings provide new insights into plant-based nanoparticle synthesis and highlight their potential for developing sustainable strategies to combat biofilm-associated infections and antimicrobial resistance.

Materials and methods

Plant material and preparation of the aqueous extract

Stachys spectabilis Choisy ex DC. was collected from Metan Mountain (Bingöl, Türkiye; 2000 m altitude). Botanical identification was verified by an institutional taxonomist, and a voucher specimen was deposited for reference. Fresh leaves were washed, air-dried and ground into a fine powder. A total of 3 g of powdered material was extracted with 100 mL of deionized water at 40 °C for 2 h. The mixture was filtered through Whatman No. 1 filter paper to obtain a clear aqueous extract, which served as the reducing and stabilizing agent for nanoparticle synthesis (Fig. 1) [22].

Fig. 1.

Schematic representation, basic reaction steps and mechanisms of green synthesis of AgNPs using Stachys spectabilis leaves

Green synthesis and characterization of silver nanoparticles

The aqueous extract was combined with an equal volume (100 mL) of 1.0 mM AgNO₃ solution and incubated at 40 °C for 1 h under continuous stirring. Nanoparticle formation was preliminarily confirmed by a visible color change of the reaction mixture. The product was centrifuged, washed with deionized water and lyophilized to obtain powdered AgNPs.

A comprehensive characterization of the synthesized AgNPs was performed in our previous study [18]. Briefly, UV–Vis spectroscopy confirmed nanoparticle formation through the appearance of a characteristic surface plasmon resonance peak at 498 nm. X-ray diffraction (XRD) analysis demonstrated a face-centered cubic crystalline silver structure, while transmission electron microscopy (TEM) revealed predominantly spherical nanoparticles with an average particle size of 10.8 nm. Energy-dispersive X-ray spectroscopy (EDX) confirmed the elemental composition of silver, and zeta potential analysis indicated moderate colloidal stability with a surface charge of − 12.8 mV. Representative characterization findings relevant to the biological assays performed in this study are summarized herein, whereas detailed spectra and instrumental analyses have been reported previously [22].

Bacterial strains and culture preparation

Antibacterial and antibiofilm assays were conducted using the following ATCC reference strains:

• Staphylococcus aureus ATCC 25,923,

• Pseudomonas aeruginosa ATCC 27,853,

• Escherichia coli ATCC 25,922, and.

• Klebsiella pneumoniae ATCC 700,603.

Frozen stocks stored at − 80 °C were revived on 5% sheep blood agar and Eosin Methylen-blue Agar and incubated at 37 °C for 24 h. Each strain was subcultured twice before use. Bacterial suspensions were prepared in physiological saline and adjusted to 0.5 McFarland turbidity.

Disc diffusion assay

The antibacterial activity of AgNPs was assessed using the disc diffusion method on Mueller–Hinton agar (MHA). A 100 µL aliquot of each bacterial suspension was spread evenly onto the agar surface. Sterile 5-mm discs were impregnated with 20 µL of AgNP suspension (0.03 mg/mL) and placed onto the inoculated plates. After gentle pressing, plates were incubated at 37 °C for 24 h. Inhibition zone diameters were measured in millimeters. All assays were performed in triplicate under aseptic conditions.

Broth microdilution assay for MIC and MBC determination

MICs were determined using 96-well microtiter plates. Each well received 100 µL of brain–heart infusion (BHI) broth, followed by two-fold serial dilutions of AgNPs starting at 1024 µg/mL (final concentrations: 512, 256, 128 and 64 µg/mL). A 10 µL aliquot of bacterial suspension (0.5 McFarland) was added to each well. Following incubation at 37 °C for 24 h, the lowest concentration showing no visible growth was recorded as the MIC.

For determination of minimum bactericidal concentration (MBC), 10 µL from each non-growth well was streaked onto MHA plates and incubated at 37 °C for 24 h. The lowest concentration showing no colony formation was recorded as the MBC. All experiments were conducted in triplicate.

Crystal violet assay for biofilm inhibition

Biofilm inhibition was evaluated using a modified crystal violet microtiter plate assay, following widely accepted standardized protocols reported in the literature [23, 24]. Briefly, 180 µL of tryptic soy broth was dispensed into each well of a sterile, flat-bottom 96-well microplate, followed by AgNP dilutions ranging from 512 to 128 µg/mL. Subsequently, 20 µL of bacterial suspension adjusted to 0.5 McFarland was added to each well. Plates were incubated at 37 °C for 24 h under static conditions to allow initial biofilm development.

