Open Veterinary Journal, (2026), Vol. 16(4): -2061

Original Article

10.5455/OVJ.2026.v16.i4.10

MIC and MBC of silver nanoparticles in antibiotic-resistant Salmonella sp. from dairy cattle waste

Sheila Marty Yanestria¹, Mustofa Helmi Effendi^2,3*, Freshinta Jellia Wibisono¹, Dyah Widhowati⁴, Marek Yohana Kurniabudhi⁵, Junianto Wika Adi Pratama⁵, Aswin Rafif Khairullah⁶, John Yew Huat Tang³, Dea Anita Ariani Kurniasih⁷, Bima Putra Pratama⁸, Riza Zainuddin Ahmad⁶, Saifur Rehman⁹ and Soebagio Soebagio¹⁰

¹Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya, Indonesia

²Division of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

³School of Food Industry, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin (Besut Campus), Besut, Terengganu, Malaysia

⁴Department of Microbiology, Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya, Indonesia

⁵Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya, Indonesia

⁶Research Center for Veterinary Science, National Research and Innovation Agency (BRIN), Bogor, Indonesia

⁷Research Center for Public Health and Nutrition, National Research and Innovation Agency (BRIN), Bogor, Indonesia

⁸Research Center for Process Technology, National Research and Innovation Agency (BRIN), South Tangerang, Indonesia

⁹Department of Pathobiology, Faculty of Veterinary and Animal Sciences, Gomal University, Dera Ismail Khan, Pakistan

¹⁰Faculty of Engineering, Universitas Wijaya Kusuma Surabaya, Surabaya, Indonesia

*Corresponding Author: Mustofa Helmi Effendi. Division of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia. Email: mhelmieffendi [at] gmail.com

Submitted: 01/12/2025 Revised: 22/02/2026 Accepted: 03/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Salmonella sp. is an important pathogen often found in dairy farm waste and poses health risks to humans and the environment. The increasing resistance of Salmonella sp. to various antibiotics due to uncontrolled use of antibiotics in the livestock industry is a serious infection control challenge.

Aim: This study aimed to evaluate the antimicrobial activity of Silver Nanoparticles (AgNPs) against antibiotic-resistant Salmonella sp isolates originating from dairy farm waste.

Methods: Silver nanoparticles with a concentration of 100 ppm (≈100 µg·ml⁻¹) were synthesized using the Nd:YAG laser method with polyvinylpyrrolidone (PVP) as a stabilizer and tested against five Salmonella sp. isolates resistant to penicillin, streptomycin, erythromycin, tetracycline, and oxytetracycline. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests were performed using the microdilution method.

Results: The colloidal AgNPs inhibited visible growth of all tested Salmonella sp. isolates at 100 ppm, with the penicillin-resistant isolate having the lowest MIC value (50 ppm). However, bactericidal activity was observed only for the penicillin-resistant isolate at 100 ppm; MBCs for other isolates exceeded 100 ppm. These findings indicate that AgNP is effective as a bacteriostatic agent against antibiotic-resistant Salmonella sp., but its bactericidal activity is not optimal.

Conclusion: Factors, such as PVP stabilization and low silver ion (Ag⁺) release, likely limit the ability to kill bacteria. Therefore, the optimization of AgNP formulations to increase Ag⁺ release and penetration into bacterial membranes, as well as further studies on environmental safety, are needed before their widespread application in livestock waste treatment.

Keywords: Antibiotics, Public health, Salmonella sp., Silver nanoparticles, Waste.

