Analytical grade chemicals with maximum purity were used in this study. Silver nitrate (AgNO₃) and bacterial culture media were purchased from Himedia Laboratories Ltd., India. Uropathogens responsible for UTIs such as S. aureus, K. pneumoniae, E. coli, P. aeruginosa, and E. faecalis, were granted by the K.A.P. Viswanatham Medical College, Tiruchirappalli – 620 001, Tamil Nadu.

Fresh T. ornata seaweeds (Figure 1) were collected from the Mandapam coastal area of the Gulf of Mannar located at the Bay of Bengal region (78°8’ E latitude and 9°17’ N longitude) of Tutucorin, Tamil Nadu, India. The seaweed samples were hand-picked and washed thoroughly in filtered seawater, tap water and distilled water to clean up adhering epiphytes, debris, and other impurities. Seaweed thalli with distinct morphology were transported immediately in separate polyethylene bags to the laboratory and shade dried for 3–5 days at room temperature (Harinee et al., 2019). The dried seaweed was then ground to fine powder with electric blender and stored in air tight container for further use.

Aqueous seaweed extract was made by adding 50 g of dried seaweed powder to 400 ml distilled water and heated to boiling until it was reduced to half its original volume. Once it cooled, the extract was filtered using Whatman grade 42 filter paper (Whatman, GE Healthcare, UK) and stored at 4°C for further studies (Harinee et al., 2019; Bhuyar et al., 2020).

Seaweed extract-mediated AgNPs were synthesized according to Azeem et al. (2021) with modifications. T. ornata extract (To-AE) (5 ml) was stirred with 95 ml of 1 mM AgNo₃ solution after adjusting the pH to 10 by adding 1 M NaOH. At alkaline pH the AgNPs formation gets accelerated. Upon completion of the reaction, a change in color was observed. The product was centrifuged at 10,000 rpm for 20 min. The pellets were collected and washed with distilled water to obtain pure NPs.

The characterization of To-AgNPs is critical for elucidating their functional properties because their physicochemical properties have profound effects on behavior, biodistribution, safety, and efficacy. An array of analytical techniques was employed for characterizing the samples, including UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffractometry (XRD), SEM, and dynamic light scattering (DLS) (Chandraker et al., 2022a,b). A UV-vis spectrophotometer (Lambda 35, Perkin Elmer) was used to confirm the NPs formation. The absorption spectrum of the AgNPs was recorded over a wavelength range of 300–800 nm. The type of seaweed biomolecules responsible for the reduction of AgNPs was determined by FTIR spectroscopy. Surface capping of the To-AgNPs by phytochemical constituents of seaweed extract was also confirmed by FTIR (Spectrum RX-I, Perkin Elmer) with a wavelength of 4,000–400 cm^–1. The size and morphological characteristics of the NPs were determined by SEM (EVO-18, Carl Zeiss) and TEM (FEI-TECNAI, G2-20 Twin microscope). XRD (Rigaku ULTIMA III) was used to determine the crystalline nature and lattice structure of the synthesized NPs, with Cu- kα anode radiation (λ = 1.54056 Å) operated at a voltage of 45 kV and a current of 30 mA, over a 2θ collection range of 20–80° with a step size of 0.02° and scan rate of 4°/min. To-AgNPs particle size distribution and zeta potential were determined using DLS (Micromeritics, Nano Plus).

DPPH radical scavenging activity of To-AE and To-AgNPs was evaluated by the method of Keshari et al. (2018). Briefly, 3 ml samples at different concentrations (10, 20, 40, 60, 80, and 100 μg/ml) were mixed with 1 ml of 0.1 mM DPPH in methanol and incubated for 30 min in the dark. The positive control was ascorbic acid. The absorbance of the samples and control was recorded at 517 nm spectroscopically and radical scavenging activity was calculated using the formula below.

A0 and A1 are the absorbance of the control and experimental samples, respectively.

