This study revealed that 45.24% (95/210) of the dairy cattle milk samples were positive for mastitis. Of these mastitis samples, 11.58% (11/95) were positive for S. agalactiae and 9.47% (9/95) were positive for K. pneumoniae. Regarding the resistance of these bacteria, there was variation in the responses of K. pneumoniae and S. agalactiae against antibiotics in general (Table 1). For penicillin, tetracycline, and amoxicillin, S. agalactiae had considerably higher zones than K. pneumoniae. On the other hand, K. pneumoniae showed significant differences in zones of inhibition to those of S. agalactiae against imipenem and erythromycin (Figure 1). There were minor variations in antibacterial responses between both bacteria against ceftriaxone. Standard deviation from the mean value of the ZOI was higher for K. pneumoniae against cetirizine (6.14 mm) and imipenem (5.12 mm) than S. agalactiae against ceftriaxone (4.97 mm), penicillin (3.56 mm), and erythromycin (3.24 mm). This reflects the fact that the latter pathogen is much more inclined toward a single susceptibility category, whereas the former exhibits variable responses against antibiotics. The response of S. agalactiae (2.75 ± 0.83 mm) was nearly 10 times lower than that of K. pneumoniae (21.5 ± 2.96 mm) against aztronam.

The FTIR pattern of nanoparticles is shown in Figure 2A. The characteristic peak of the Mg-O bond was observed at a wavelength of 680 nm. This confirms that the metal-oxygen bond was present in the nanoparticles. Surfactant sodium dodecyl sulfate is an organic molecule and contains a C-H bond. Its band was observed at ~1,400–1,500 cm⁻¹ due to the adsorption of surfactant on the surface of the nanoparticles. Water molecules present in the atmosphere are quickly adsorbed on the surface of the nanoparticles as soon as they are exposed to the atmosphere. The band at ~3,700 cm⁻¹ shows the presence of an O-H bond due to water molecules.

EDX analysis of nanoparticles is shown in Figure 2B. Characteristic peaks of magnesium and oxygen are present in the plot. This confirms that magnesium and oxygen are present in the nanoparticles, which are MgO. Carbon tape was used for sample handling; hence, a peak of carbon can be observed.

STEM analysis of the nanoparticles is shown in Figure 2C. Spherical or oval-shaped nanoparticles were formed, ranging from 50 to 300 nm in size. The surface of the nanoparticles appeared to be smooth. The size distribution plot was drawn using data from STEM and shows that most of the nanoparticles ranged from 50 to 150 nm in size (Figure 2D), although a few nanoparticles were larger. Very small nanoparticles may have been attached to the surface of the other nanoparticles, thus making them larger than the others. XRD produced diffracted peaks for the nanoparticles that were narrow at higher temperatures. The Miller index at 2 theta angles 43.0°, 46.0°, 63.0°, 75.0°, and 78.0° were noted at 111, 200, 220, 311, and 222, respectively. These nanoparticles belonged to the face-centered cubic structure and Fm-3m space group, thus confirming the synthesis of MgO nanoparticles. SEM analysis showed a rounded cubical-shaped structure with corners that were not pointed but rather rounded or curved. The boundaries of these nanoparticles were smooth and clear. The size of the prepared nanoparticles ranged between 80 and 200 nm (approximately), and they had a well-dispersed shape rather than an aggregate form (Figure 2).

The cytotoxicity study revealed a significant difference between MgO-treated groups and the controls (Table 2). DNA damage was significantly (p < 0.05) lower in the MgO-treated groups than in the positive control after 24 and 48 h of contact time. However, DNA damage was directly proportional to the concentration of MgO nanoparticles, with a significant difference between each other, except between concentrations of 2.5 mg/mL and 5 mg/mL after 48 h of contact time. On the other hand, the phase index increased with an increase in the concentration of MgO up to 2.5 mg/mL; the phase index decreased beyond this concentration. This finding thus suggests that MgO nanoparticles are safe as their toxicity is significantly lower than the positive control.

The mean and standard deviation of the comparison of the MIC between K. pneumoniae and S. agalactiae showed a non-significant difference. GT, GA, GTM, and GAM had means and standard deviations (μg/mL) of 39.06 ± 0.00 and 19.53 ± 0.00; 78.13 ± 0.00 and 39.06 ± 0.00; 26.04 ± 11.28 and 9.766 ± 0.000; and 78.13 ± 0.00 and 26.04 ± 11.28, respectively, at 24 h against K. pneumoniae and S. agalactiae, respectively (Table 6).

China in a study published in year 2022 has reported 105 strains of S. agalactiae out of 313 clinical mastitis samples thus accounting for 33.55% prevalence on phenotypic basis of identification. The prevalence in the current study was lower than Kurjogi and Kaliwal (13), i.e., 28.10%. The pathogen is said to cause infection not only in bovines but also in humans and aquatic animals (3), so the rise in prevalence is justifiable as the pathogen spreads at one health interface. The current study showed a high prevalence of S. agalactiae compared with the study by Permatasari et al., (14). A study from Bangladesh reported 62.5% Klebsiella sp. in mastitic milk (15), which is much higher than that observed in the current study.