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Introduction

Salmonella sp. is a major pathogen that is frequently detected in dairy farm waste and can cause human disease through contact with the food chain and the environment (Ayuti et al., 2024). The presence of Salmonella sp. in liquid and solid waste threatens public health and the ecosystem. This contamination can also affect the water and soil quality around farm areas (Pham-Duc et al., 2020). Salmonella sp. in farm waste can be a source of infection for humans and animals (Black et al., 2021). Several studies have proven the presence of Salmonella sp. in dairy farm waste. A study in Nigeria reported the presence of Salmonella sp. in waste and dairy products from dairy farms with a prevalence of approximately 37% (Owhe-Ureghe et al., 2022). A study in Algeria found that the prevalence of Salmonella sp. was approximately 31.11% from samples taken from various sources on farms, including waste (Ghougal et al., 2021). A study in South Africa reported the prevalence of Salmonella sp. in dairy farm waste in the range of 30%–40%, which is related to sanitation conditions and farm waste management (Manyi-Loh and Lues, 2023).

The uncontrolled use of antibiotics in dairy farming has led to the emergence of Salmonella sp. resistant to various antibiotics (Sobur et al., 2019). These resistant bacteria can spread through farm waste and contaminate the surrounding environment, increasing the risk of difficult-to-treat infections. This resistance can also spread through the transfer of resistance genes to other microorganisms in the environment, which has negative implications for the control of infectious diseases (Yanestria et al., 2022). Antibiotic resistance in farm waste poses a serious threat to global health and the environment (He et al., 2020).

Conventional waste treatment methods, such as composting and anaerobic digestion, have not completely eliminated resistant bacteria from dairy farm waste (Wallace et al., 2018). Several studies have found that resistant bacteria are still detected after treatment, indicating the need for more effective and innovative waste treatment technologies (Bairán et al., 2020; Meradji et al., 2025). The use of additives in the treatment process is needed to improve the reduction of resistant bacteria in livestock waste (Enami et al., 2024).

Silver nanoparticles have potent antibacterial activity against Salmonella sp., including antibiotic-resistant strains commonly found in livestock environments (Arshad et al., 2022). A study showed that biosynthetically synthesized silver nanoparticles effectively inhibited multidrug-resistant Salmonella enteritidis and Salmonella typhimurium isolated from poultry, with a minimum inhibitory concentration (MIC) as low as 5 µg/ml and a minimum bactericidal concentration (MBC) of 6–8 µg/ml, confirming their efficacy against resistant strains (Elez et al., 2021; Mohammed, 2022).

Studies using various biosynthetic methods have confirmed that silver nanoparticles can disrupt the cell membrane of Salmonella sp., causing cytoplasmic leakage and inducing cell death, underscoring their potent bactericidal properties (Saqib et al., 2022; El Rabey et al., 2024). Silver nanoparticles were synthesized using an Nd:YAG laser because this method produces particles with a narrow size distribution, high purity, and no harmful chemical residues, making it safer for environmental applications than conventional chemical methods. Polyvinylpyrrolidone (PVP) was chosen as a stabilizer to prevent agglomeration and maintain colloidal dispersibility, although PVP attached to the particle surface can inhibit the release of silver ions (Ag⁺), which are the main antimicrobial agents (Ferreira et al., 2023). This study aimed to evaluate the antimicrobial activity of AgNPs against antibiotic-resistant Salmonella sp isolates originating from dairy farm waste. These findings collectively support the integration of silver nanoparticles as a promising solution to combat antibiotic-resistant Salmonella sp. in dairy cow wastewater, potentially improving wastewater treatment effectiveness and reducing the risk of environmental pollution.

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Materials and Methods

Research design

This study was conducted in July 2025. Silver nanoparticles were tested on Salmonella sp isolates from dairy cattle waste that were resistant to the antibiotics penicillin, streptomycin, erythromycin, tetracycline, and oxytetracycline, each amounting to 1 isolate. The antibiotic-resistant Salmonella sp. isolates were obtained from the Veterinary Public Health Laboratory, Faculty of Veterinary Medicine, Wijaya Kusuma University Surabaya, Indonesia. This isolate was tested through isolation and identification stages (morphological test, Gram staining, IMViC, and urease biochemistry) to detect Salmonella sp.