The anti-uropathogenic activity of To-AE and To-AgNPs was studied by the well diffusion method (Faraja et al., 2018). Overnight cultures of UTIs causing bacteria, such as E. coli, E. faecalis, K. pneumoniae, S. aureus, and P. aeruginosa were used for the study. Mûller Hinton agar was prepared and poured into 4 petri plates and allowed to cool down. Once the medium got solidified, agar plates were swabbed with the inocula of the test pathogens. Wells (6 mm diameter) were cut out of the agar, and 50 and 100 μl of To-AE and the To-AgNPs were placed into each well and incubated at 37°C for 24 h. At the end of the incubation the diameters of the clear zone of inhibition of growth were measured.

The MIC and MBC of To-AgNPs were determined according to CLSI guidelines (Clinical and Laboratory Standards Institute [CLSI], 2015). MIC tests were performed as a standard macro broth dilution method, whereas MBC tests were carried out on the MHA plates. Bacteria for the experiment were 10⁶ CFU/ml, and two fold serial dilutions of To-AgNPs were prepared by mixing 1 ml of the working solution with 1 ml of bacterial inoculum in MHB. The concentration gradients of To-AgNPs were 2–0.0156 mg/ml. Additionally, a negative control (medium only) and a positive control (medium and bacterial inoculum) were maintained. Then the tubes were incubated for 24 h at 37°C. A MIC value was determined with the lowest concentration of To-AgNPs that inhibited bacterial growth. The MBC test was performed by plating the suspension from each tube which showed no visible bacterial growth, onto a MHA plate which was then incubated for 24 h. A MBC value was taken as the lowest concentration to kill the bacteria.

Confocal microscopy (Carl Zeiss, LSM710, Jena, Germany) was used to examine antimicrobial activity of To-AgNP against S. aureus and E. coli (Henciya et al., 2020). To-AgNPs were used at a concentration of 0.125 mg/mL for E. coli and 0.0625 mg/ml for S. aureus based on MIC results. Cultures without treatment were used as controls. Acridine orange and ethidium bromide (AO-EtBr) was used to stain the treated and untreated bacterial cells, and live and dead cells were observed under the confocal microscope.

The visualization of impact of To-AgNPs on the structure and morphology of S. aureus and E. coli cells was performed by SEM microscopy according to the following procedure (Zawadzka et al., 2016). Overnight cultures of treated and untreated samples were centrifuged at 6000 rpm for 15 min and pellets were collected thereafter. After being washed thrice with sterile PBS the pellets were suspended in cold glutaraldehyde and incubated for 2 h at 4°C. Bacterial cells were then washed three times with sodium buffer and dehydrated with a series of ethanol solutions (30-100%). A subsequent SEM examination was carried out by coating washed bacterial cells on SEM stubs (EVO-18, Carl Zeiss, Germany).

A statistical analysis of the data was performed using GraphPad Prism Software (Version 9.0.0). The normality distribution of the antioxidant activity and antimicrobial activity assay data was examined using the Shapiro–Wilk test. One-way ANOVA was used to analyze data, which passed the Shapiro–Wilk test. The Tukey–Kramer post-hoc test was used to further analyze the data for multiple comparisons. All the experiments were performed in triplicates and the data obtained were reported as means ± standard deviation, and statistically significant differences between the treatment and control groups were considered at a P-value ≤ 0.05.

The aqueous extracts of seaweed T. ornata were obtained using the hot extraction method. A color change from dark yellow to brown was observed after the addition of To-AE to AgNO₃ solution, which indicated the reduction of silver ions (Ag⁺) to silver (Ag⁰) upon excitation of surface plasmon resonance (SPR). To obtain pure NPs, the solution was centrifuged at 10,000 rpm for 20 min, and pellets were washed with distilled water.

To-AgNPs formation was confirmed using a UV–visible spectrophotometer. The UV absorption band for the To-AgNPs, at 420 nm, is shown in Figure 2.

