Enhanced antibacterial and anti-biofilm activity of Tamarix aphylla ethanolic extract and its silver nanoparticles

Ethical compliance statement

This study utilized anonymized bacterial isolates obtained from routine diagnostic procedures at the Microbiology Laboratory of King Saud Hospital, Unayzah, Saudi Arabia. No personally identifiable information was collected, and all samples were used exclusively for research purposes. In accordance with institutional guidelines, ethical approval and informed consent were not required for studies involving fully anonymized, residual clinical specimens.

Processing and extraction of smoked ethanolic extract from Tamarix leaves (EETL)

To prepare EETL, fresh leaves (2 kg) were collected, cleaned thoroughly under running water to remove any dirt or contaminants, and air-dried in a shaded area at room temperature. Once dried, leaves were burnt to smoke that was collected in jars to obtain a sticky substance that was dissolved in 99% ethanol. Under reduced pressure, the filtrate was concentrated using a rotary evaporator at 40–45°C until all ethanol was fully evaporated, leaving behind the EETL. For future experimentation, the extract was stored in a sealed container at 4°C.

Analysis of total flavonoid contents (TFC) in EETL

TFC in EETL was analyzed using the aluminum chloride colorimetric assay, as described in a previous study.^[11] This method involves preparing a quercetin standard, a known flavonoid, by dissolving it in methanol to prepare a concentrated solution in 0–100 µg/mL range. For TFC measurement, 0.5 mL of the EETL sample was first combined with 0.16 mL of 5% NaNO₂ solution, then 0.16 mL of 10% AlCl₃ solution was added, followed by 1 mL of 1 M NaOH solution. The reaction mixture was thoroughly mixed and incubated briefly to allow the color to develop. The absorbance at 510 nm was determined using a spectrophotometer. Flavonoid concentration in the EETL was calculated using a quercetin standard curve, with results presented as milligrams of quercetin equivalents (mg QE) per gram of EETL (mg QE/g EETL). This method provided an accurate and reproducible estimate of the flavonoids concentration in the extract.

Evaluation of total phenolic content (TPC) in EETL

The Folin-Ciocalteu (FC) assay was employed to determine the TPC in EETL, following a previously established protocol.^[12] The standard curve for analysis was developed using gallic acid solutions ranging from 1.4 µg/mL to 1000 µg/mL. To perform the assay, 1.6 mL of either EETL, or gallic acid solution was mixed with 0.2 mL of a five-fold diluted FC reagent, followed by 0.2 mL of 10% sodium carbonate. The reaction was allowed to proceed by keeping the mixture at room temperature in a dark environment for 30 min to ensure the development of the characteristic blue color. Using a spectrophotometer, the absorbance at 765 nm was measured, and the TPC was determined from the standard curve. TPC was expressed in milligrams of gallic acid equivalents per gram of extract (mg GAE/g EETL), indicating the phenolic content’s contribution to the extract’s antioxidant activity.

Total antioxidant activity of EETL

The antioxidant activity of EETL was assessed through its 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity following a previously published protocol with slight adjustments.^[12] The samples and ascorbic acid were dissolved in dimethyl sulfoxide, also serving as the blank. A 20 µL aliquot of each sample was added to a 96-well microplate, followed by 180 µL of 100 mM DPPH solution, freshly prepared on the day of the experiment. For optimal stability, the mixtures were left in the dark at room temperature for 30 min to limit DPPH degradation. Absorbance was measured at 517 nm using a Biotech microplate spectrophotometer. The IC₅₀ value, which denotes the EETL concentration required to scavenge 50% of DPPH radicals, was calculated. A standard curve was generated using EETL (10–500 µg/mL), while ascorbic acid served as the reference standard with concentrations in the range from 1 µg/mL to 20 µg/mL.

