Green-synthesised silver nanoparticles using sewage-derived fish gut bacteria with antibacterial potential against AmpC-β producing Escherichia coli

Microbial-mediated green synthesis of silver nanoparticles (AgNPs) represents an eco-friendly alternative to conventional chemical methods by utilising biological reducing and stabilising agents. In this study, gut bacteria isolated from sewage-exposed fish were used to synthesise AgNPs, and the resulting nanoparticles were evaluated for their antimicrobial and antioxidant activities. Bacterial biomass was used to reduce Ag + ions from a 1 mM AgNO 3 solution. Nanoparticle formation and characterisation were confirmed using UV–Visible spectroscopy, Field Emission Scanning Electron Microscopy (FE-SEM), Transmission Electron Microscopy (TEM), and X-ray Diffraction (XRD). A distinct color change from pale yellow to brown and a characteristic absorption peak at approximately 350–400 nm confirmed AgNP synthesis. TEM analysis revealed predominantly spherical nanoparticles ranging from 10 to 30 nm in size. The biosynthesised AgNPs exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria including AmpC-producing Escherichia coli, producing inhibition zones in the range of 24–31 mm. The synthesised nanoparticles displayed strong dose-dependent antioxidant activity, in terms of DPPH and ABTS + radical scavenging activity, with IC 50 of 18 μg/mL and 39 μg/mL, respectively. These findings indicate the potential of sewage fish gut bacteria as a sustainable source for producing bioactive AgNPs with promising applications as an antibacterial agent.

Antimicrobial resistance (AMR) has emerged as a major global public health challenge, decreasing the effectiveness of existing antibiotics and exacerbating the prevalence of infectious diseases (Dilawari et al., 2025; Patra et al., 2025). Among resistant pathogens, AmpC β-lactamase-producing Escherichia coli is of particular concern due to its ability to hydrolyse a broad range of β-lactam antibiotics, including cephalosporins and monobactams (Jameel et al., 2014). The plasmid-mediated nature of AmpC enzymes further facilitates the rapid horizontal transfer of resistance genes in both clinical and environmental settings (Haenni et al., 2014). Consequently, the increasing prevalence of AmpC-producing E. coli has significantly reduced available therapeutic options, revealing the urgent requirement for alternative antimicrobial strategies (Jacoby, 2009).

Nanotechnology has emerged as a promising approach for combating multidrug-resistant bacteria. Various nanomaterials, including gold nanoparticles, silver nanoparticles, magnetic nanoparticles, and nanozymes, have shown potential biomedical applications (Kaushal et al., 2023; Ahmed et al., 2025; Makkar et al., 2025). Among them, AgNPs are particularly important because of their broad-spectrum antimicrobial activity, high surface-area-to-volume ratio, and multi-target mode of action (Karataş et al., 2026). AgNPs display antibacterial effects through membrane disruption, reactive oxygen species generation, protein inactivation, and interference with DNA replication, thereby reducing the likelihood of resistance development (Das G. et al., 2019). The conventional physical and chemical synthesis methods often involve toxic chemicals, high energy consumption, and environmental risks (Saxena et al., 2025). As an alternative to these limitations, a sustainable and environmentally friendly approach, namely, green synthesis, has emerged as a sustainable alternative (Samuel et al., 2022).

Green synthesis utilises biological systems such as plants, enzymes, and microorganisms to reduce metal ions into nanoparticles in an environmentally sustainable manner (Cardoso et al., 2025). Among biological agents, bacteria are considered suitable for nanoparticle biosynthesis because of their rapid growth, metabolic diversity, and ability to produce reducing and stabilising biomolecules (Adeleke et al., 2024). Nevertheless, most studies have focused on well-characterised microbial strains, while environmentally adapted microorganisms remain largely unexplored (Iravani, 2014; Alsaiari et al., 2023).

The gut microbial flora of fish living in the sewage waters is one such untapped reservoir of microbial diversity (Xiong et al., 2019). These are environments that are characterised by complex combinations of pollutants, such as heavy metals, pharmaceuticals and organic waste (Mohammed and Swalaha, 2025). The microorganisms that survive in those environments are exposed to extreme selective forces, which frequently lead to some distinct physiological and metabolic adaptations, such as toxic compound resistance and elevated enzyme activity (Suzzi et al., 2022). These characteristics render them especially interesting to be used in the synthesis of nanoparticles because they can be equipped with new biomolecules that could reduce and stabilise metal ions, including silver (Ying et al., 2022).

Therefore, the present study is designed to investigate the potential of gut bacteria isolated from sewage-grown fish for the green synthesis of AgNPs, evaluate their antibacterial activity against AmpC β-lactamase-producing E. coli, and characterise the synthesised nanoparticles. This work integrates environmental microbiology and nanobiotechnology to explore sustainable strategies for combating antibiotic resistance. The authors have intentionally focused on the green synthesis approach because it is widely recognized as an environmentally sustainable, cost-effective, and biocompatible alternative to conventional chemical synthesis.