Following incubation, non-adherent cells were removed by washing the wells three times with phosphate-buffered saline. Biofilms were fixed with 200 µL of methanol for 15 min, stained with 150 µL of 0.1% crystal violet for 15 min at room temperature, and rinsed three times with sterile saline to remove unbound dye. The retained crystal violet was solubilized in 95% ethanol for 15 min, and absorbance was measured at 630 nm using a microplate reader.

Biofilm inhibition was calculated according to the following equation:

All assays were performed in triplicate to ensure reproducibility.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA in SPSS v24. A significance level of α = 0.05 was applied, and p-values below this threshold were considered statistically significant.

Results

Disc diffusion assay

The antibacterial activity of the AgNPs was initially examined using the disc diffusion method. Among the tested strains, only E. coli and P. aeruginosa exhibited measurable inhibition zones; however, the zone diameter (approximately 6 mm) corresponded largely to the intrinsic diameter of the disc, indicating minimal nanoparticle diffusion through the agar matrix. This limited diffusion capacity likely explains the small zone sizes despite the substantial inhibitory effects observed in MIC assays (Fig. 2).

Fig. 2.

Inhibition zones of AgNPs obtained by the disc diffusion assay

Broth microdilution assay (MIC and MBC)

The antibacterial potency of the synthesized AgNPs was quantified through MIC and MBC measurements. A clear concentration-dependent inhibitory effect was observed. Complete growth inhibition of P. aeruginosa, E. coli and K. pneumoniae occurred at 2048 µg/mL, while S. aureus exhibited a lower MIC of 1024 µg/mL.

Bactericidal concentrations were consistently higher than MICs for all strains, demonstrating a typical inhibitory–bactericidal threshold separation. The MIC and MBC values are summarized in Table 1, and the concentration–response patterns are illustrated in Fig. 3.

Table 1.

Antimicrobial activity of AgNPs against tested bacterial strains

Test Bacteria	MIC (µg/mL)	MBC (µg/mL)	Inhibition Zone (mm)
S. aureus	1024	2048	0
E. coli	2048	4096	6 ± 0.5
P. aeruginosa	2048	4096	6 ± 0.5
K. pneumoniae	2048	4096	0

Fig. 3.

MIC and MBC values of AgNPs against tested microorganisms

Antibiofilm activity

AgNPs exhibited notable antibiofilm properties against all tested strains. The highest inhibition of biofilm biomass was observed in P. aeruginosa (59.7%), followed by E. coli (57.0%), S. aureus (54.9%) and K. pneumoniae (40.1%). Although all species demonstrated measurable reductions, the differences likely reflect species-specific variations in biofilm structure, extracellular polymeric substance composition and surface adhesion characteristics. The inhibition profiles are presented in Fig. 4. Statistically significant differences were observed among AgNP concentrations for all tested bacterial species (p < 0.05), indicating a concentration-dependent inhibition of biofilm formation.

Fig. 4.

Inhibitory effect of AgNPs on biofilm formation by E. coli, P. aeruginosa, S. aureus and K. pneumoniae. Data are presented as mean ± SD of three independent experiments. Different letters above the bars indicate statistically significant differences between concentrations according to one-way ANOVA followed by Tukey’s multiple comparison test (p < 0.05)

Discussion

Stachys species are well known in traditional medicine for their diverse biological activities, including anti-inflammatory, antibacterial, antioxidant and anticancer effects, largely attributed to their rich content of phenolic acids, flavonoids and terpenoids. These compounds have been associated with hepatoprotective, neuroprotective, cardioprotective and antimicrobial properties [25]. Such phytochemical diversity positions the genus as a valuable source of bioactive molecules and provides a strong rationale for exploring Stachys-based nanomaterials in antimicrobial applications.