Dairy cow waste was collected from the livestock area of Grati District, Pasuruan Regency, East Java. The wastewater was collected directly from the drainage channels or wastewater storage tanks of each farm using sterile pipettes. To minimize external contamination, samples were taken at a depth of 5–10 cm below the wastewater surface using aseptic techniques. Each sample was labeled with the farm’s identity, date, time, and environmental conditions. All samples were transported to the laboratory in a cooler containing ice packs maintained at 4°C for a maximum of 2 hours after sampling.

Silver nanoparticle formulation

Colloidal AgNPs were produced using a silver metal plate with a purity of 99.9% and dimensions of 5 × 10 × 20 mm³. PVP was the liquid medium used to make colloidal AgNPs. An Nd:YAG laser with a wavelength of 1,064 nm and a pulse width of 7 ns was used as the radiation source. The laser energy was set at 30 mJ with a repetition rate of 10 Hz using the LaserExec II software. A transmission electron microscope was used in this study. Transmission Electron Microscopy (TEM, JEOL) Equipped with Energy Dispersive X-ray Spectroscopy and an ultraviolet-visible (UV-Vis) absorption spectrometer (Shimadzu 1240 SA). In this experiment, a silver mirror was used to direct the pulsed laser beam, and a quartz lens with a focal length of 30 mm was used to focus the beam on a silver metal plate in a petri dish containing a liquid medium for 11 hours. The color of the liquid medium changed from transparent to light yellow before finally becoming brownish yellow (Avicenna et al., 2021).

Salmonella sp. culture medium and cultivation

Ampoules of antibiotic-resistant Salmonella sp. were revived by inoculating them into 10 ml of tetrationate broth and incubating them at 37°C for 24 hours. The surface of the selective agar medium, Xylose Lysine Deoxycholate (XLD), was streaked with a loop containing the bacterial suspension and then incubated for 24 hours at 37°C (Mathew et al., 2023).

Minimum inhibitory concentration

Bacterial inoculum preparation was carried out by culturing Salmonella sp isolates in Mueller–Hinton Broth (MHB) at 37°C for 18 hours with 150 rpm agitation until an optical density (OD600) of 0.5–0.6 McFarland standard was achieved, resulting in a bacterial concentration of approximately 1.5 × 10⁸ CFU/ml.

The standard broth dilution method (CLSI M07-A8) was used to determine the MIC to assess the antimicrobial performance of silver nanoparticles. This was done by assessing the visible growth of microorganisms in MHB. The microdilution process was performed in 96-well microplates. Silver nanoparticles were serially diluted twofold at concentrations from 100 to 0.19 ppm to measure the MIC in MHB. This was done by adding adjusted bacterial concentrations (10⁸ CFU/ml, McFarland standard 0.5). The control consisted of only the inoculated broth and was incubated for 24 hours at 37°C for 24 hours. The MIC endpoint was the lowest silver nanoparticle concentration at which bacterial growth was not visible. To confirm the MIC value, visual turbidity was recorded both before and after incubation (Rodríguez-Melcón et al., 2021). This test was performed in duplicate.

Minimum bactericidal concentration

Following the MIC of the silver nanoparticles, 10 µl aliquots from each well that did not exhibit any discernible bacterial growth were seeded onto Mueller–Hinton Agar plates and cultured for 24 hours at 37°C. The MBC endpoint was reached when 99.9% of the bacterial population was eliminated at the lowest antimicrobial agent concentration. The researchers checked for the presence or absence of bacteria on agar plates before and after incubation (Parvekar et al., 2020). This test was performed in duplicate.

Ethical approval

The Ethical Clearance Committee of the Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Indonesia, approved this research (Ethics number: 108-KKE/2025). This study was conducted in accordance with institutional guidelines and ethical standards for research involving microbiological work and environmental sampling. All procedures involving the collection, transportation, and handling of environmental samples from the dairy farm wastewater were performed following the biosafety protocols established by the institution and the relevant national regulations in Indonesia.