Sample collection and bacterial isolation

Fish samples of Channa punctata were obtained from sewage-affected water bodies across Guwahati metropolitan city, Assam, India, between September 2023 and February 2024. A total of five specimens were collected from different locations, including Ahomgaon (26.106,989° N, 91.720,866° E) and Pamohi Boragaon (26.111,766° N, 91.708,557° E). All specimens were found non-living (dead) at the time of collection but were fresh, showing no signs of decomposition. Therefore, no live animal handling or experimental intervention was involved in this study.

Morphometric parameters such as total length, gut length, and body weight were recorded. Gut contents were aseptically removed and homogenised in sterile saline solution. The homogenates were serially diluted up to 10 −7, and 100 µL aliquots from 10 −6 and 10 −7 dilutions were spread on Tryptic Soy Agar (TSA) (Himedia, M1365-100G) plates. The plates were incubated aerobically at 37 °C overnight. Morphologically distinct colonies were isolated, purified, and preserved in 25% glycerol (Himedia, TC503-500 ML) (Dhayalan et al., 2022).

Screening of antagonistic activity of the bacterial isolates

The representative colonies obtained from the fish gut were initially screened for their antagonistic activity through Streak-on-lawn inhibition assay and agar well diffusion methods.

Streak-on-lawn inhibition assay

The antagonistic activity of the isolated bacterial strains was initially tested against the E. coli (ATCC 11229) using a streak on lawn inhibition assay, a modification of the cross-streak assay. A lawn culture of E. coli was prepared on a Mueller-Hinton Agar (Himedia, M173-500G) plate, and the isolated bacterial strain was streaked as a straight line on the agar surface. The plates were then incubated at 37 °C for 24 h. Antagonistic activity was assessed by observing the presence of inhibition zone surrounding the test strains (Lertcanawanichakul and Sawangnop, 2008).

Agar well diffusion method

Antagonistic activity of the study isolates was also further carried out by agar well diffusion method using E. coli (ATCC 11229). Wells of size 5 mm were made on Mueller-Hinton Agar (MHA) (Himedia, M173-500G). 100µL of the cell-free supernatant was added to each well and incubated at 37 °C for 24 h. After incubation at 37 °C for 24 h, the zone of inhibition was measured (Valgas et al., 2007; Balouiri et al., 2016).

Identification of the bacterial isolates

Bacterial isolates exhibiting antagonistic activity were subjected to molecular identification using 16S rDNA sequencing, following the method followed by Ingti et al. (2017).

Green synthesis of silver nanoparticles (AgNPs)

Silver nanoparticles were synthesised using a biological reduction method following Kalimuthu et al. (2008), Gurunathan et al. (2009) and Koilparambil et al. (2016) with slight modifications.

Preparation of crude bacterial extract

An isolated bacterial strain (RGUS1) having antagonistic activity was selected for the synthesis of AgNPs. The isolates were initially inoculated into 200 mL of sterile Luria broth (Himedia, M575-500G) and kept on a shaking incubator (ORBITEK, Scigenics Biotech) at 37 °C. After 24 h of growth, the culture was centrifuged at 10,000 rpm for 10 min to separate the biomass and supernatant (Priyadarshi et al., 2022). Approximately 3 gm of wet biomass was washed three times with sterile distilled water to remove adhering contaminants. After removing contaminants, the biomass was collected for the synthesis of silver nanoparticles.

Preparation of silver nitrate (AgNO3) solution

For the green synthesis of silver nanoparticles, a 10 −3 M silver nitrate solution was prepared by dissolving 0.0169 g of silver nitrate (Sigma-Aldrich, v800358-25G; Batch No. 0000003756) in 100 mL of double-distilled water (ddH 2 O).

Biomass-mediated synthesis of AgNPs

For the synthesis of silver nanoparticles, approximately 3 gm of wet microbial biomass was resuspended in 100 mL of 10 −3 M silver nitrate (AgNO 3) solution. The reaction mixtures were incubated at room temperature (25 °C ± 2 °C) on a shaking incubator (ORBITEK, Scigenics Biotech) at 200 rpm for 24 h. Following incubation, the reaction mixtures were kept at room temperature (25 °C ± 2 °C), maintaining light intensity of 230 lx (LX-101 lx Meter) and 50% ± 5% relative humidity for 48 h. A visible colour change from light pinkish to dark brown was observed, indicating the reduction of silver ions and the formation of silver nanoparticles. Following synthesis, the nanoparticles were collected by centrifugation at 10,000 rpm for 10 min and subsequently washed three times with 5 mL of distilled water to eliminate residual reagents.

Characterisation of synthesised silver nanoparticles

The initial assessment of Ag + ion reduction was carried out after 12 h of reaction by diluting the nanoparticle solution in a 1:4 ratio. The absorption spectra were then recorded over a wavelength range of 300–800 nm using a UV–Visible Spectrophotometer (Systronics-2201) (Das et al., 2025).

The shape and surface morphology of the synthesised nanoparticles were examined using Field Emission Scanning Electron Microscopy (FESEM). AgNPs colloidal solution was drop casted onto a silicone waffer and dried for further observation under FESEM (Garg et al., 2023). The analysis was performed with a FEI-Quanta FEG 200F instrument, operated at an accelerating voltage of 30 kV and a magnification of 1000X to 20000X. Energy Dispersive X-ray (EDX) analysis was conducted to identify the elemental composition of the synthesised nanoparticles using EDAX TSL AMETEK systems (Ali I. A. M. et al., 2023).