Previous studies conducted in the USA on Klebsiella spp. showed 5–19.5% resistance against the tetracycline group and up to 6.9% resistance against β-lactam. Similarly, up to 32% resistance against cefquinome, kanamycin, ceftiofur, polymyxin B, and tetracycline was found against Klebsiella spp (14). However, our findings reported otherwise by demonstrating the effective response of K. pneumoniae against penicillin, tetracycline, and amoxicillin. Contrary to the findings of our study, S. agalactiae showed 90% resistance against chloramphenicol, 78% against clindamycin, and 72% against enrofloxacin in previous studies, while the most sensitive response was shown against gentamicin (15).

The characterization of nanoparticles in the current study were in line with previously reported by Zaheer et al. (16). Similar findings were made in recent studies. Some studies reported smooth surfaces with a well-dispersed form, whereas others showed some nanoparticles clumped together. The average size of spherical nanoparticles ranged between 7 and 38 nm. Azam et al., (17) reported nanoparticles that ranged between 30 and 80 mm and were compactly aggregated. MgO nanoparticles have shown significant antibacterial activity, reducing growth by more than 90% at doses >5 mg/mL (18). In our study, there was a significant reduction in MIC with GAM after 8 h of incubation for each preparation, which was sustained until 16 h of incubation; this is in line with the findings from a previous study in which MgO was tested against S. aureus (19). Similar to our study, Aymen et al., (5) reported that MgO showed significant differences in MIC (P < 0.05) at the 20th hour of incubation compared with the 4th hour. According to Dehkordi et al. (20), CuO and MgO nanoparticles inhibit a wide variety of bacterial species. Siddiqi and Husen (19) reported that the antibacterial activity of ZnO nanoparticles was 1.8 ± 0.1 mg/mL. Additionally, the antibacterial activity of Fe₂O₃ has been shown to be 0.3125 ± 0.00 mg/mL (21, 22).

The antibacterial activity of ampicillin and florfenicol has been reported to increase when combined with silver nanoparticles against gram-positive and gram-negative bacteria. The enhancement in antimicrobial potential of antibiotics and nanoparticles stabilized in sodium alginate was also noted against bacterial pathogens (23, 24). They reported a significant reduction in the growth of bacteria with sodium alginate gel in combination with propolis. The current study was in line with the findings of (25), who reported a time-dependent increase in the antibacterial activity of sodium alginate-stabilized composites of antibiotics and MgO nanoparticles. The composites significantly reduced the MIC after incubation for 8–12 h. By forming reactive oxygen species (ROS), magnesium oxide inactivates pathogens. However, the adsorption process and direct cell membrane penetration may also be effective means of inactivating bacteria (26).

One theory for the primary mechanism of bacterial lysis is the action of ROS produced by MgO nanoparticles. This idea is reinforced by the inclusion of ascorbic acid, a ROS scavenger, in the formula (27). Therefore, it can be inferred that these ROS play a significant role in the breakdown of microorganisms and that the bacterium continues to grow and reproduce normally in their absence. According to the literature, MgO nanoparticles can control ROS formation by adjusting their size, shape, surface charge, surface area, ion release, and alkalinity (28, 29). A review on MgO nanoparticles as an antibacterial (29) reads alkaline earth metallic oxides being covered with OH⁻ layers. As MgO solution is naturally alkaline and superoxide ions are chemically stable in alkalis. The concentrated layers of superoxides may be present on the surface of the MgO powder while equilibrium of superoxide with hydroperoxyl radical (HO2⋅) is maintained as O2− + H⁺ ← HO2⋅. The HO2⋅ is created when the MgO powder interacts with substances such as a bacterial cell would improve antibacterial activity. Additionally, the Mg²⁺ ions produced from the mitochondria might deactivate cellular enzymes or inhibit mitochondrial respiration, which raises the levels of ROS in the mitochondria (30). The increased ROS generation puts the cells under oxidative stress, oxidizing the components of their membranes and ultimately jeopardizing their integrity, resulting in necrosis. The mechanism underlying the ability of these nanoparticles to kill cells is not yet fully understood. To increase the antibacterial activity of nano-MgO, other polymeric additions have been used, such as chitosan (31), polylactic acid (32), or starch (33). However, the mechanism behind their synergistic effects is also not yet known. It is evident that the innovation of this work rests in its original synthesis method, its in-depth understanding of the quantity of ROS created, and the reduction of activity in the presence of a radical scavenger.