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Results

AgNPs were successfully synthesized using PLA in PVP media and showed excellent characterization with an average diameter of 11.62 ± 1.8 nm measured by TEM from 247 particles analyzed with a coefficient of variation of 15.5%. UV-Visible Spectroscopy analysis showed a surface plasmon resonance peak at 418 nm with an absorbance intensity of 0.786 AU, confirming the formation of pure silver without silver oxide contamination. Fourier Transform Infrared Spectroscopy analysis confirmed the presence of (PVP K30) as a capping agent on the AgNP surface with a strong peak at 1,680 cm⁻¹ (C=O stretching), peaks at 2,950, 1,550, and 1,250 cm⁻¹ indicating intact PVP backbone without degradation, and an estimated coating thickness of ~2.6 nm, which functions as a stabilizing layer. These silver nanoparticles with an average diameter of 11.62 nm showed excellent physicochemical characterization, monodisperse size distribution, high purity, and excellent colloidal stability, and were ready for antimicrobial testing (MIC/MBC) against antibiotic-resistant Salmonella sp. isolates from dairy cow waste.

The results of reviving antibiotic-resistant Salmonella sp. bacteria on the surface of selective agar media, namely XLD, in the form of pink bacterial colonies with a black center and a reddish or slightly transparent zone around the colony. Salmonella sp. did not ferment the lactose and sucrose in the media, so the media did not turn yellow. This can be seen in Figure 1.

Fig. 1. The result of Salmonella sp. cultivation on XLD media.

Furthermore, each isolate of Salmonella sp. resistant to penicillin, streptomycin, erythromycin, tetracycline, and oxytetracycline was tested for MIC with silver nanoparticles using the standard broth microdilution method (Table 1). After 24 hours of incubation under aerobic conditions at 37°C, turbidity was observed in the microplate wells. The MIC test showed that Salmonella sp isolates resistant to penicillin, streptomycin, and erythromycin began to show no turbidity in wells containing 50 ppm silver nanoparticles. Meanwhile, Salmonella sp isolates resistant to tetracycline and oxytetracycline began to show no turbidity in wells containing 100 ppm silver nanoparticles. The most effective inhibitory effect of silver nanoparticles at a concentration of 100 ppm against antibiotic-resistant Salmonella sp. isolated from dairy cow waste was observed, although growth inhibition was already observed in 3 of the 5 isolates at 50 ppm.

Table 1. MIC and turbidity for different concentrations of silver nanoparticles.

Next, an MBC test was performed on clear wells, indicating no bacterial growth. Aliquots from clear wells were planted on Mueller–Hinton agar media to determine whether the bacteria had died. The results of the MBC test are shown in Figure 2 and Table 2. The results of the MBC test showed that most of the tested bacteria showed bacterial growth at a concentration of 100 ppm, the highest concentration tested in this study. Only penicillin-resistant Salmonella sp. did not show bacterial growth at 100 ppm. The bactericidal effect of silver nanoparticles on antibiotic-resistant Salmonella sp isolates isolated from dairy cow waste was less than optimal.

Fig. 2. The result of the MBC method on Mueller Hinton Agar plates showed that silver nanoparticles have an antibacterial effect against penicillin-resistant Salmonella sp. at a concentration of 100 ppm.

Table 2. MBC of silver nanoparticles.

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Discussion

The MIC results showed that silver nanoparticles at a concentration of 100 ppm were the highest effective concentration capable of inhibiting the growth of all Salmonella sp isolates resistant to various antibiotics, such as penicillin, streptomycin, erythromycin, tetracycline, and oxytetracycline. This confirms that silver nanoparticles at this concentration can be used as an alternative for inhibiting the growth of resistant bacteria from dairy cow waste (Motrenko et al., 2025). This is consistent with other reports showing that silver nanoparticles at concentrations of around 20–100 ppm have a significant inhibitory effect on S. enterica bacteria, including those resistant to conventional antibiotics (Abdelsattar et al., 2021). Previous studies have also shown that silver nanoparticles are effective against multidrug-resistant S. enteritidis and S. typhimurium from diarrheal sheep and goats in both in vitro and in vivo experiments (Farouk et al., 2020).

