This study was approved by the Ethical Clearance Committee of the Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Indonesia (Ethics number: 97-KKE/2025). All procedures were conducted in accordance with institutional ethical guidelines for microbiological research and environmental sampling. The collection, transportation, and handling of dairy farm wastewater samples followed established institutional biosafety protocols and relevant national regulations in Indonesia.

The study was conducted from June to August 2025 at the Veterinary Public Health Laboratory, Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Indonesia.

A cross-sectional study was conducted to evaluate the antimicrobial effectiveness of AgNPs synthesized using PLA against antibiotic-resistant E. coli isolates obtained from dairy cow waste in the livestock area of Grati District, Pasuruan Regency, East Java.

Wastewater samples (5 mL per farm) were collected from 50 smallholder dairy farms housing approximately 2–10 dairy cows each. Samples were obtained directly from drainage channels or wastewater storage tanks using sterile pipettes. To minimize external contamination, sampling was performed at a depth of 5–10 cm below the wastewater surface under aseptic conditions. Each sample was labeled with farm identity, date, time, and environmental conditions. All samples (n = 50) were transported to the laboratory in insulated containers with ice packs maintained at 4°C and processed within 2 h of collection.

AgNPs were synthesized from silver metal plates, 5 × 10 × 20 mm³, 99.9% purity (Sigma-Aldrich, St. Louis, MO, USA), using polyvinylpyrrolidone (PVP) (Merck KGaA, Darmstadt, Germany) as the liquid medium. An Nd:YAG laser, 1064 nm wavelength, 7 ns pulse width, 20 Hz (Polaris II, New Wave Research, Fremont, CA, USA) was used as the radiation source, with laser parameters controlled using LaserExec II software (New Wave Research, Fremont, CA, USA). The laser energy was set at 30 mJ with a repetition rate of 10 Hz. Characterization was performed using a ultraviolet–visible (UV–Vis) spectrophotometer (Shimadzu Corporation, Kyoto, Japan), transmission electron microscopy (TEM; JEOL Ltd, Tokyo, Japan) coupled with energy-dispersive X-ray spectro-scopy, and Fourier transform infrared spectroscopy (FTIR) (Shimadzu Corporation, Kyoto, Japan) [19].

During synthesis, the laser beam was directed by a silver mirror and focused through a quartz lens (30 mm focal length) onto the silver target immersed in a liquid medium in a Petri dish for 11 h. The solution color gradually changed from transparent to light yellow and then to brownish yellow with increasing laser exposure. TEM, particle size analysis, UV–Vis spectrophotometer, and FTIR spectroscopy were used to assess morphology, particle size distribution, optical plasmon resonance, and surface chemistry [19]. The AgNP suspension was stored in sealed amber glass bottles at 4°C–8°C to maintain stability and prevent aggregation. Only a single batch was used in this study. The freshly synthesized AgNP colloid was diluted in sterile 0.9% NaCl to obtain an initial stock concentration of 1000 ppm.

Wastewater samples (1 mL) were inoculated into 9 mL of buffered peptone water (Himedia, Mumbai, India) and incubated at 37°C for 18–24 h for pre-enrichment. Subsequently, 0.1 mL of enriched culture was streaked onto MacConkey agar (Oxoid, Basingstoke, UK) and incubated at 37°C for 18–24 h under aerobic conditions. Lactose-fermenting colonies exhibiting pink to red coloration, smooth surface, round shape, and convex edges were selected for further analysis. Presumptive E. coli isolates were confirmed using morphological examination, Gram staining, and standard biochemical tests (Indole, methyl red, Voges–Proskauer, citrate) [20, 21].

Confirmed E. coli isolates were subjected to AST using the Kirby–Bauer disk diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI) M100, 34th edition standards [22]. Four antibiotic disks (Oxoid) were used for screening: penicillin (10 IU/disk), streptomycin (10 µg/disk), erythromycin (15 µg/disk), and tetracycline (30 µg/disk). Resistance breakpoints were defined as ≤11 mm for streptomycin and tetracycline, and ≤13 mm for penicillin, according to the CLSI 2024 guidelines [22]. As E. coli is not routinely tested for erythromycin under CLSI standards, a general resistance cutoff of ≤13 mm was applied. A total of 20 antibiotic-resistant E. coli isolates (five per antibiotic category) were selected for MIC and MBC assays [22, 23]. E. coli ATCC 25922 (Oxoid) was used as the quality control strain.