Chemical analysis of EETL by liquid chromatography (LC) electrospray ionization time-of-flight mass spectrometry (MS)

Experimental setup and data acquisition method

An Exion LC system (AB Sciex, USA) with an autosampler, pre-column filter disks (0.5 µm × 3.0 mm, Phenomenex, USA), and an Xbridge C18 column (3.5 µm, 2.1 × 50 mm, Waters, USA) was used for small molecule separation at 40°C and a 300 µL/min flow rate. The mobile phase for positive mode consisted of solution A (5 mM ammonium formate in 1% methanol, pH 3.0 with formic acid) and solution B (100% acetonitrile), while for negative mode, solution C (5 mM ammonium formate in 1% methanol, pH 8 with sodium hydroxide) was used. Gradient elution was applied as follows: 0–20 min, 10% B; 20.01–25 min, 90% B; 25.01–28 min, and 10% B followed by equilibration with 90% B.

Processing of LC-MS data

MS-DIAL 3.70 was used for untargeted small molecule detection, referencing either the ReSpect positive (2737 entries) or ReSpect negative (1573 entries) database depending on the acquisition mode. Peak detection was performed with MS1 and MS2 tolerances of 0.01 Da and 0.05 Da, a minimum peak height of 100 amplitudes, a mass slice width of 0.05 Da, smoothing over two scans, and a minimum peak width of six scans. Identification was based on MS1/MS2 tolerances of 0.2 Da, while alignment settings included a retention time tolerance of 0.05 min and an MS1 tolerance of 0.25 Da. Processed data were re-evaluated in PeakView 2.2 with MasterView 1.1 (AB SCIEX), selecting aligned features from the total ion chromatogram with a Signal-to-Noise ratio >5 and a sample-to-blank intensity ratio >5.

Preparation of Ag-NPs Formulation of EETL (Ag-NPs-EETL)

EETL (0.25 g) was dissolved in 10 mL double double-distilled water, and silver nitrate (0.1 M, 10 mL aq. solution) was vigorously stirred at room temperature (22 ± 2°C) for an hour and kept overnight in the dark.^[13] A color change from brown to light brown was observed, which was indicative of the formation of the nanoparticles. Ag-NPs-EETL formulation was filtered through a 0.2-micron filter paper and purified by centrifugation. The synthesized Ag-NPs-EETL formulation was then dried or suspended in deionized water for further analysis.

Characterization of Ag-NPs-EETL by ultraviolet (UV)-visible absorbance spectroscopy

The reduction process of Ag⁺ ions was analyzed using a UV-Vis spectrophotometer of the reaction mixture at different wavelengths (200–800 nm) at different functional times. The result showed that Ag-NPs-EETL formulation had a characteristic absorption maxima peak (λ_max) at ~ 430 nm.

Fourier-transform infrared (FT-IR) analyses

The FT-IR spectra of Ag-NPs-EETL and the extract were recorded to identify the functional groups involved in nanoparticle synthesis and stabilization. For sample preparation, the synthesized Ag-NPs-EETL were centrifuged at 10,000 rpm for 15 min to remove unreacted components, washed with deionized water, and dried at 40°C to obtain a fine powder. The extract was used in its dried form without further modification. A small quantity of each sample was combined with potassium bromide (KBr) in a 1:100 ratio (Sample: KBr), ground into a fine powder, and pressed into transparent pellets using a hydraulic press. FT-IR spectra were obtained using a Perkin Elmer FT-IR spectrometer (UK) across a wavelength range of 4000–400 cm^-1 at a 4 cm^-1 resolution. The spectra were analyzed to identify characteristic peaks, revealing functional groups responsible for reducing silver ions (Ag⁺) and capping the nanoparticles for stabilization. This analysis provided insights into the biomolecular interactions between the extract and Ag-NPs-EETL.