High-resolution transmission electron microscopic (HR-TEM) imaging was done using FEI Tecnai G2, F30 instrument, operated at an accelerating voltage of 300 kV. HR-TEM analysis was done by the drop cast method onto 300 mesh size carbon-coated copper grids. The grid was further air-dried and then observed under HR-TEM (Priyadarshi et al., 2025). This was used to determine the size, shape and morphology of the synthesised silver nanoparticles. Particle size distribution of more than 500 individual nanoparticles was done using Origin (version b9.5.5.409). Polydispersity of the nanoparticles was determined according to the equation: Polydispersity (%) = (σ/x̄) × 100, in which σ is the standard deviation of particle size distribution, and x̄ is the mean particle size (Das et al., 2021).

To determine the X-ray diffraction (XRD) pattern, dried and centrifuged silver nanoparticle crystals were utilised. The analysis was conducted using a BRUKER AXS D8 ADVANCE instrument (model: BRUKER KAPPA APEX II), operating at a current of 30 mA and a voltage of 40 kV. The sample was irradiated with Cu Kα radiation, and data were recorded at a scanning rate of 5° per minute across the 2θ range. The resulting diffraction pattern was compared with standard reference data from the JCPDS database to identify the crystalline structure (Das and Mandal, 2020; Ali I. A. M. et al., 2023).

The hydrodynamic diameter and size distribution of the synthesised nanoparticles were determined using Dynamic Light Scattering (DLS) (Malvern Zetasizer Nano ZS90_Malvern Panalytical, UK). The nanoparticle suspension was appropriately diluted with deionised water to minimise multiple scattering effects and analysed at 25 °C using a clear disposable zeta cell. Measurements were recorded for 60s, with a measurement position of 5.50 mm and an attenuator setting of 8. The average hydrodynamic diameter (Z-average), polydispersity index (PDI), and particle size distribution based on intensity were calculated automatically by the instrument software.

Antimicrobial studies of the synthesised nanoparticles

Antimicrobial activity was evaluated against Bacillus subtilis (ATCC 11774), E. coli (ATCC 11229), Staphylococcus aureus (ATCC 11632), and Pseudomonas aeruginosa (ATCC 15442), phenotypically confirmed AmpC β-lactamase E. coli isolated form ground water.

The silver nanoparticles synthesised from the bacterial cell pellet were tested for their antimicrobial activity by the well diffusion method, following the method of Koilparambil et al. (2016). A lawn culture of the bacterial strain was individually prepared using a sterile cotton swab, followed by making small wells of size 5 mm on Mueller-Hinton Agar (Himedia, M173-500G). 50µL of AgNO 3, AgNPs, Cefotaxime and bacterial crude extract without AgNO 3 were added into the wells. The plates were then kept in an incubator at 37 °C for 24 h.

Minimum inhibitory concentration (MIC)

The MIC of the synthesised silver nanoparticles (AgNPs) was determined using the agar dilution method adapted from Wiegand et al. (2008) with minor modifications. All procedures were carried out under sterile conditions to prevent contamination. The bacterial strains E. coli (ATCC 11229), S. aureus (ATCC 11632), B. subtilis (ATCC 11774), P. aeruginosa (ATCC 15442), and AmpC β-lactamase-producing E. coli selected for the study were standardised to a 0.5 McFarland turbidity standard. The standardised bacterial suspensions were then inoculated onto Mueller–Hinton Agar (MHA) (HiMedia, M173-500G) plates supplemented with AgNPs at two-fold serial concentrations ranging from 2 to 512 μg/mL. MHA plates containing ampicillin at corresponding concentrations were used as controls. All plates were incubated at 37 °C for 24 h. Following incubation, the plates were examined for visible bacterial growth. The lowest concentration of AgNPs that completely inhibited visible bacterial growth was recorded as the MIC. All assays were performed in triplicate.

Antioxidant activity

The antioxidant potential of the synthesised silver nanoparticles and the corresponding bacterial extract was evaluated using DPPH and ABTS + radical scavenging assays following standard protocols (Das D. et al., 2019). In the DPPH assay, ascorbic acid was used as the reference standard, whereas butylated hydroxytoluene (BHT) served as the standard for the ABTS + assay. Radical scavenging activity was calculated as a percentage inhibition using the equation: where A 0 represents the absorbance of the control, and A 1 represents the absorbance of the sample. Antioxidant activity was expressed as the IC 50 value, defined as the concentration required to achieve 50% free radical scavenging (Das D. et al., 2019).

Statistical analysis

The graphs were made using OriginLab software (https://www.originlab.comorigin). The results of the inhibition zone are expressed as mean ± standard deviation (SD) of three independent replicates. Different letters (a,b,c,….) indicate statistically significant differences among treatments at p < 0.05 according to one-way ANOVA trailed by Tukey’s post hoc test.