Bacterial inocula were prepared by culturing selected E. coli isolates in Mueller–Hinton broth (MHB) (Oxoid, Basingstoke, UK) at 37°C for 18 h with agitation at 150 rpm until an OD₆₀₀ of 0.5–0.6 McFarland standard was achieved, corresponding to approximately 1.5 × 10⁸ CFU/mL. MIC determination was performed using the broth microdilution method according to CLSI M07-A8 guidelines [24]. Serial twofold dilutions of AgNPs (100–0.195 ppm) were prepared in 96-well microplates (OneMed, Sidoarjo, Indonesia) containing MHB inoculated with standardized bacterial suspensions (10⁸ CFU/mL). Plates were incubated at 37°C for 24 h. The MIC endpoint was defined as the lowest AgNP concentration showing no visible bacterial growth, as assessed by visual turbidity before and after incubation. Negative and positive controls were included, and all assays were performed in duplicate.

MBC determination was conducted using the conventional agar diffusion method. Aliquots (10 µL) from MIC wells without visible growth were spread onto Mueller–Hinton agar (Himedia) plates and incubated at 37°C for 24 h. The MBC endpoint was defined as the lowest AgNP concentration resulting in the complete absence of bacterial growth on agar plates, indicating total bacterial killing [25]. All tests were performed in duplicate.

MIC and MBC values were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test to identify differences among antibiotic resistance groups. Statistical significance was set at p < 0.05. All analyses were performed using IBM SPSS Statistics version 29 (IBM Corp., Armonk, NY, USA).

AgNPs were successfully synthesized using PLA in PVP media and demonstrated excellent physicochemical characteristics. TEM analysis of 247 particles showed an average particle diameter of 11.62 ± 1.8 nm, with a coefficient of variation of 15.5%, indicating a narrow and monodisperse size distribution. UV–Vis spectrophoto-meter revealed a distinct surface plasmon resonance peak at 418 nm with an absorbance intensity of 0.786 AU, confirming the formation of metallic silver without detectable silver oxide contamination. FTIR analysis verified the presence of PVP K30 as a capping and stabilizing agent on the AgNP surface, characterized by a strong peak at 1680 cm^-1 (C = O stretching) and additional peaks at 2950, 1550, and 1250 cm^-m, indicating an intact PVP backbone without degradation. The estimated coating thickness of approximately 2.6 nm contributed to colloidal stability. Overall, the synthesized AgNPs exhibited high purity, uniform morphology, and stability, making them suitable for subsequent antimicrobial evaluation (MIC/MBC) against antibiotic-resistant E. coli isolates from dairy cow waste.

The antimicrobial activity of AgNPs was evaluated against antibiotic-resistant E. coli isolates recovered from dairy cattle waste. The isolates comprised streptomycin-resistant (S1–S5), erythromycin-resistant (E1–E5), penicillin-resistant (P1–P5), and tetracycline-resistant (T1–T5) groups.

Figure 1 presents the MIC results determined using the standard broth microdilution method. Following MIC determination, MBC assessment was performed by subculturing wells without visible growth onto Mueller–Hinton agar. As illustrated in Figure 2, AgNPs exerted a bactericidal effect at 100 ppm against isolate P1, whereas isolate P2 continued to grow at the same concentration.

Results of the minimum inhibitory concen-tration assay of silver nanoparticles against antibiotic-resistant Escherichia coli were determined using the standard broth microdilution method. K− indicates the negative control with no bacterial growth (clear wells), whereas K+ indicates the positive control with bacterial growth (turbid wells).

Results of the minimum bactericidal concentration assay of silver nanoparticles against antibiotic-resistant Escherichia coli determined using the conventional agar diffusion method on Mueller–Hinton agar. P denotes penicillin-resistant isolates.

The MIC and MBC values for all antibiotic-resistant E. coli isolates tested against AgNPs are summarized in Table 1. MIC values ranged from 37.5 to 100 ppm across all isolates. In contrast, MBC values were generally higher, with most isolates exhibiting MBC values exceeding 100 ppm, the highest concentration tested in this study. These findings indicate that AgNPs exerted a strong inhibitory (bacteriostatic) effect but comparatively weaker bactericidal activity against antibiotic-resistant E. coli isolated from dairy cattle waste.

After 24 h of incubation, streptomycin-resistant E. coli required the highest AgNP concentrations for growth inhibition, with MIC values ranging from 62.5 to 100 ppm. In contrast, erythromycin-resistant E. coli showed the lowest MIC values among the resistance groups, ranging from 37.5 to 50 ppm. Across all isolates, MBC values exceeded corresponding MIC values and ranged between 100 and >100 ppm. Overall, 75% (15/20) of the isolates exhibited MBC values >100 ppm. Only five isolates showed an MBC of 100 ppm, comprising three erythromycin-resistant and two penicillin-resistant E. coli isolates. Due to the predominance of MBC values >100 ppm, further MBC interpretation was limited to descriptive analysis.