To measure the antibacterial activity of EETL and AgNPs-EETL

The antibacterial properties of EETL and Ag-NPs-EETL were examined through agar well diffusion against S. aureus, MRSA, E. coli, and colistin-resistant E. coli. Bacterial cultures were cultivated in nutrient broth, and 8 mm diameter wells were aseptically created in the agar using a sterile cork borer. Different amounts (100 and 200 µg) of EETL or AgNPs-EETL were carefully pipetted into each well. To enable compound diffusion, the plates were maintained at 37°C for 24 h. The zones of inhibition around each well were subsequently measured in mm, providing an assessment of the antibacterial activity of EETL and Ag-NPs-EETL.^[13]

Macrodilution method to determine minimum inhibitory concentration (MIC)

To assess the MIC of WSEE, the protocol from the Clinical and Laboratory Standards Institute was adopted.^[14] MIC of EETL and Ag-NPs-EETL against bacterial strains, S. aureus, MRSA, E. coli, and CR- E. coli was determined using macrodilution method. Stock solutions of EETL and Ag-NPs-EETL were serially diluted in nutrient broth to achieve concentrations from 2 µg/mL to 1000 µg/mL. In sterile test tubes, 1 mL of each bacterial suspension, adjusted to 0.5 McFarland standard (1 × 10⁸ colony-forming unit [CFU]/mL), was added to 1 mL of the diluted extract or nanoparticle solutions. A negative control (broth without extract or nanoparticles) and a positive control (broth with bacterial suspension only) were included in the study. The samples were incubated at 37°C for 18–24 h to allow proper growth. After incubation, the tubes were visually examined for turbidity. The MIC was determined as the lowest concentration of EETL or Ag-NPs-EETL that exhibited no visible bacterial growth.

Time-dependent bactericidal activity assay

The time-kill assay was performed to evaluate the bactericidal activity of EETL and Ag-NPs-EETL against bacterial strains.^[15] Bacterial cultures were grown in nutrient broth to the mid-logarithmic phase and adjusted to a turbidity equivalent to 0.5 McFarland standard (1 × 10⁸ CFU/mL). In sterile test tubes, the bacterial suspensions were exposed to predetermined concentrations of EETL and Ag-NPs-EETL based on their MIC values. Tubes containing bacterial suspensions without treatment served as growth controls, while tubes with broth only acted as sterility controls. The test mixtures were incubated at 37°C under shaking conditions to ensure uniform exposure. At predetermined time intervals (0, 4, 8, 12, and 24 h), aliquots of 100 µL were withdrawn from each test tube, serially diluted in Phosphate buffered saline (PBS), and plated onto nutrient agar. Following a 24-h incubation at 37°C, the CFUs were enumerated, and the log reduction in CFU/mL over time was calculated to determine the bactericidal or bacteriostatic effect of EETL or Ag-NPsEETL. A 3-log reduction in CFU/mL compared to the initial inoculum was defined as bactericidal activity. The results were plotted as CFU/mL against time to visualize the kinetics of bacterial killing.

Evaluation of the anti-biofilm activity of EETL and AgNPs-EETL formulation against drug-resistant bacteria

The effect of EETL and Ag-NPs-EETL on biofilm formation was assessed by the crystal violet staining method.^[15] Bacterial strains (S. aureus, MRSA, E. coli, and CR-E. coli) were cultured to mid-logarithmic phase, diluted to 1 × 10⁶ CFU/mL, and treated with 100 and 200 µg of EETL or Ag-NPs-EETL in 96-well microplate, with untreated wells serving as a negative control. By including these controls, the assay ensured that any observed inhibition of biofilm formation was attributable to the tested extracts or nanoparticles and not due to external factors or experimental error. Non-adherent cells were discarded after 24 h by PBS washing, and biofilm staining was performed using 0.1% crystal violet. The retained crystal violet was eluted with 200 µL of ethanol, and the absorbance was assessed at 570 nm through a microplate reader. This assay was executed for all bacterial strains, enabling a comparative evaluation of EETL and Ag-NPs-EETL efficacy against biofilm formation. The percentage inhibition of biofilm formation was evaluated using the formula:

Biofilm formation (%) = (1-Absorbance of treated wells/Absorbance of control well) × 100.