Screening of antagonistic activity of the bacterial isolates and identification of bacteria

Five specimens of C. punctata were collected from sewage water at different locations in Guwahati city, Kamrup, Assam, India. Bacterial strains were isolated from the gastrointestinal tracts of C. punctata. Each representative isolate was screened for antagonistic activity against E. coli ATCC 11229. Out of 47, one isolate (RGUS1) showed antagonistic activity in both the cross-streak as well as agar well diffusion method (Figure 1) and was selected for downstream study. The selected isolate RGUS1 was further subjected to molecular identification by 16s ribosomal RNA sequencing and showed high sequence similarity (99.27%) with Bacillus tropicus, a closely related members of the B. cereus group. The sequence was submitted to the GenBank database bearing an accession number PZ529495. A phylogenetic analysis of the sequence was performed to assess the genetic relationships of the isolate, and the findings are provided in Supplementary Figure S1.

Synthesis and characterisation of silver nanoparticles

Based on the level of antagonistic activity observed, the bacterial strain RGUS1 was selected for nanoparticle biosynthesis, as it was assumed that this bacterium may produce secondary metabolites that act as reducing and stabilising agents, facilitating the conversion of silver ions (Ag +) into silver nanoparticles (Ag 0). The preliminary screening of silver nanoparticle formation was done by observing the colour change, which occurred after 72 h of incubation in the presence of light from colourless to light pinkish and finally to dark brown. This suggests that the colour change observed in the bacterial biomass samples was due to the formation of silver nanoparticles.

UV–visible spectroscopy analysis

The formation of silver nanoparticles synthesised using Bacillus extract was confirmed by UV–Visible spectroscopy. The spectrum (Figure 2) exhibited a distinct surface plasmon resonance (SPR) absorption peak at approximately ∼350–400 nm, which is characteristic of silver nanoparticles. The presence of this SPR band in the visible region confirms the reduction of Ag + ions to metallic Ag 0 nanoparticles by biomolecules present in the Bacillus extract. The broad nature of the absorption peak indicates the formation of nanoscale silver particles with possible variation in particle size distribution, which is typical for biologically synthesised nanoparticles.

Field emission scanning electron microscopy (FESEM) analysis

FESEM was used to analyse the morphology of silver nanoparticles prepared from Bacillus extract. The synthesised particles were primarily irregular to semi-spherical in shape with rough uneven surfaces, as seen from the micrograph (Figure 3a). These nanoparticles were aggregated and clustered together forming macrostructures due to the capping and interaction of biomolecules present in the bacterial extract. This can also happen during sample drying and preparation for microscopic analysis. Despite this agglomeration, the FESEM image unequivocally confirms the formation of silver particles in the nanoscale formed by the bacterial extract-mediated reduction process.

Energy dispersive X-ray (EDX) analysis

The elemental composition of the biosynthesised silver nanoparticles was examined through EDX spectroscopy (Figure 3b). Results of EDX showed the presence of Ag next to C, O, N and S. A fingerprint peak centred around ∼3 keV was identified, affirming metallic silver and confirming the formation of silver nanoparticles. Carbon, oxygen, nitrogen and sulfur can be associated with biomolecules (proteins, enzymes and other metabolites present in Bacillus extract) that help to reduce and stabilise the synthesised nanoparticles. The strong silver signal, with smaller peaks attributable to additional elements, is evidence of biosynthesis and stabilisation of silver nanoparticles mediated by a bacterial extract.

High-resolution transmission electron microscopy (HR-TEM) analysis

HR-TEM analysis was performed to examine the morphology and size distribution of Bacillus extract-synthesised silver nanoparticles. HR-TEM micrograph showed that nanoparticles were almost spherical and slightly oval and well dispersed with only minor aggregation in some areas (Figure 3c). The particles had well-defined boundaries, which confirmed that the biosynthesis of nanoscale silver particles was successful. Particle size distribution analysis states that the majority of nanoparticles were found to distribute between 10 and 30 nm with an average particle size of 16.56 ± 4.6 nm determined through Gaussian fitting using Origin software (Figure 3d). The obtained standard deviation (σ) of 4.6 nm and full width at half maximum (FWHM) of 10.85 nm confirmed that the particle size distribution was relatively uniform. The polydispersity index calculated was 0.2777 (27.77%), which shows a moderately polydispersed nanoparticle population. The variation of the particle size studied could be due to agglomeration or self-aggregation of nanoparticles during nano-formulation or sample preparation. The area around the centre was a little fainter than that of the flower in the centre, which could indicate an organic layer that would be coating these nanoparticles.

X-ray diffraction (XRD) analysis

X-ray diffraction (XRD) analysis confirmed the crystalline nature of Bacillus extract-synthesised silver nanoparticles. The XRD pattern showed four strong diffraction peaks at the 2θ values of 38.24°, 44.39°, 64.51° and 77.44° that correspond to (111), (200), (220) and (311) crystallographic planes, respectively. Such diffraction planes correspond to the face-centred cubic (FCC) structure of metal silver. The (111) plane was found to be the most intense among the various peaks, confirming that synthetic nanoparticles are mainly oriented along this plane. The diffraction pattern obtained is closely matched with the JCPDS database reported standard silver diffraction data file no. 04-0783 and indicates formation of crystalline AgNPs through bacterial-mediated synthesis. The sharp and distinct peaks also indicate high crystallinity of the synthesised nanoparticles (Figure 3e).