The biomolecule nano-capping of the extract around the synthesized To-AgNPs confirmed by FT-IR spectroscopy led to greater stability of the synthesized NPs. To-AE was shown to contain functional groups such as alkanes, methylene, amines, and carboxylic acids that are potential reducing agents for the synthesis of AgNPs (Cho et al., 2005). The FTIR analysis of To-AE indicated the presence of carboxylic acids and amines. Figure 3 shows the molecular arrangement of various functional groups in the To-AE and the powdered To-AgNPs. A comparison of transmission peaks revealed that suppressed/increased peaks in colloidal solutions were due to metal NPs bound to bioorganic molecules. The shifted peak at 3,439.87 represents O–H stretching modes formed by flavonoid and phenolic groups in the phytoconstituents. The band at 2,075.00 cm^–1 confirmed the presence of nitrile carbons and C=O (1,650.78, 1,643.72, and 1,633.77 cm^–1) was due to a free carboxylic acid group. Another band at 1022.43 cm^–1 can be ascribed to the C–OH vibrations of alcohol groups in the To-AE. The FT-IR spectra of To-AgNPs also indicated the presence of functional groups like phenolic O–H (3,391.54 cm^–1), nitrile carbons (2,882.07 cm^–1), and C=O (1,608.92 cm^–1).

The intensities of diffracted X-rays were recorded from 20° to 80°. Four Bragg reflections at 38.07°, 43.97°, 64.36°, and 77.32° represented the lattice planes of (1 1 1), (2 0 0), (2 2 0), and (3 1 1), respectively (Figure 4), which confirmed the cubic structure of AgNPs. According to the Joint Committee on Powder Diffraction Standards (JCPDS) – 040783, the XRD results verified that the To-AgNPs formed by seaweed extract reduction of Ag⁺ ions were crystalline (Chandraker et al., 2022c). Additionally, peaks were observed at 28.57° and 32.15°, suggesting that bio-organic crystallization occurred on AgNPs surfaces.

The SEM image provided an overview of the surface morphology, size and shape of the biosynthesized To-AgNPs (Merin et al., 2010). As shown in Figure 5, spherical and some irregular AgNPs with average size of 73.98 nm with diameters ranging from 64.67 nm to 81.28 nm were observed. This result strongly supported the notion that To-AE may act as reducing and capping agents in the production of To-AgNPs. As shown in TEM images (Figure 6), most of the particles are almost spherical, while others displaying irregular shapes, which is consistent with SEM results.

Hydrodynamic size distribution and the polydispersity index (PdI) of AgNPs were determined by DLS analysis (Adebayo-Tayo et al., 2019). The experiment also provided information about the population of particles in a short time. The results showed that the cumulant diameter of AgNPs at optimum conditions was 128.3 nm with a PdI of 0.313 (Figure 7A). Observation of the particle’s zeta potential reveals a sharp peak with a negative value (−63.3 mV), indicating the surface of the To-AgNPs was negatively charged and stable (Figure 7B).

Ascorbic acid showed the highest antioxidant activity, followed by To-AgNPs and To-AE. Based on the concentrations, 100 μg/ml of ascorbic acid, To-AE, and To-AgNPs demonstrated excellent radical scavenging activity, 95.12, 61.27, and 70.09%, respectively (Figure 8). The Shapiro–Wilk normality test showed that the data had a significance of P < 0.05 (Figure 9A). A one-way ANOVA was applied to examine differences between the treatment groups (To-AE, To-AgNPs) and controls (ascorbic acid), which were statistically significant (DF = 5, F = 5.037, P = 0.0212). Based on a Post-hoc Tukey’s test, To-AE and To-AgNPs showed highly significant positive correlations with ascorbic acid, P = 0.0013 and P = 0.0007, respectively, and To-AgNPs showed a higher radical scavenging activity than To-AE (Figure 9B).

Antimicrobial activity tests were performed against bacteria that cause UTIs including K. pneumoniae, P. aeruginosa, E. faecalis, E. coli, and S. aureus. As shown in Figure 10 and Supplementary Figure 1, To-AgNPs were more effective than AgNO₃ solutions, and To-AE. Additionally, DMSO, as a negative control, did not show any inhibition zone. An antibiotic disk of 30 mcg Cefotaxime (CTX) was used as a positive control because it exhibited broad-spectrum antibacterial activity in most uropathogens. Among the selected uropathogens S. aureus (15.75 ± 0.35 mm) and E. coli (15 ± 0.7 mm) were more susceptible to To-AgNPs. A maximum inhibition zone was observed against Gram-positive S. aureus, while Gram-negative P. aeruginosa (9 ± 0.7 mm) had a minimum zone of inhibition.