Statistical analysis

To assess the statistical significance of the differences in biofilm formation among the various formulations, a one-way analysis of variance was conducted using GraphPad Prism software, version 5.0 (GraphPad Software Inc., San Diego, USA).

Phytochemical Profiling and Identification of EETL by LC–ESI-TOF–MS

Utilizing ESI-MS in both positive and negative modes, the study identified the presence of many important compounds in EETL, as shown in Table 1.

Spectrophotometric characterization of Ag-NPs by UV-vis analysis

The synthesis of Ag-NPs was confirmed by monitoring the reduction of Ag⁺ ions through UV-Vis spectroscopy using a Thermo-Scientific Evolution 201 spectrophotometer. The absorbance spectrum was recorded at different time points in the 200–800 nm range [Table 2, Supplementary Figure 1]. A prominent absorption maximum (λmax) at approximately 430 nm, characteristic of the surface plasmon resonance (SPR) of Ag-NPs, was observed, indicating successful nanoparticle formation.

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Figure 1:

Antibacterial activity of ethanolic extract of Tamarix aphylla leaves (EETL) and its silver nanoparticle formulation (Ag-NPs-EETL), as determined by the agar well diffusion method. The assay was conducted against four bacterial strains: Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli, and carbapenem-resistant E. (CR-E. coli). Clear zones of inhibition were observed around the wells containing EETL and Ag-NPs-EETL, indicating their antibacterial efficacy.

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FT-IR analyses

The FT-IR spectrum of EETL alone showed absorption peaks at 3250, 2925, 1730, 1410, 1390, 1090, and 610 cm⁻¹ [Table 3, Supplementary Figure 2a]. The peak at 3250 cm⁻¹ reflects O-H stretching, while peaks at 2925 cm⁻¹ and 1730 cm⁻¹ correspond to C-H stretching and ester carbonyl groups, respectively. Peaks at 1410 cm⁻¹ and 1390 cm⁻¹ represent bending vibrations of phenolic compounds, and the peak at 1090 cm⁻¹ indicates C-O stretching. The additional peak at 610 cm⁻¹ signifies aromatic C-H bending vibrations.

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Figure 2:

Time-kill kinetics of EETL and Ag-NPs-EETL against Staphylococcus aureus, MRSA, Escherichia coli and CR-E coli. Control (◆), EETL-100 µg/mL (●), EETL-200 µg/mL (◼), Ag-NPsEETL-100 µg/mL (○), and Ag-NPs-EETL-100 µg/mL (□). The data are displayed as mean ± SD of three separate values. EETL: Ethanolic extract of Tamarix leaves, Ag-NPs: Silver nanoparticles, MRSA: Methicillin-resistant S aureus, SD: Standard deviation.

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The formation and stabilization of AgNPs were confirmed using FT-IR spectroscopy, highlighting the role of functional groups from the extract molecules in the reduction of silver ions (Ag⁺). The FT-IR spectrum of the synthesized nanoparticles, recorded using a Perkin Elmer FT-IR spectrometer (UK), revealed prominent absorption peaks at 3290, 1690, 1370, 1090, and 520 cm⁻¹ [Table 3, Supplementary Figure 2b]. These peaks correspond to specific functional groups involved in capping and stabilizing the nanoparticles. For instance, the peak at 3290 cm⁻¹ indicates O-H stretching vibrations from hydroxyl groups, while the peak at 1690 cm⁻¹ is attributed to C=O stretching from carbonyl groups, likely originating from phenolic or flavonoid compounds in the extract. Peaks at 1370 cm⁻¹ and 1090 cm⁻¹ suggest the presence of C-H bending and C-O stretching vibrations, respectively, indicating alcohol and ether functionalities. The peak at 520 cm⁻¹ suggests metal-ligand interactions, confirming the bonding between silver ions and biomolecules from the extract.