Previous studies have extensively examined the antibacterial activity of Stachys extracts and essential oils. Reported antimicrobial effects vary considerably depending on plant species, extraction method, solvent composition and phytochemical profile. Aybey [26] reported inhibition zones ranging from 10 to 26 mm using solvent-derived extracts, highlighting the influence of extraction conditions on biological activity. In general, crude plant extracts often exhibit variable antimicrobial efficacy, whereas nanoparticle-mediated formulations may enhance the delivery, stability and bioavailability of bioactive constituents. In the present study, the absence of inhibition zones for S. aureus and K. pneumoniae in the disc diffusion assay is consistent with the known limited diffusion capacity of nanoparticle suspensions and plant extracts in agar matrices. Importantly, green-synthesized nanoparticles often demonstrate stronger antimicrobial performance than crude extracts, a trend supported by recent reports. For example, AgNPs produced using Raphanus sativus leaf extract displayed potent dose-dependent antibacterial activity against clinically relevant pathogens [27], reinforcing our observation that nanoparticle formation markedly enhances antimicrobial efficacy. The substantially lower MIC values obtained for S. spectabilis-derived AgNPs compared with crude extracts further highlight the advantages of nanoparticle-mediated transformation. The discrepancy between disc diffusion and MIC outcomes also supports the notion that, while AgNPs diffuse poorly in solid media, their antibacterial activity may be more effectively expressed in liquid environments where direct particle-cell interactions are maximized. The biological activity of silver nanoparticles is strongly influenced by their physicochemical properties, including particle size, morphology and surface charge. In the present study, previously reported characterization data demonstrated that the synthesized AgNPs were predominantly spherical with an average particle size of 10.8 nm and a zeta potential of − 12.8 mV [18]. The relatively small particle size increases the surface-area-to-volume ratio and enhances interactions with bacterial cell envelopes, facilitating membrane disruption and silver ion release. Furthermore, the spherical morphology may promote homogeneous contact with bacterial surfaces, while the negative zeta potential suggests moderate colloidal stability that can support nanoparticle dispersion in liquid media. These characteristics may partly explain the antibacterial and antibiofilm activities observed against both Gram-positive and Gram-negative pathogens.

Although the MIC values observed in the present study were higher than those reported for some green-synthesized AgNPs, direct comparisons among studies should be interpreted with caution. Antibacterial activity is strongly influenced by nanoparticle synthesis conditions, plant-derived capping agents, particle aggregation, surface chemistry and differences in experimental protocols. Therefore, variations in MIC values among studies do not necessarily reflect differences in nanoparticle quality or biological relevance. Despite the relatively high MIC values, the synthesized AgNPs demonstrated consistent concentration-dependent antibacterial effects and substantial antibiofilm activity against all tested bacterial species.

Interestingly, our results differ from those of Lazarević et al. [28] who reported greater susceptibility of Gram-positive bacteria to Stachys officinalis essential oil. In contrast, the AgNPs synthesized in this study exhibited comparable or even stronger inhibitory effects against Gram-negative strains. This divergence likely reflects the distinct mechanisms of action associated with nanoscale silver rather than plant-derived extracts. Although Gram-negative bacteria possess an outer membrane that typically restricts the penetration of many antimicrobial agents, AgNPs can bypass this barrier through unique physicochemical interactions. Electrostatic attraction between the negatively charged lipopolysaccharide layer and AgNPs promotes localized membrane destabilization and increased permeability. Additionally, outer membrane porins may permit partial entry of silver ions or small nanoparticle aggregates into the periplasmic space, where they induce oxidative stress, disrupt enzymatic pathways and interfere with DNA replication. Similar mechanisms have been proposed in other studies evaluating biogenic AgNPs, including those synthesized using carob leaf extract [12], supporting the interpretation that membrane-level interactions underlie the heightened antimicrobial response observed in E. coli and P. aeruginosa.

The antibiofilm findings of this study align with the inhibition levels reported by Hamid et al. [29], who observed 40–60% reductions in biofilm biomass across multiple bacterial species using green-synthesized AgNPs. Notably, the lower biofilm inhibition observed for K. pneumoniae compared with the other tested species may reflect species-specific differences in extracellular polymeric substance composition, biofilm density, capsular structure or nanoparticle–matrix interactions. K. pneumoniae is known to produce dense polysaccharide-rich biofilms and a prominent capsular layer, both of which can limit nanoparticle penetration and reduce direct interactions with bacterial cells. In contrast, the higher inhibition rates observed for P. aeruginosa and E. coli may reflect greater susceptibility of their developing biofilm matrices to nanoparticle-mediated disruption. These observations are consistent with previous reports demonstrating variable responses of bacterial biofilms to silver nanoparticle treatment depending on biofilm composition and structural organization.