One-way ANOVA followed by Tukey’s multiple comparison test revealed no statistically significant differences in mean MIC values among the antibiotic resistance groups (F (3,36) = 2.20; p = 0.105). Post hoc Tukey’s Honestly Significant Differences analysis confirmed that none of the pairwise comparisons reached statistical significance (p > 0.05). Descriptively, streptomycin-resistant isolates exhibited the highest mean MIC values (75.0 ± 33.3 ppm), whereas erythromycin-resistant isolates showed the lowest mean MIC values with the narrowest variability (45.0 ± 10.5 ppm). These data are presented in Table 2.

The lowest MIC value observed was 37.5 ppm, which inhibited erythromycin-resistant E. coli isolates (E2 and E5), one penicillin-resistant isolate (P2), and one tetracycline-resistant isolate (T3). At this concentration, streptomycin-resistant E. coli isolates were not inhibited (Figure 3). Growth inhibition of streptomycin-resistant isolates was first observed at an MIC of 62.5 ppm. Therefore, 62.5 ppm was identified as the most effective concentration for consistently inhibiting the growth of antibiotic-resistant E. coli isolated from dairy cattle waste across resistance profiles.

Minimum inhibitory concentration of silver nanoparticles against antibiotic-resistant Escherichia coli. Data bars represent the mean of duplicate measurements for each isolate. S = Streptomycin-resistant, E = Erythromycin-resistant, P = Penicillin-resistant, T = Tetracycline-resistant.

AgNPs have been extensively investigated as alternative antimicrobial agents against ARB due to their broad-spectrum activity. In the present study, the antimicrobial efficacy of AgNPs was evaluated against E. coli isolates resistant to streptomycin, erythromycin, penicillin, and tetracycline recovered from dairy cow waste. The observed MIC values ranged from 37.5 to 100 ppm, indicating effective growth inhibition within this concentration range. A serial twofold dilution assay (100–0.195 ppm) using a standardized bacterial inoculum of 10⁸ CFU/mL was applied, consistent with commonly adopted nanomaterial antimicrobial testing protocols, thereby ensuring data reliability and comparability with previous studies [26, 27].

The lowest MIC values were recorded for erythromycin-resistant E. coli (37.5–50 ppm), whereas streptomycin-resistant isolates required higher AgNP concentrations (62.5–100 ppm) for growth inhibition [28]. These differences likely reflect variability in resistance mechanisms, including efflux pump activity or antibiotic target modifications, which may also influence nanoparticle–cell interactions [27]. Streptomycin-resistant E. coli may possess enhanced protective mechanisms against metal nanoparticles, such as altered membrane permeability or more efficient efflux systems, reducing nanoparticle penetration and intracellular accumulation [29, 30].

One-way ANOVA followed by Tukey’s multiple comparison test showed no statistically significant differences in MIC values among the antibiotic resistance groups, indicating that variations in AgNP concentration within the tested range did not significantly alter overall inhibitory efficacy. This suggests a relatively narrow effective concentration window for AgNP-mediated growth inhibition, within which concentration-dependent differences are not statistically distinguishable [31]. Streptomycin-resistant E. coli required an MIC of 62.5 ppm for inhibition, whereas erythromycin-, penicillin-, and tetracycline-resistant isolates were inhibited at concentrations below 62.5 ppm. Accordingly, 62.5 ppm was identified as the most effective concentration for consistent inhibition of antibiotic-resistant E. coli isolated from dairy cattle waste.

MBC values exceeded MIC values for most isolates and frequently surpassed the maximum tested concentration of 100 ppm, indicating limited bactericidal activity under the conditions tested. These findings support the notion that AgNPs primarily exert a bacteriostatic effect against these isolates rather than direct bactericidal action [32]. Similar observations have been reported previously, where bacterial survival and metabolic activity persisted under sublethal AgNP exposure, suggesting that higher concentrations or combination strategies may be required to achieve complete bacterial killing [33]. The antimicrobial mechanisms of AgNPs, including membrane disruption, Reactive oxygen species (ROS) generation, and interactions with proteins and DNA, contribute to growth inhibition and, at higher doses, cell death [34].