Selected area electron diffraction (SAED) analysis

The characteristic crystalline structure of the synthesised silver nanoparticles was confirmed by the selected area electron diffraction (SAED) pattern that formed on HR-TEM study (Figure 3f). The SAED pattern exhibits rings of alternating bright and dim along with several distinguishable diffraction spots, which confirms that the crystalline structure corresponds to the (111), (200), (220) and (311) planes of silver. The generated diffraction rings indicate the polycrystalline character of the synthesised nanoparticles. The strong diffraction ring corresponding to (111) plane validates the XRD results, confirming that nanoparticles are primarily aligned along the (111) direction of the crystallographic plane.

Dynamic Light Scattering analysis

The DLS analysis demonstrated that the synthesised nanoparticles possessed a Z-average hydrodynamic diameter of 38.48 nm with a polydispersity index (PDI) of 0.436 (Supplementary Figure S2; Supplementary Table S1). The measurement quality was classified as good, indicating reliable data acquisition. The intensity-based particle size distribution revealed three distinct populations (Supplementary Table S2). The predominant fraction exhibited an average particle diameter of 59.86 nm, accounting for 90.9% of the total scattering intensity, indicating that the majority of nanoparticles were within the nanoscale range. A minor population of very small particles with an average diameter of 4.36 nm contributed 6.1% of the intensity, whereas a third population centred around 4005 nm represented only 3.0% of the intensity, suggesting the presence of a small number of larger aggregates. The intercept value of 0.841 further confirmed good signal quality during the measurement.

Antimicrobial activity

The antimicrobial activity of the synthesised silver nanoparticles was evaluated using the agar well diffusion assay against Escherichia coli (ATCC 11229), S. aureus (ATCC 11632), B. subtilis (ATCC 11774), P. aeruginosa (ATCC 15442), and an AmpC β-lactamase-producing E. coli isolate obtained from groundwater. The antibacterial efficacy of AgNPs was compared with AgNO 3, ampicillin, and bacterial crude extract using 50 µL of each treatment. Among all treatments, AgNPs exhibited the highest antibacterial activity against all tested organisms. The maximum zone of inhibition was observed against E. coli (31.33 ± 2.8 mm), followed by B. subtilis (29 ± 3.8 mm), AmpC β-lactamase-producing E. coli (28.33 ± 3.6 mm), S. aureus (27.33 ± 2.8 mm), and P. aeruginosa (24 ± 2.4 mm) (Table 1). In contrast, the crude extract showed no inhibitory activity against any of the tested organisms.

The minimum inhibitory concentration analysis further confirmed the potent antibacterial activity of the synthesised AgNPs. The lowest MIC value was recorded against Staphylococcus aureus (6 μg/mL), whereas Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa showed MIC values of 32 μg/mL. The highest MIC value for AgNPs was observed against the AmpC β-lactamase-producing E. coli isolate (128 μg/mL), although it remained substantially lower than that of ampicillin (256 μg/mL), indicating the enhanced efficacy of AgNPs against multidrug-resistant bacteria. Antibiotic susceptibility profiling further revealed that the AmpC β-lactamase-producing E. coli isolate was resistant to cefotaxime, amikacin, gentamycin, ciprofloxacin, and trimethoprim-sulfamethoxazole, while remaining susceptible only to cefepime and imipenem.

Antioxidant activity

The antioxidant capacity of samples was estimated with 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay for a concentration range of 10–30 μg/mL. The percent inhibition values of all the test samples increased in a concentration-dependent manner as the dose escalated. At all tested concentrations, ascorbic acid (standard) produced the highest antioxidant activity, with inhibition increasing from ∼48% in 10 μg/mL to ∼60% in 30 μg/mL (Figure 4a). Compared to the standard, silver nanoparticles showed strong scavenging activity of ∼38%, as compared to ∼59% among similar ranges, which was comparable to the standard at higher concentrations. The activity of the bacterial extract was relatively low (the inhibition value increased from 32% to ∼53%). Polynomial regression analysis showed an excellent model fit for all samples (R 2 = 0.991–0.999), confirming a dose-response relationship between wt and Av in all ratios observed. The activity trend was also supported by the IC 50 values. The most effective radical scavenging efficiency was observed for ascorbic acid with an IC 50 of ∼11 μg/mL (Figure 4b). The IC 50 obtained with silver nanoparticles was moderate (∼18 μg/mL), but the highest value for the bacterial extract (∼24 μg/mL) indicated relatively lower antioxidant activity.