Salmonella sp isolates resistant to penicillin, streptomycin, and erythromycin had the lowest MIC value of 50 ppm, whereas Salmonella sp isolates resistant to tetracycline and oxytetracycline required higher concentrations of silver nanoparticles to inhibit their growth. This phenomenon indicates a significant variation in the sensitivity of isolates and silver nanoparticles. This variation may be caused by differences in the resistance mechanisms of each isolate, which affect their susceptibility to the antimicrobial effects of silver nanoparticles (Khalifa et al., 2025). Penicillin-resistant isolates with low MICs may still be susceptible to the mechanisms of action of silver, such as cell membrane damage and induction of oxidative stress, which are not directly related to beta-lactam resistance. Thus, silver nanoparticles respond better to penicillin-resistant isolates than to other isolates with different resistance mechanisms (Elez et al., 2021; Dias De Emery et al., 2023).

Isolates that require higher concentrations of AgNPs typically have more complex defenses, such as the production of beta-lactamase enzymes capable of breaking down beta-lactam antibiotics, modification of penicillin-binding proteins, and the ability to form biofilms that serve as a physical barrier against nanoparticle penetration (Muteeb et al., 2023). Furthermore, changes in the cell wall or membrane structure can reduce the effectiveness of silver nanoparticle interactions with bacterial targets, requiring higher concentrations (Abdelsattar et al., 2021). Genetic factors between isolates also influence the expression of resistance genes and defense proteins, which impact sensitivity to silver nanoparticles (Mahfouz et al., 2025). The observation that tetracycline- and oxytetracycline-resistant isolates still showed growth at a concentration of 50 ppm but were inhibited at 100 ppm indicates that antibiotic resistance does not always correlate directly with resistance to silver nanoparticles. The mechanism of action of AgNPs is physically and chemically different from that of traditional antibiotics, allowing AgNPs to overcome some of these resistance mechanisms (Nuti et al., 2024).

In MBC measurements, silver nanoparticles were not always able to kill bacteria at a concentration of 100 ppm, except for the penicillin-resistant Salmonella sp. isolate that showed no growth at that concentration. Although silver nanoparticles are effective as growth inhibitors, their bactericidal (bacteria-killing) effect is less than optimal for most of the isolates tested (Khan et al., 2023). This phenomenon was also found in a previous study that reported that although silver nanoparticles are effective in significantly reducing bacterial counts, their full bactericidal ability requires different conditions or formulations (Pal et al., 2007).

This study also demonstrates the importance of silver particle characterization, as the physicochemical properties of nanoparticles, such as size, stabilizer, and synthesis method, significantly influence their antimicrobial activity. The use of PVP as a stabilizer and the Nd:YAG laser method provides the advantages of uniform particle size distribution and stability, which can enhance the interaction of AgNPs with bacterial membranes (El-Tantawy et al., 2023). These physicochemical characteristics enhance the interaction of AgNPs with bacterial membranes, effectively inhibiting the growth of resistant bacteria. However, despite their effectiveness in inhibiting growth, silver nanoparticles are less than optimal in killing resistant bacteria (Xu et al., 2020). This is due to several factors. The Nd:YAG laser method produces nanoparticles with good size control, but the release of Ag⁺ from the particle surface is relatively limited (Hamad and Atiya, 2025). Ag⁺ ions are key agents in the bactericidal mechanism because they can permanently damage bacterial proteins and DNA (More et al., 2023). If this ion release is insufficient, the effectiveness of killing bacteria is also limited.

Furthermore, the presence of a PVP layer as a stabilizer that wraps the nanoparticles can hinder direct contact of Ag⁺ with the bacterial cell membrane, although it increases stability and prevents particle aggregation (Liao et al., 2019a). As a result, deeper penetration and damage within the bacteria are less successful with PVP-coated silver nanoparticles than with unstabilized nanoparticles or those using other stabilizers. Concentration can also be a limiting factor because a higher concentration is usually required to achieve a more optimal killing effect, which in clinical applications must be balanced with the risk of toxicity to host cells and the surrounding environment (Zalewska-Piątek and B, 2023). Therefore, although the use of PVP and the Nd:YAG laser method offers advantages in terms of stability and particle size distribution, these factors together with the biological characteristics of resistant bacteria cause the silver nanoparticles produced by this method to be more effective as growth inhibitors (bacteriostatic) than as killing agents (bactericidal) at the concentrations used (Liao et al., 2019b; Tričković et al., 2023).

