The rising threat of antimicrobial resistance (AMR) is a global concern in both human and veterinary medicine, with multidrug-resistant (MDR) pathogens such as Staphylococcus aureus and Staphylococcus pseudintermedius presenting significant challenges. Background/Objectives: This study evaluates the effectiveness of amoxicillin against these MDR pathogens in canine isolates using pharmacokinetic and pharmacodynamic parameters. Methods: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and mutation prevention concentration (MPC) were assessed. Additionally, time-kill assays and post-antibiotic effect (PAE) assessments were performed. Epidemiological cutoff (ECOFF) values were established for both species to guide therapy. Results: S. aureus had a higher resistance rate (35.89%) than S. pseudintermedius (15.27%), with MIC50 values of 0.50 μg/mL and 0.25 μg/mL, respectively. The MPC analysis revealed that S. pseudintermedius required higher antibiotic concentrations (16.11 μg/mL) to prevent mutations compared to S. aureus (2.20 μg/mL). Time-kill assays indicated that higher amoxicillin dosages caused faster bacterial reduction. The PAE analysis showed extended post-treatment bacterial suppression at elevated doses, particularly against S. aureus. Conclusions: Species-specific amoxicillin dosing strategies are necessary due to differing resistance and susceptibility profiles between S. aureus and S. pseudintermedius. High-dose amoxicillin therapy is recommended to achieve optimal therapeutic outcomes for resistant SA, while slightly adjusted dosing can manage S. pseudintermedius infections. These findings provide essential insights for veterinary antimicrobial stewardship, underscoring the need for tailored therapeutic approaches to minimize AMR development while ensuring effective infection control.

The growing challenge of antibiotic resistance and inflammation-related disorders calls for safe, multi-target therapeutic strategies. Morus alba (MOAL) and Pinus densiflora (PIDE) are known for their medicinal properties, yet their combined potential with methyl gallate (MG) has not been fully explored. In this study, the phytochemical composition of MOAL and PIDE was characterized using GC–MS, and their combined antimicrobial, antioxidant, and anti-inflammatory activities were evaluated. Hydroethanolic extracts were prepared and assessed for antioxidant activity (DPPH assay), antibacterial activity (disk diffusion, MIC, time kill), and nitric oxide (NO) suppression in Lipopolysaccharide (LPS)-stimulated macrophages, alongside MTT cytotoxicity screening. MOAL exhibited a higher extraction efficiency, reaching 500 mg/mL at 4 h, whereas Pinus achieved 450 mg/mL at the same time point. Both exhibited a diverse and abundant phytochemical profile. The optimized blend (MOAL:PIDE:MG, 1:1:0.1) demonstrated significantly enhanced bioactivity, with over 90% DPPH scavenging with the low IC₅₀ value (66.62 mg/mL), potent inhibition of both Gram-positive and Gram-negative bacteria, and the strongest effect against Staphylococcus aureus (264 μg/mL). Time-kill assays confirmed rapid bactericidal action, and NO production was reduced by approximately 75% without cytotoxicity. Molecular docking identified a lead multi-target compound exhibiting strong binding affinities to COX-2, TNF-α, and Keap1, supporting its observed anti-inflammatory and antioxidant potential. These findings highlight the promise of synergistic phytochemical formulations as broad-spectrum, multifunctional therapeutic candidates, supporting further in vivo and clinical validation.

Employing affordable and uncomplicated sample preparation techniques to recommend the most efficient antibacterial therapy could help reduce antibiotic-resistant bacteria. This study evaluated the suitability of immunoassays and microbiological assays as alternatives for liquid chromatography/mass spectrometry (LC/MS) in determining plasma tylosin concentrations after intramuscular administration at a dose of 20 mg/kg to both healthy and diseased pigs in clinical veterinary practice. The diseased pigs were confirmed using the target genes Actinobacillus pleuropneumoniae (apxIVA) and Pasteurella multocida (kmt1). The methods showed good linearity, precision, and accuracy. In both healthy and diseased pigs, a significant correlation was observed between LC/MS and the microbiological assay (Pearson correlation coefficient: 0.930, p < 0.001 vs. Pearson correlation coefficient: 0.950, p < 0.001) and between LC/MS and the enzyme-linked immunosorbent assay (ELISA) (Pearson correlation coefficient: 0.933; p < 0.001 vs. Pearson correlation coefficient: 0.976, p < 0.001). A strong correlation was observed between the microbiological assay and the ELISA in both healthy and diseased pigs (Pearson correlation coefficient: 0.911; p < 0.001 vs. Pearson correlation coefficient: 0.908, p < 0.001). A Bland-Altman analysis revealed good agreement between the methods, i.e., 95% of the differences were within the limits of agreement. Therefore, the microbiological assay and the ELISA, which demonstrated sufficient precision and accuracy, can be viable alternatives to LC/MS when it is unavailable.

Tylosin (TYL), a macrolide antibiotic, is commonly used to treat respiratory infections in weaned piglets. Despite its utility, TYL administration is associated with adverse side effects, including immune dysregulation and gut microbiota imbalance. This study evaluated whether methyl gallate (MG), a natural polyphenolic compound with antimicrobial and immunomodulatory properties, could mitigate these side effects of TYL. Forty weaned piglets were randomly assigned to a saline control group, a TYL (10 mg/kg) group, an MG (10 mg/kg) group, and a TYL + MG (10 mg/kg each) group. Changes in body weight, hematological parameters, plasma cytokines, culturable fecal bacterial populations, and 16S rRNA-based gut microbiota composition were evaluated in all weaned piglets. There were no significant differences (p > 0.05) in ADG, ADFI, or FE among the treatment groups. Hematological analysis revealed significant changes in neutrophil and eosinophil counts, showing significant fluctuations in the MG-treated group compared with the control. TYL administration significantly decreased TNF-α and IL-1β levels while increasing IL-10 and IL-1ra concentrations, indicating immunomodulatory effects. In contrast, MG significantly increased adaptive immune-associated cytokines, including IL-2, IL-4, IL-6, and IL-12. Culture-based fecal microbiological analysis revealed that TYL reduced Lactobacillus abundance and lowered the culture-based Lactobacillus/Enterobacteriaceae ratio, suggesting acute dysbiosis. Co-administration of MG partially attenuated these microbiota-associated alterations and promoted the recovery of bacterial populations. Moreover, 16S rRNA sequencing demonstrated that similar relative abundances of Proteobacteria were observed in the TYL and TYL + MG groups. Therefore, no clear reduction in relative abundance of Proteobacteria was demonstrated under the present experimental conditions. These findings support the possibility that MG may partially attenuate selected TYL-associated immunological and gut microbiota-related changes under the conditions of the present exploratory study. A graphical summary of the study design and the principal findings is presented in the Graphical