EETL and Ag-NPs-EETL showed antibacterial activity

The results demonstrated that EETL and Ag-NPs-EETL were effective against S. aureus, MRSA, E. coli, and CR- E. coli [Figure 1]. At doses of 100 µg and 200 µg, EETL demonstrated significant antibacterial activity against S. aureus, producing inhibition zones measuring 12 mm and 21 mm, respectively. Furthermore, EETL demonstrated effectiveness against MRSA, producing inhibition zones of 14 mm and 24 mm at doses of 100 µg and 200 µg, respectively [Figure 1]. The activity of EETL was also investigated and found to be effective against E. coli and CR-E. coli. EETL at 100 µg and 200 µg showed 15 mm and 25 mm of inhibition zones against E. coli. EETL at 100 µg was not effective against CR-E. coli, whereas at 200 µg, it exhibited an inhibition zone of 12 mm.

At comparable doses, Ag-NPs-EETL exhibited remarkable antibacterial activity against S. aureus, with inhibition zones of 28 mm at 100 µg and 36 mm at 200 µg, demonstrating a dose-dependent enhancement of efficacy. Similarly, Ag-NPs-EETL was highly effective against MRSA, producing inhibition zones of 24 mm and 33 mm at 100 µg and 200 µg, respectively, indicating strong activity against this resistant strain [Figure 1]. Against E. coli, Ag-NPs-EETL showed inhibition zones of 21 mm and 36 mm at 100 µg and 200 µg, respectively, further confirming its broad-spectrum antibacterial potential. Notably, Ag-NPs-EETL also exhibited significant activity against colistin-resistant E. coli (CR-E. coli), with inhibition zones of 20 mm and 32 mm at 100 µg and 200 µg, respectively [Figure 3]. These findings highlight the superior efficacy of the Ag-NPs-EETL formulation across a range of pathogenic and drug-resistant bacterial strains.

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Figure 3:

Inhibition of biofilm formation by EETL and Ag-NPs-EETL against (a) Staphylococcus aureus, ***P < 0.001 in comparison to control; ●P < 0.05 EETL-100 versus Ag-NPs-EETL-100 (b) MRSA, ***P < 0.001 in comparison to control (c) Escherichia coli, ***P < 0.001 in comparison to control and (d) CR-E. coli, ***P < 0.001 in comparison to control; ●P < 0.05 EETL-100 versus Ag-NPs-EETL-100; ^●●_P < 0.01 EETL-200 versus Ag-NPs-EETL-200. Error bars denote the standard deviations derived from three replicates. ***P < 0.001 compared to control; ●P < 0.05 EETL-100 versus Ag-NPs-EETL-100; ^●●_P < 0.01 EETL-200 versus Ag-NPs-EETL-200. EETL: Ethanolic extract of Tamarix leaves, Ag-NPs: Silver nanoparticles. MRSA: Methicillin-resistant S aureus, CR-E: Colistin-resistant E-coli.

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MICs comparison of EETL and Ag-NPs-EETL against bacterial pathogens

The MICs of EETL and Ag-NPs-EETL were determined against different bacterial strains. EETL exhibited MICs of 200 µg/mL against both S. aureus and MRSA, demonstrating its potent antibacterial activity. The MIC of EETL was 150 µg/mL for E. coli but increased to 400 µg/mL for CR-E. coli, indicating lower effectiveness against the resistant strain.

In contrast, Ag-NPs-EETL displayed significantly lower MICs, highlighting its enhanced antibacterial potency. For S. aureus and MRSA, the MIC was found to be 100 µg/mL, reflecting its strong activity against drug-resistant bacterial pathogens. Ag-NPs-EETL also exhibited superior efficacy against E. coli, with an MIC of 50 µg/mL, and against CR-E. coli with an MIC of 100 µg/mL, demonstrating its potential to overcome resistance mechanisms of the pathogens. These outcomes indicated that Ag-NPs-EETL formulation significantly enhanced the antibacterial activity of EETL, particularly against MRSA and CR-E. coli, making Ag-NPs-EETL a promising candidate for combating MDR bacterial infections.