Bacterial biofilm formation and antibiotic resistance profiles play a critical role in determining sensitivity to AgNPs. Biofilm-producing bacteria exhibit increased resistance due to the extracellular matrix acting as a physical barrier that limits nanoparticle penetration, along with adaptive responses such as upregulation of efflux pump–related genes associated with metal resistance [35, 36]. These adaptations may promote cross-resistance to both antibiotics and AgNPs, highlighting the need for combination therapies or biofilm-targeted approaches [37, 38]. The substantial variability in MIC values observed within resistance groups can be attributed to multiple interacting factors, including heterogeneous resistance mechanisms, media-induced changes in AgNP stability and Ag⁺ ion availability [39], intrinsic heteroresistance within bacterial populations [40], and differences in biofilm-forming capacity among isolates [41].

AgNPs exert antimicrobial activity through ROS generation, membrane damage, increased permeability, and interference with bacterial proteins and DNA. Because these mechanisms differ from the specific targets of conventional antibiotics, AgNPs generally remain effective against ARB and present a lower risk of resistance development [26, 42]. Nevertheless, adaptive resistance may emerge through mutations affecting efflux systems, membrane proteins, and stress response pathways following prolonged AgNP exposure [43, 44].

The relatively high MIC values observed in this study may be partly explained by AgNP aggregation within the PVP matrix. Although PVP enhances colloidal stability, it may reduce Ag⁺ ion release and diminish antimicrobial efficacy. Under biorelevant conditions, AgNPs may aggregate to the micron scale, substantially reducing biological activity by decreasing surface area and particle–cell contact [45]. Previous studies have demonstrated that aggregation can alter MIC values by up to two orders of magnitude [46]. The MIC range observed here (37.5–100 ppm) is consistent with this phenomenon, as non-aggregated AgNPs of similar size typically exhibit MICs in the μg/mL range. Reduced Ag⁺ bioavailability, limited particle–bacteria interactions, and heterogeneous surface properties likely contributed to the predominance of MBC values >100 ppm and the identification of 62.5 ppm as the optimal inhibitory concentration [45–47].

The effectiveness of AgNPs compared with conventional antibiotics highlights their potential as alternative antimicrobial agents against antibiotic-resistant E. coli. However, these findings also underscore the need for improved nanoparticle formulations or synergistic combinations with antibiotics to enhance bactericidal activity [28, 48]. Strategies to overcome aggregation include replacing PVP with alternative stabilizers such as dextran or plant-derived compounds, or applying surface coatings such as polydopamine or mesoporous silica to improve Ag⁺ release and antimicrobial performance [41, 49]. Such approaches have been shown to reduce MIC values to more competitive ranges (5–20 ppm) [39].

In this study, AgNPs effectively inhibited the growth of 20 antibiotic-resistant E. coli isolates from dairy farm waste, with MIC values ranging from 37.5 to 100 ppm. Erythromycin-resistant isolates exhibited the highest sensitivity, whereas streptomycin-resistant isolates required higher concentrations, reflecting heterogeneous resistance patterns. These MIC values are comparable to or lower than those reported in several published studies [50, 51]. For example, Trzcińska-Wencel et al. [50] reported MIC values of 16–64 μg/mL against E. coli ATCC with MBCs of 32–512 μg/mL, while Tufail et al. [51] reported MICs of 3.3–3.6 μg/mL for biogenic AgNPs. Rana et al. [52] reported an MIC of 1 mg/mL against E. coli MTCC 1698. The MIC values observed here (0.0375–0.1 mg/mL) are particularly noteworthy given that they were obtained using field-derived antibiotic-resistant isolates rather than more susceptible reference strains.

Evaluating E. coli isolates from dairy cow waste has direct relevance to veterinary medicine and livestock waste management. The application of antimicrobial agents such as AgNPs in waste treatment systems may reduce environmental dissemination of resistant bacteria and limit exposure risks to animals and humans. However, further research is required to optimize dosing strategies while considering long-term environmental safety and potential nanoparticle residues in animal products [53, 54].

This study aligns strongly with the One Health framework, which integrates human, animal, and environmental health. Dairy cattle waste represents a significant reservoir of ARB capable of transmission through multiple environmental pathways [55, 56]. Inadequately treated agricultural waste can contaminate soil, surface water, and drinking water sources, posing sustained public health risks [57, 58]. The demonstrated antimicrobial activity of AgNPs against antibiotic-resistant E. coli supports the role of nanotechnology in strengthening One Health–based AMR mitigation strategies, particularly by reducing pathogen spillover from livestock systems to the wider human population [59]. By identifying 62.5 ppm as an effective inhibitory concentration across resistance profiles, this study offers a scalable technological option for integration into modern livestock waste management and cross-sectoral AMR risk reduction programs [60].