The ABTS radical cation decolourisation assay clearly demonstrated the concentration-dependent antioxidant activity of all tested samples (10–100 μg/mL) (Figure 4c). Percentage inhibition was observed to increase with dose, confirming radical scavenging ability. Among the extractions tested, butylated hydroxytoluene (reference standard) exhibited the highest antioxidant activity, with ∼37% inhibition at 10 μg/mL, increasing to ∼95% at 100 μg/mL. Silver nanoparticles were also found to be significantly active, the percentage of inhibition ranging from ∼29% at a lower concentration to ∼95% at a higher concentration. In contrast, with lower concentrations of the bacterial extract, a significantly lower scavenging efficiency was observed (∼13% at 10 μg/mL; ∼21% at 15 μg/mL), but higher doses approached similar maximal inhibition (∼94%,100 μg/mL). The trend in activity was unchanged at intermediate concentrations. At 50 μg/mL, inhibition values were ∼74% (BHT), ∼68% (silver nanoparticles), and ∼65% (bacterial extract). At this concentration (25 μg/mL), the inhibitions were ∼67%, ∼57% and ∼35%, indicating that AgNP showed greater ability to quench radicals when compared to the use of crude extract alone. The dose–response results were further supported by IC 50 values. Among them, BHT had the lowest IC 50 of ∼34 μg/mL, indicating the highest antioxidant activity. Silver nanoparticles showed a moderate IC 50 of ∼39 μg/mL, followed by the bacterial extract of ∼42 μg/mL, which is a comparatively low scavenging activity (Figure 4d).

In the present investigation, B. tropicus (RGUS1) isolated from the gut of Channa punctata, collected from sewage water, was used to reduce Ag + ions to metallic Ag, as it displays antagonistic activity against Escherichia coli (ATCC 11229). Species of the genus Bacillus are widely recognised for producing extracellular enzymes, peptides, and secondary metabolites that probably function as reducing and stabilising agents during the biosynthesis of nanoparticles (Iravani, 2014; Singh et al., 2016).

The visible colour changes from light pinkish to dark brown after 72 h of incubation, indicating the reduction of Ag + to Ag 0 nanoparticles, which is consistent with earlier reports of AgNP formation (Rai et al., 2009). UV–Visible spectroscopic analysis of the biosynthesised silver nanoparticles showed surface plasmon resonance (SPR) in and around ∼350–400 nm, which is a common identification feature of silver nanoparticles (Wiley et al., 2006; Narenkumar et al., 2018). The appearance of SPR bands of AgNPs depends on particle size and shape, as well as the surrounding medium (Kuppurangam et al., 2015; Das D. et al., 2019). The presence of only one broad absorption peak indicates the formation of mostly spherical and monodisperse nanoparticles, which is typical for green-synthesised nanoparticles (Awwad et al., 2013; Singaravel and Murugan, 2025). However, the broadening of the SPR suggests polydispersity and interaction of nanoparticles with biomolecules in the bacterial extract, acting as stabilising agents (Tolaymat et al., 2010).

FESEM micrograph revealed that the synthesised nanoparticles were mostly irregular to semi-spherical in morphology with significant aggregation, a characteristic that is commonly observed in biologically synthesised nanoparticles. This means aggregation can occur through both biomolecule interactions between nanoparticle and bacterial extract, and due to drying effects in nanosample preparations (Bélteky et al., 2019; Dhaka et al., 2023). The morphological characteristics have been described in microbial-mediated biosynthesis of silver nanoparticles, where proteins and metabolites secreted by microorganisms regulate the growth, morphology, and stabilisation of nanoparticles (Vanlalveni et al., 2024; Akdaşçi et al., 2025). The clustered appearance observed on FESEM image may be indicative of the presence of organic capping agents from bacterial extract, which mediates nanoparticle stabilisation but also leads to agglomeration (Awwad et al., 2013). The elemental composition of the obtained nanoparticles was further verified by energy-dispersive X-ray (EDX) analysis. A prominent silver (Ag) signal at ∼3 keV in the EDX spectrum is a characteristic indicator of metallic silver and indicates successful reduction of Ag + ions to form elemental silver nanoparticles. This strong signal is a very typical spectral sign of silver attributed to its surface plasmon resonance and electron transition (Das and Mandal, 2020). Besides silver, small peaks relating to carbon (C), oxygen (O), nitrogen (N) and sulfur (S) were also observed. These elements are likely present due to biomolecules including proteins, enzymes and other organic compounds found in the Bacillus extract, which contribute towards reduction and stabilisation of nanoparticles during their biosynthesis (Sidhu et al., 2022; Bibi et al., 2025). The carbon and oxygen signals can be associated with the organic compounds stabilising or capping the nanoparticle surface, while nitrogen (N) and sulphur (S) probably stem from amino acids and proteins excreted by bacterial cells (Akhbari et al., 2010; Rodrigues et al., 2024). Functional groups, including amines, carboxyls, and thiols in biomolecules, bind to the nanoparticle surface, preventing aggregation among nanoparticles that go beyond a certain threshold in addition to providing an overall stability advantage (Fahim et al., 2024; Jiang et al., 2025). Similar elemental profiles have been reported in numerous studies based on bacteria-mediated production of AgNPs (Yusof et al., 2020; Huq and Akter, 2021; Singh and Mijakovic, 2022).