As shown in Figure 11A, Shapiro–Wilk normality test results indicate statistical significance for normality distribution. In vitro antibacterial activity was compared with one-way ANOVA statistical analysis, and statistically significant results were obtained (P < 0.05). A Tukey’s test was conducted for multiple comparison of To-AE, To-AgNPs, AgNO₃, positive control (Cefotaxime), and negative control (DMSO) inhibitory diameters for antimicrobial activity. In Figures 11B, C, it is seen that all treatment groups had significantly different antibacterial activities except for AgNO₃, which was not significant when compared with Cefotaxime (mean difference (MD) = 3.2, P = 0.1198), To-AgNPs (MD = 1.65, P = 0.0076) and To-AE (MD = –3.8, P = 0.019), as well as Cefotaxime with To-AgNPs also not significant (MD = 1.55, P = 0.6588). Based on the ANOVA results, it can be concluded that To-AgNPs had significantly higher antibacterial activity than all other groups except Cefotaxime. Cefotaxime serves as a standard to correlate the lead agent between the groups.

To-AgNPs showed potent activity against S. aureus, E coli, K. pneumoniae, and E. faecalis with MIC values 0.0625, 0.125, 0.25, and 0.25 mg/ml, respectively. On the other hand, inhibitory activity was not detected against P. aeruginosa. The MBC against S. aureus, E. coli, K. pneumoniae, and E. faecalis, 0.125, 0.25, 0.5, 0.5 mg/mL, respectively. These results showed that the To-AgNPs were highly effective against S. aureus and E. coli.

Confocal microscopy analysis of S. aureus and E. coli treated individually with a lethal dose (MIC) of To-AgNPs revealed the dead and surviving bacteria. In Figure 12, acridine orange and ethidium bromide stainings showed large number of dead cells (red image) and fewer surviving bacteria with physical deterioration and disintegration (green image) in both treated groups, but no dead cells are seen in the control groups.

To further confirm the results observed with CLSM, SEM was performed on the same samples (Figure 13). The untreated E. coli cells appeared intact with no signs of ruptures and collapses, while the To-AgNPs-treated E. coli cells appeared shorter and compact or completely deformed. For S. aureus, the untreated cells appeared as smooth surfaces without any damage to their exterior structures, whereas, the treated cells displayed alterations and deformities of their surfaces and corrugated outer membranes.

In the present study, an aqueous extract of T. ornata, obtained by hot extraction, was used to synthesize AgNPs. Biomolecules present in T. ornata function as reducing and capping agents during AgNP synthesis. The addition of 1 M NaOH enhanced the reducing power of functional groups in To-AE and accelerated the formation of small and stable AgNPs. When AgNO₃ solution was added to the To-AE and the pH was adjusted to 10, there are color changes from dark yellow to reddish brown, resulting from the reduction of Ag⁺ to Ag⁰ due to surface plasmon resonance. Confirmation of AgNPs formation is commonly done by UV-Vis Spectroscopy. The conduction and valence bands in AgNPs are located very close to each other, allowing electrons to move freely. As these electrons oscillate collectively in resonance with the light wave, they generate an SPR absorption band (λ_max at 400–420 nm). AgNPs absorb light depending on their size, dielectric medium, and chemical environment (Salleh et al., 2020). The typical band with λ_max at 400-420 nm indicates spherical-shaped AgNPs (Raja et al., 2017).

The FTIR spectrum of To-AE and To-AgNPs showed the presence of various functional groups such as flavanoid, phenolic, nitrile carbons and carboxylic acid groups which is also present in AgNPs, indicating the reduction of functional groups during AgNPs synthesis. These functional groups may donate electrons to silver ions, reducing them to silver atoms, which then aggregate to form AgNPs. They also act as a stabilizer by adsorbing onto the surface of the AgNPs and preventing them from aggregating. Researchers observed a similar phenomenon when investigating possible biomolecules responsible for AgNPs synthesized from other seaweed extracts (Krishnan et al., 2015).