Earlier studies suggest that Stachys extracts primarily disrupt mature biofilms rather than inhibit early-stage adhesion [26]. In contrast, the present study revealed significant inhibition during initial biofilm formation, indicating that nanoparticle-mediated delivery enhances the accessibility and antimicrobial activity of bioactive constituents on bacterial surfaces.

The consistent antibiofilm efficacy observed across all tested strains is particularly relevant given the clinical challenges posed by biofilm-associated infections. Variability in antimicrobial activity reported among different Stachys species [30] highlights the importance of phytochemical composition, synthesis protocols and nanoparticle physicochemical properties such as size, charge and surface functionality in determining biological performance. Metal nanoparticles are known to exert multi-targeted bio-interactions, including membrane oxidation, protein inactivation and metabolic disruption [31], which is consistent with the broad-spectrum inhibition documented here. The synergistic contribution of S. spectabilis derived reducing agents and the intrinsic antimicrobial properties of nanosilver likely underlie the antibiofilm and antibacterial effects observed.

Although the precise mechanisms underlying the antibacterial and antibiofilm effects of AgNPs are not fully elucidated, multiple pathways have been proposed. Silver ions released from AgNPs may bind to sulfur- and phosphorus-containing biomolecules, disrupt membrane permeability, inactivate essential enzymes and induce reactive oxygen species–mediated oxidative stress. Additionally, AgNPs can interfere with quorum sensing, suppress exopolysaccharide synthesis and destabilize biofilm structure [29, 32]. These multifaceted mechanisms likely contribute to the strong antimicrobial and antibiofilm performance demonstrated in this study.

Collectively, the findings demonstrate that Stachys spectabilis–derived AgNPs exhibit substantial antimicrobial and antibiofilm activity and expand current understanding of Stachys-based nanomaterials. The results highlight the potential of green-synthesized nanoparticles as promising candidates for strategies aimed at managing antimicrobial resistance and biofilm-associated infections.

Conclusion

This study provides the first evidence that silver nanoparticles synthesized using Stachys spectabilis extract display significant antimicrobial and antibiofilm activities against major Gram-positive and Gram-negative pathogens. Although the disc diffusion assay showed limited inhibition due to restricted nanoparticle mobility in agar matrices, MIC and biofilm assays demonstrated concentration-dependent antibacterial activity and substantial suppression of early-stage biofilm formation. These findings suggest a potential advantage of green-synthesized AgNPs over crude plant extracts, emphasizing the enhanced biological activity achieved through nanoparticle-mediated delivery.

The broad-spectrum antibiofilm activity observed across all tested species is particularly relevant given the increasing global burden of biofilm-associated infections and antimicrobial resistance. The ability of S. spectabilis–derived AgNPs to inhibit both planktonic growth and early biofilm development suggests their potential applicability in biomedical contexts where biofilm control is critical, including wound dressings, implant surfaces, catheter coatings and other medical device interfaces.

Given their eco-friendly synthesis, potential biocompatibility advantages and strong antibacterial performance, S. spectabilis-mediated AgNPs represent a promising alternative platform for the development of next-generation antimicrobial materials. Future work should include cytotoxicity assessments, evaluation in complex biological systems and integration into polymeric matrices or nanofiber scaffolds to explore their translational potential. Collectively, these findings underscore the value of plant-based nanotechnological approaches in addressing the ongoing challenges posed by multidrug-resistant pathogens and biofilm-related infections.

Abbreviations

NPs

Nanoparticles

AgNPs

Silver Nanoparticles

MICs

Minimum Inhibitory Concentrations

MHA

Mueller–Hinton Agar

BHI

Brain–Heart Infusion

MBC

Minimum Bactericidal Concentration

Authors' contributions

AKT: Investigation, Data curation, Formal analysis, Writing – Original draft preparation; ENG: Data curation, Laboratory analysis; RE: Supervision, Project administration, Data curation, Formal analysis; MM: Investigation, Writing – Reviewing and Editing. All authors read and approved the final manuscript.

Funding

There is no funding for this research.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.