HR-TEM micrographs confirmed that the synthesised nanoparticles were mostly spherical, with a slight oval morphology, and some regions had agglomerated into small clusters. This type of morphology was commonly reported for biologically synthesised AgNPs, where some biomolecules in microbial extracts may affect the nucleation and nanoparticle growth during the reduction (Akdaşçi et al., 2025). The slight aggregation observed in certain regions may be due to interparticle interactions or organic molecules that aggregate on the surface of nanoparticles (Jiang et al., 2025). The analysis of particle size distribution suggested that most of the nanoparticles were 10–30 nm in size, with the average particle size calculated from Gaussian fitting being 16.56 ± 4.6 nm. The calculated polydispersity value of 27.77% implies a moderate level of polydispersion. This variability in particle size is common with biological synthesis methods due to the heterogeneous nature of the biomolecules involved in nanoparticle reduction and stabilisation (Das et al., 2017; Das et al., 2021). The HR-TEM images also revealed that the edges of the nanoparticles appeared lighter than their central part due to the presence of an organic layer surrounding the particles (Das et al., 2021). This observation demonstrates that the biomolecules present in the Bacillus extract serve as a natural capping agent that stabilises the nanoparticles, thus preventing their excessive aggregation (Singh and Mijakovic, 2022). Proteins and amino acid residues of bacterial extracts interacted with the nanoparticle surface due to functional groups such as amine, carboxyl, or thiol groups, which help them in stabilising the nanoparticles (Das and Mandal, 2020; Mikhailova, 2024).

Dynamic Light Scattering (DLS) analysis confirmed the successful synthesis of nanoparticles with a Z-average hydrodynamic diameter of 38.48 nm, demonstrating that the synthesised particles are predominantly within the nanoscale range. The hydrodynamic diameter measured by DLS was larger than the average particle size obtained from transmission electron microscopy (TEM) (16.56 ± 4.6 nm). This difference is expected because DLS determines the hydrodynamic diameter of particles dispersed in solution, which includes the particle core along with its surrounding hydration layer and any surface-bound biomolecules or capping agents (Das et al., 2025). In contrast, TEM measures the actual physical core size of dried nanoparticles. Such discrepancies between DLS and TEM measurements are commonly reported for biologically synthesized nanoparticles due to the presence of stabilizing organic molecules adsorbed on the nanoparticle surface (Das D. et al., 2019). The PDI value of 0.436 obtained from DLS indicates a moderately polydisperse nanoparticle suspension. This observation is consistent with the TEM particle size distribution, which showed that most nanoparticles ranged from 10 to 30 nm. While DLS characterises the size distribution of nanoparticles in suspension by hydrodynamic diameter, TEM directly measures the physical dimensions of individual particles. Therefore, the slightly higher polydispersity observed by DLS likely reflects variations in the hydration shell and the presence of a small proportion of larger aggregates in solution rather than substantial differences in the primary particle size (Das and Mandal, 2020).

X-ray diffraction (XRD) revealed the crystalline nature of silver nanoparticles. The peaks detected at 2θ degrees of 38.24°, 44.39°, 64.51° and 77.44° are assigned to (111), (200), (220) and (311) lattice planes of metallic silver in the face-centred cubic form, respectively (Ali Md. H. et al., 2023). These diffraction peaks exhibit a strong correlation with the standard diffraction pattern of silver retrieved from the JCPDS file no. 04-0783, indicating the successful formation of crystalline silver nanoparticles (Vijayan et al., 2014; Niu et al., 2025). The highest intensity was obtained for the (111) plane among the observed peaks, suggesting that the nanoparticles preferred an orientation along this plane (Das et al., 2017). Similar diffraction patterns have been largely documented for biogenic silver nanoparticles prepared from the extracts of both plants and microbial extracts (Ahmed et al., 2020; Das et al., 2021; Hossain et al., 2023; Al-asbahi et al., 2024). The SAED pattern displayed clear concentric diffraction rings corresponding to the crystalline planes, (111), (200), (220) and (311), therefore supporting the crystallinity of nanoparticles (Ghodake et al., 2020). The ring-like diffraction pattern is a feature of polycrystalline nanoparticles, supporting the conclusion from XRD analysis. The sharp diffraction ring related to the (111) plane validates the favourable orientation of the nanoparticles along this crystallographic plane (Maiti et al., 2014; Gevorgyan et al., 2022).

The synthesised AgNPs exhibited strong antimicrobial activity against test organisms, including AmpC-producing E. coli. The AmpC-producing E. coli strain displayed resistance to multiple antibiotics, which include cefotaxime, amikacin, gentamicin, and ciprofloxacin. The observed antibacterial efficacy of AgNPs against this multidrug-resistant strain may be attributed to the unique mode of action, such as disruption of bacterial cell membrane integrity, generation of reactive oxygen species, interaction with thiol-containing proteins, and interference with DNA replication (Rai et al., 2009; Durán et al., 2016). While β-lactam antibiotics are degraded by the enzymes AmpC β-lactamases, the antimicrobial activity of AgNPs may be due to damage in cell membrane integrity, generating reactive oxygen species, interacting with thiol groups on proteins, and interfering with DNA replication (Rai et al., 2009; Moirangthem et al., 2026).

Interestingly, differential MIC values observed among test organisms may be attributed to variations in cell wall structure and permeability between Gram-positive and Gram-negative bacteria. Gram-negative bacteria have a different outer membrane structure with lipopolysaccharides, which can affect the penetration of the nanoparticles, while the thick peptidoglycan layer of Gram-positive bacteria may also affect the susceptibility (Morones et al., 2005). It is also recorded that the MIC of the AmpC-producing isolate has a higher MIC compared to the susceptible E. coli strain, which also suggests that there might be other mechanisms, such as efflux pump or reduced outer membrane permeability, that might have limited the entry of the nanoparticle (Blair et al., 2015). AgNPs are known to have antibacterial effect by multiple non-specific mechanism like reactive oxygen generation, membrane disruption, release of Ag +, enzyme deactivation and disruption of cellular respiration. The significant antibacterial activity against AmpC-producing E. coli suggests that biosynthesised AgNPs may be used as alternatives or adjuncts to conventional antibiotics, especially to resistant strains. Although the precise mechanism underlying their antibacterial action needs to be studied.