Time-Kill assay results

The time-kill kinetics assessed the antibacterial efficacy of EETL and Ag-NPs-EETL against S. aureus, MRSA, E. coli, and CR-E. coli by tracking the reduction in bacterial CFUs over time [Figure 2]. The results demonstrated time- and dose-dependent declines in bacterial CFUs with both the treatments, with Ag-NPs-EETL showing greater efficacy as compared to the EETL product.

For S. aureus, EETL at 100 µg/mL, achieved a reduction of over 2 log₁₀ CFUs/mL at 24 h, while at 200 µg/mL, it reduced CFUs by more than 3 log₁₀ [Figure 2]. The Ag-NPs-EETL displayed enhanced activity, with both 100 µg/mL, and 200 µg/mL concentrations achieving over 3 log₁₀ reductions in CFUs within the same time frame. Against MRSA, EETL at 200 µg/mL reduced CFUs by more than 3 log₁₀ after 24 h of incubation. In contrast, Ag-NPs-EETL was more potent, with 100 µg/mL achieving a 3 log₁₀ reduction and 200 µg/mL, causing a >4 log₁₀ reduction at 24 h measuring point [Figure 2].

The activity of EETL and Ag-NPs-EETL was also tested against E. coli. EETL at 100 µg/mL, and it reduced CFUs by over 2 log₁₀ at 24 h, while 200 µg/mL achieved a reduction greater than 3 log₁₀. The Ag-NPs-EETL demonstrated superior efficacy, with both 100 µg/mL, and 200 µg/mL, achieving reductions >3 log₁₀ and 4 log₁₀, respectively, at the 24-h mark [Figure 2].

The CR-E. coli was less susceptible to EETL, with neither 100 µg/mL, nor 200 µg/mL achieving significant reductions in the CFUs at 24 h [Figure 2]. However, Ag-NPs-EETL showed notable activity, with 200 µg/mL reducing CR-E. coli CFUs by over 3 log₁₀ within 24 h [Figure 2]. These findings highlight the enhanced bactericidal activity of Ag-NPsEETL as compared to the EETL, particularly against resistant strains, such as MRSA and CR-E. coli. The nanoparticle formulation demonstrated the potential to overcome resistance mechanisms and offered a promising approach for managing MDR bacterial infections.

Anti-biofilm activity of EETL and Ag-NPs-EETL

The anti-biofilm activity of EETL and its Ag-NPs formulation (Ag-NPs-EETL) was evaluated against S. aureus, MRSA, E. coli, and CR-E. coli using the crystal violet staining method. EETL exhibited moderate biofilm inhibition, showing a reduction of 54% and 73% at 100 µg/mL and 200 µg/mL, respectively, against S. aureus [Figure 3a]. Similarly, EETL inhibited biofilm formation in MRSA by 60% and 72% at the same concentrations [Figure 3b]. Against E. coli, EETL showed biofilm inhibition rates of 56% and 80%, while against CR-E. coli, the inhibition was limited to 24% and 36%, indicating reduced efficacy against colistin-resistant strain [Figure 3c and d].

In contrast, the Ag-NPs-EETL demonstrated significantly enhanced biofilm inhibition across all bacterial strains. Against S. aureus, biofilm formation was inhibited by 66% and 92% at 100 µg/mL and 200 µg/mL, respectively [Figure 3a]. For MRSA, biofilm inhibition rates were 74% and 92%, demonstrating strong activity even against resistant strains [Figure 3b]. Ag-NPs-EETL also exhibited superior efficacy against E. coli, with inhibition rates of 72% and 91% at the same concentrations [Figure 3c]. Notably, CR-E. coli, which was less susceptible to EETL, showed inhibition rates of 56% and 74% with Ag-NPs-EETL, highlighting its effectiveness against drug-resistant biofilms [Figure 3d].