The high MIC values in this study may also be due to the possible formation of micro-sized aggregates in the PVP matrix. Although PVP offers colloidal stability, its antibacterial activity is diminished, and Ag+ ion release is decreased. Under biorelevant circumstances, AgNPs tend to agglomerate at the micron scale, which almost eliminates biological activity (Ferreira et al., 2023). Extreme aggregation decreases the specific surface area and interaction contact with the target microorganisms. Aggregation in culture media can alter MIC values by up to two orders of magnitude (Bélteky et al., 2019). The MIC in this study (50–100 ppm) was consistent with the aggregation hypothesis because AgNPs of 11–12 nm without aggregation usually show much lower MICs in micrograms/ml. The low activity is caused by decreased Ag+ ion bioavailability, reduced particle-bacteria contact, and heterogeneous changes in surface properties (Bélteky et al., 2019; Ferreira et al., 2023). This finding, with 80% of isolates showing MBC >100 ppm and optimal effectiveness only at 62.5 ppm, indicates that aggregation limited the dose-response system in this study.

Overall, silver NPs offer a potential solution for combating antibiotic-resistant Salmonella sp bacteria in dairy farm waste. However, the limited bactericidal effectiveness of most isolates indicates the need for further research to improve bactericidal effectiveness, focusing on developing formulations that enhance Ag⁺ release, modifying nanoparticle surfaces for better penetration, and combining them with other antimicrobial agents to more comprehensively address bacterial resistance mechanisms (Mateo and Jiménez, 2022). The safety of silver nanoparticles in the environment and their potential for toxic accumulation require further consideration before large-scale implementation (Ferdous and Nemmar, 2020).

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Conclusion

The results of this study indicate that silver NPs have a good inhibitory effect, especially at an MIC of 100 ppm. However, the bactericidal effect of silver nanoparticles against antibiotic-resistant Salmonella sp. isolated from dairy cow waste was less effective. Although the Nd:YAG laser method and the use of PVP provide advantages in terms of stability and particle size, these factors, together with the bacterial biological defense mechanisms, result in silver nanoparticles having a more dominant bacteriostatic effect than bactericidal effect under current conditions. Further research is recommended to focus on developing formulations that can increase the release of Ag⁺, modifying the surface of nanoparticles for more effective penetration, and combining them with other antimicrobial agents to overcome bacterial resistance more comprehensively. In addition, environmental safety aspects and the potential for toxic accumulation must also be considered before large-scale application.

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Acknowledgments

The authors thank the Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, for providing the necessary facilities for the study.

Conflict of interest

The authors declare no conflict of interest.

Funding

This research was successfully implemented thanks to the "Institute for Research and Community Service of Wijaya Kusuma University Surabaya" and the "Research and Community Service Information Base (BIMA)" Program funded by the Ministry of Higher Education, Science, and Technology (Kemendikdiktisaintek), in accordance with the Announcement of Funding Recipients Number 0070/C3/AL.04/2025 dated May 23, 2025.

Author’s contributions

S.M.Y., S.R., and M.H.E.: Conceived, designed, and coordinated the study. F.J.W., R.Z.A., and D.W.: designed data collection tools, supervised field sample and data collection, and performed laboratory work and data entry. M.Y.K., S.S., and J.W.A.P.: Validation, supervision, and formal analysis were performed. A.R.K. and J.Y.H.T.: Reagents, materials, and analysis tools were contributed. D.A.A.K. and B.P.P.: Carried out the statistical analysis and interpretation and participated in the preparation of the manuscript. All authors have read, reviewed, and approved the final version of the manuscript.

Data availability

All data are available in the revised manuscript.

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