The biosynthesised silver nanoparticles exhibited highly dose-dependent antioxidant activity in the DPPH and ABTS radical scavenging assays (Bedlovičová et al., 2020). These antioxidant properties mainly result from the presence of phytochemical functional groups capable of reducing metal ions, which then remain adsorbed on the surface of nanoparticles as stabilising redox-active moieties (Mhlongo et al., 2025).

The DPPH assay, which is a common model for the evaluation of free radical scavenging reaction because the DPPH radical has high stability together with its strong absorbance at 517 nm, allows for rapid assessment of the hydrogen or electron-donating capacity of antioxidants (Bhakya et al., 2016; Gulcin and Alwasel, 2023). In this study, a concentration-dependent scavenging activity was observed in all samples throughout the assay range (10–30 μg/mL). Ascorbic acid displayed the highest activity, as expected due to its recognised antioxidant power (Bala et al., 2011). The radical quenching capability of silver nanoparticles was significant, and at elevated concentrations approached that of the standard, while the bacterial extract was comparatively less effective. The comparison of the crude extract and silver nanoparticles revealed a much higher amount of redox activity due to their nanoscale implementation, which brings out more surface area with better interaction with free radicals (Bedlovičová et al., 2020). There have been previous reports on similar amplifications of DPPH scavenging activity by the green-synthesised silver nanoparticles (Dipankar and Murugan, 2012; Knurek et al., 2019). Analysis with polynomial regression demonstrated great fitting of a model (R 2 = 0.991–0.999), corroborating the high dose–response relationship between concentration and radical scavenging activity. The IC 50 values provide additional evidence for the activity pattern, as a smaller IC 50 correlates with greater antioxidant power (Jumina et al., 2019). Among the assayed samples, the standard showed the least IC 50, followed by silver nanoparticles and bacterial extract, reiterating the efficiency enhancement of the silver nanoparticles in comparison to bacterial extract.

The strong antioxidant capacity of the synthesised nanoparticles was also shown with respect to the ABTS radical cation decolourisation assay. The formation of the ABTS + chromophore from this oxidation reaction is due to the hydrogen-donating and chain-breaking antioxidant capacity of the test samples (Moteriya et al., 2017; Das D. et al., 2019). All samples showed concentration-dependent inhibition from 10 to 100 μg/mL. Although the reference standard (BTH) exhibited maximum activity, silver nanoparticles showed significant radical scavenging efficiency with virtually equimolar maximal inhibition at higher concentrations. While the bacterial extract had some lower activity at minimal concentrations, its inhibition followed a statistically significant increase and approached maximal levels at high concentrations. Similar strong ABTS + scavenging activity of green-synthesised silver nanoparticles has previously been described (Shanmugam et al., 2016). The IC 50 analysis confirmed these observations, with the standard exhibiting a lower IC 50 followed by silver nanoparticles and bacterial extract. A comparatively lower IC 50 value for nanosilver than crude extract suggests better antioxidant activity following green nanoparticle synthesis due to the synergistic action of metallic core and surface-adsorbed phytochemicals (Das D. et al., 2019).

The study shows an effective green synthesis of silver nanoparticles using B. tropicus isolated from the gut microbiota of C. punctata. Visual observation, UV–Vis spectroscopy, electron microscopy, energy dispersive X-ray, X-ray diffraction and selected area electron diffraction analyses confirmed the formation of stable, predominantly spherical crystalline AgNPs (16.56 ± 4.6 nm) with biomolecule-mediated capping and moderate polydispersity in the range of 10–30 nm. The study demonstrates the synthesis of silver-core nanostructures at the nanoscale that exhibited broad-spectrum antibacterial activity, including efficacy against AmpC-producing E. coli. The study demonstrates the potential of synthesised nanoparticles in combating antimicrobial resistance through multimodal mechanisms such as membrane disruption and oxidative stress induction. Moreover, the dose-dependent antioxidant activity in DPPH and ABTS assays indicates the increased redox potential through nanoscale surface effects along with biofunctional coating.

These findings indicate that biogenic AgNPs synthesised using bacterial metabolites may serve as promising candidates for applications in antimicrobial formulations, wound management, surface disinfection, and biofilm control. However, the present study is limited to physicochemical characterisation and in vitro biological assessments. Comprehensive investigations on cytotoxicity, biocompatibility, biodistribution, bioavailability, and long-term environmental and biological safety were not conducted. Therefore, further in vitro and in vivo studies are necessary to evaluate the safety profile, therapeutic efficacy, and translational potential of these nanoparticles before their application in biomedical and clinical settings. Such studies will be essential for establishing the feasibility of utilising these biogenic AgNPs in nanomedicine and infection-control strategies.