The XRD pattern showed sharp Bragg peaks at 38.07°, 43.97°, 64.36°, and 77.32°, indicating the crystalline nature of To-NPs. Some unassigned peaks were also in their vicinity, possibly due to the capping agent that stabilized the nanoparticles. AgNPs synthesized from the seaweed extract of Enteromorpha compressa (Ramkumar et al., 2017), also showed similar results.

The shape and size of the AgNPs were clarified by SEM analysis. The morphology of To-AgNPs was clearly seen in the SEM images with an average size of 73.98 nm with diameters ranging from 64.67 to 81.28 nm. In addition, most of the silver nanoparticles produced were spherical, while some were irregular shaped. TEM was also used to determine the size, shape, and crystallinity of To-AgNPs. In agreement with SEM results, most of the particles appear almost spherical on TEM images, while some have irregular shapes. The lighter areas in the image suggest that Ag⁰ is surrounded by bioactive compounds contained in TO-AE. In an experiment using Sonchus arvensis leaf extract-mediated AgNPs, Chandraker et al. (2019) observed similar semi-transparent surrounding zones of capping phytochemical constituents.

The DLS measured size was larger than the SEM size since DLS measures the hydrodynamic diameter, which is the diameter of the particle, hydration layer, and other surface ions and molecules that move alongside the AgNPs (Fissan et al., 2014). According to Du et al. (2015), PDI value of <0.5 is preferred for the nanoparticle solutions to be monodispersed. In the present study, a PDI value of 0.313 was obtained, which confirmed the monodispersity of To-AgNPs. The stability of nanoparticles in aqueous solutions can be understood using the zeta potential, a measure of surface charge potential. Nanoparticles with a zeta potential of more than +30 mV or less than –30 mV are said to be particularly stable in the dispersion medium (Erdogan et al., 2019). To-AgNPs’ zeta potential value were found to be –63.3 mV. The results demonstrate that To-AgNPs are negatively charged and more stable, allowing them to maintain their structural integrity over an extended period of time.

Antioxidant and free radical scavenging properties of AgNPs depend on the bioactive compounds present in seaweeds, and they can improve by increasing the concentration of AgNPs. In the present study, the DPPH antioxidant activities of To-AE, To-AgNPs and the standard ascorbic acid were analyzed at various concentrations (10, 20, 340, 60, 80 and 100 μg/ml). It was found that the radical scavenging activity of both To-AE and To-AgNPs were significantly different to those of standard ascorbic acid. To-AgNPs showed higher antioxidant activity (70.09%) compared to To-AE (61.27%). It was also found that, for all groups, there was an increasing trend in radical scavenging activity with concentration. Alharbi et al. (2020) reported that T. ornata extract contained excellent antioxidants. Polyphenols, sulfated polysaccharides and terpenoids present in T. ornata were probably responsible for the antioxidant properties (Vijayabaskar and Shiyamala, 2012). Chakraborty and Joseph (2016) reported that phenolic compounds in T. ornata were the effective antioxidants that were scavenging DPPH free radicals. Our FTIR results also proved the presence of phenolic compounds in To-AE and To-AgNPs that might be responsible for its antioxidant property. Also, AgNPs are intended to transport electrons to the reactive media in order to neutralize the unstable DPPH free radicals (Chandraker et al., 2022a). Prior to the present study were the seaweed Ecklonia cava (Venkatesan et al., 2016), Desmarestia antarctica, and Iridaea cordata (González-Ballesteros et al., 2021) mediated AgNPs studied for their antioxidant activity and provided similar results.