The findings of the current study were in line with those of Ge et al. (26), who found that MgO nanoparticles were only slightly toxic to human cells at low concentrations (0.3 mg/mL). As a result of advanced strategies, antimicrobial activities have been improved and undesirable nanoparticle side effects have been reduced. The toxcicity of nanoparticles not only depend upon physical and chemical characteristics of nanoparticles but also on cell types being tested (34). This outcome is a result of the packaging of multiple antimicrobial agents into one nanoparticle, the coating of nanoparticles with biodegradable materials, and the engineering of target-specific nanoparticles for delivery to infection sites (35). However, further studies are required on the development of effective regimens against pathogens.

For the telophase test, small onions were merged in solutions with different concentrations for 48 h. The test involved the following three groups: (1) a negative control group, in which only distilled water was used; (2) a positive control group, in which methyl methanesulfonate (MMS) (10 μg/mL) was used; and (3) a treatment group, in which different concentrations of MgO nanoparticles (M) were used. The onions were incubated in a dark room at room temperature for two different periods, 24 h and 48 h, and the concentrations used in the treatment groups were 1.25, 2.5, and 5 mg/mL. The root tips, after incubation, were fixed in 3:1 (v/v) ethanol:acetic acid, following which root tips were washed with distilled water and finally fixed in 70% ethanol. A total of eight root tips from every treatment were hydrolyzed with 1N HCl at 60°C for 10 min and then rinsed with water. Root tips were stained with Schiff's reagent at room temperature for 30 min. Apical tips were crushed on slides with acetic acid (45%) and examined under a microscope using cover slips placed on the tips. To check for mitotic activity, more than 500 cells were counted for each treatment and the mitotic index and phase index were calculated as follows:

The dairy farms located at the border of Lodhran District and Bahawalpur District consented to sample collection. A dairy farm greater than n = 20 animals in production was selected for sampling. Convenient sampling techniques were used to collect milk samples from 210 cattle. Milk samples presenting clinical or subclinical mastitis were collected using standard protocols. Screening for subclinical mastitis was carried out following the protocol described by Muhammad et al. (37). S. agalactiae and K. pneumoniae were identified using pooled information obtained through a series of biochemical tests described in Bergey's Manual of Determinative Bacteriology (38).

Biochemically characterized S. agalactiae and K. pneumoniae were tested for antibiotic susceptibility using the disc diffusion method described by Bauer et al. (39). Briefly, freshly grown bacteria (24 h) were spread over Mueller Hinton agar (10⁸ CFU/mL), upon which 10 antibiotics were placed following the guidelines of the Clinical and Laboratory Standard Institute (40). ZOI were recorded following incubation at 37°C for 24 h. Means and standard deviations were measured to assess the antibacterial potential of S. agalactiae and K. pneumoniae.

MgO nanoparticles were prepared using the method described by Parashar et al. (41) using MgCl₂ 0.6H₂O, sodium dodecyl sulfate, and 2.5 M NaOH solution. The preparation protocol has been described in our previous study. XRD and SEM analysis was performed to determine the shape and dimensions of MgO nanoparticles. XRD analysis of nanoparticles was carried out using a powder diffractometer Rigaku D/max Ultima III operated at 40 kV and 0.130 A. Cu-Kα radiation was applied as a source emitting at a wavelength of 0.15406 nm, and a Quanta 250 SEM operating at 30 kV was used to obtain images of the nanoparticles (42).

Sodium alginate solution (2% m/v) and gelatin solution (2% m/v) were made in water. These solutions were mixed at an 80:20 ratio (sodium alginate:gelatin) and homogenized for 2 h at 500 rpm with a mechanical stirrer to obtain sodium alginate gel (G). MgO nanoparticles (1.5 g) were added to 20 mL of sodium alginate gel and stirred for 4 h at 500 rpm to stabilize nanoparticles in the gel. Antibiotic (0.035 g) was dissolved in 20 mL of distilled water mixed with 20 mL of gel, and stirred for 4 h at 500 rpm. Then, the product was dried and ground to a fine powder. Nanoparticles and antibiotics were mixed with 20 mL of gel and stirred for 4 h at 500 rpm (23).

The following products/composites were prepared for further study: tylosin stabilized in sodium alginate gel (GT); ampicillin stabilized in sodium alginate gel (GA); MgO nanoparticles and tylosin simultaneously stabilized in sodium alginate gel (GTM); and MgO nanoparticles and ampicillin simultaneously stabilized in sodium alginate gel (GTA).

For this trial, S. agalactiae and K. pneumoniae were selected based on their minimum ZOI against more than three different classes of antibiotics. The selected isolates were used in well diffusion and broth microdilution assays to estimate resistance modulation and determine the least effective dose of gel-based preparations, respectively.

Univariate data were analyzed through descriptive statistics and parametric tests were applied for quantitative data from two or more than two groups using a t-test and ANOVA, respectively. For comparison of means, Tukey's test was applied as a post hoc test. All the data were analyzed with a 5% probability using SPSS version 22.