Urinary tract infection-causing bacteria like P. aeruginosa, K. pneumoniae, E. faecalis, E. coli, and S. aureus were selected for the antimicrobial study of the To-AE and its biofabricated Ag-NPs. Gram-positive S. aureus exhibited the greatest zone of inhibition, followed by Gram-negative E. coli. A minimum level of activity was found in P. aeruginosa. The statistical analysis of antimicrobial activities of To-AgNPs, To-AE, AgNO₃, DMSO and Cefotaxime showed that To-AgNPs exhibited higher antimicrobial activity than To-AE and AgNO₃, which is comparable with Cefotaxime. According to Neelamathi and Kannan (2016), the crude methanolic extract of T. ornata contains hydrocarbons, acids, aldehydes, ketones, esters, alcohols, halogenated compounds, and aromatics that are likely to be responsible for its antimicrobial properties. It was reported that antimicrobial activity of T. ornata extract killed P. aeruginosa effectively (Alharbi et al., 2020) and ethanolic extracts were more effective against Gram-positive bacteria than Gram-negative bacteria (Ward and Deyab, 2016). Few reports are published on the synthesis of Sn-O₂ and Mg(OH)₂ NPs from T. ornata extract has been reported with antibacterial activity (Govindaraju et al., 2020; Sunny et al., 2022). In a study by Anuluxan et al. (2022) using AgNPs derived from T. ornata against water-contaminated bacteria, Gram-positive S. aureus, B. circulans, and E. faecalis and Gram-negative E. coli and P. aeruginosa and found that To-AgNPs demonstrated the strongest antibacterial activity against S. aureus, which was consistent with our findings.

The antibacterial effects of To-AgNPs were dose-dependent. Of the five bacterial strains tested, To-AgNPs showed potent activity against S. aureus and E. coli with MIC values of 0.0625, 0.125 mg/ml, and MBC values of 0.125, 0.25 mg/ml, respectively. There is still a lot of uncertainty about how AgNPs exert their antimicrobial effects. Possibly, the antimicrobial property is due to Ag⁺ released from To-AgNPs, which bind to cellular structural groups (carbonyl, sulfhydryl and phosphate), penetrate into cells, condense DNA and react with proteins (Leng et al., 2020). Ag⁺ ions interact with thiol groups in enzymes and proteins to attack bacteria. Additionally, Ag⁺ ions trigger the release of K⁺ ions in bacteria and target plasma or cytoplasmic membranes. They also interact with nucleic acids, inhibit cell division and prevent bacterial growth. Additionally, the capping agent of the nanoparticle also interacts with the bacterial membrane. All these phenomena lead to damage or even the death of the microorganisms. The results are in agreement with the disk diffusion results. The MIC values from prior research showed a considerable amount of fluctuations in AgNPs’s antimicrobial properties. Consequently, it is difficult to compare the data since there is no accepted method for determining AgNPs’ antibacterial activity (Loo et al., 2018).

The findings from both CLSM and SEM analysis were consistent, with both showing changes in morphology and structure in bacteria, suggesting the affinity of To-AgNPs to attach to the cell surface resulting in cell membrane degradation and deformation. According to our CLSM results, both Gram-negative E. coli and Gram-positive S. aureus were effectively killed by To-AgNPs treatment. However, S. aureus treated with To-AgNPs were almost dead compared to treated E. coli. This indicates that To-AgNPs had a greater effect on S. aureus than on E. coli. The SEM image revealed that the membranes of S. aureus were severely damaged and distorted after being exposed to To-AgNPs, resulting in cellular fluid discharge. AgNPs interfered with the permeability of cell membranes and caused mechanical damage by interacting with cell wall components. For E. coli, the surviving cells retained their rod shape with smooth surfaces. This is because Gram-positive and Gram-negative bacteria exhibit different AgNPs toxicity due to their structural differences in cell walls. The peptidoglycan layer in Gram-negative E. coli is surrounded by two lipid bilayers and lipopolysaccharides that act as antimicrobial barriers, thus preventing silver ions from entering the cell. A permeability barrier formed by the outer membrane diffusion channels and porins in E. coli also prevents more To-AgNPs entry. In contrast, Gram-positive bacteria’s cell walls are highly porous and enable the entry of foreign substances into the cell (Lee et al., 2019). These findings offer additional support for the potential and mechanism of action of To-AgNPs to inhibit uropathogens.

Recurrent UTIs may increase the risk of kidney stones. A wide variety of uropathogens may cause infection stones, including E. coli, S. aureus, P. mirabilis, K. pneumoniae, Streptococcus sp. and P. aeruginosa (Mohan et al., 2022; Raj et al., 2023). Therefore, biofabricated To-AgNPs are promising anti-uropathogenic agents and may act as stone preventers.