Background/Objectives: Antimicrobial resistance, an evolutionarily entrenched microbial capacity amplified by extensive antibiotic exposure, has increased the burden of difficult-to-treat infections caused by priority pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. In this study, we assessed whether phytochemical-rich extracts from fully ripe Rosa canina pseudo-fruits (WF) and fully developed Colchicum autumnale flowers (CA) can provide combined antioxidant, antibacterial, and antibiofilm effects against multidrug-resistant clinical isolates. Methods: Plant materials were processed using seven extraction systems spanning non-polar to polar conditions (n-hexane, ethyl acetate, n-butanol, aqueous, 40% ethanol, 60% ethanol, and enzyme-assisted hydrolysis). Fractions were quantified for total phenolics, flavonoids, and tannins, evaluated for antioxidant capacity (DPPH and FRAP), tested for antibacterial activity (disc diffusion and MIC/MBC), and assessed for inhibition of early biofilm attachment. Differences among extraction methods and fractions were analyzed using standard comparative statistics (group comparisons across solvents/fractions), and relationships between chemical composition and bioactivity were examined using correlation-based analysis. Results: Extraction strategy emerged as the main determinant of bioactivity across endpoints. The WFE/ENZ fraction maximized phytochemical recovery (TPC 203.34 ± 11.55 mg GAE/g DW; TFC 35.67 ± 3.06 mg QE/g DW; TTC 53.00 ± 2.65 mg TAE/g DW) and showed strong antioxidant performance (DPPH IC₅₀ 33.60 ± 0.02 μg/mL; FRAP A₇₀₀ 1.90 ± 0.010 at 250 μg/mL). Antibacterial effects were strongest in polar fractions, particularly hydroethanolic and enzyme-assisted extracts, while n-hexane fractions were consistently weakest. Across eight clinical isolates and three reference strains, MIC values ranged from 0.04875 to 6.25 mg/mL for WF extracts and 0.0975–12.5 mg/mL for CA extracts. In the biofilm model, suppression of early attachment was most consistent for CAE/E60–ENZ and WFE/E40–E60–ENZ fractions. Conclusions: Correlation analysis indicated that antibacterial potency aligned primarily with flavonoid levels in R. canina pseudo-fruits and with tannin content in C. autumnale material. Overall, these results support hydroethanolic and enzyme-assisted extraction as rational strategies to enrich polyphenol-dense fractions with convergent antioxidant, antibacterial, and antibiofilm activity, reinforcing plant-derived matrices as a structured discovery space for developing complementary antimicrobial solutions beyond conventional antibiotics. Notably, this is among the first studies to evaluate the antibacterial potential of C. autumnale plant material in this context and to comprehensively assess R. canina pseudo-fruit extracts against multidrug-resistant clinical.

Antimicrobial resistance (AMR) emerges and circulates across interconnected human, animal, food, and environmental reservoirs; however, food fermentation systems remain underexplored as indicators of local AMR pressure, even though artisanal dairy fermentations may function as natural sentinels of AMR. In this study, we used an artisanal dairy fermentation chain as a One Health model to investigate whether environmentally exposed lactobacilli can reflect stage-associated shifts in resistance. A total of 1.085 isolates representing 16 Lactobacillus species were recovered from the same artisanal dairy matrix at two fermentation stages: day 5 (“Tyrovolia”; n = 518) and day 30 (“Kopanisti”; n = 567). Susceptibility to 14 antibiotics was evaluated by broth micro-dilution, and L. acidophilus was further screened for selected resistance genes. Overall resistance increased significantly from 69.88% (362/518) at day 5 to 77.25% (438/567) at day 30 (p = 0.0059), while multidrug resistance rose from 37.57% to 60.73% of resistant isolates (p < 0.001). Across the 224 species–antibiotic combinations examined, 129 (57.58%) showed an increased upper MIC limit at day 30, and resistance increased significantly for 9 of the 14 antibiotics tested, with the largest rises observed for metronidazole (RR = 7.67), chloramphenicol (RR = 5.74), quinupristin/dalfopristin (RR = 4.11), vancomycin (RR = 2.78), and trimethoprim (RR = 2.43). In contrast, erythromycin and oxytetracycline resistance declined significantly at the ripened stage. In L. acidophilus, 21 resistance genes were detected in 14/70 day-5 isolates and 19 genes in 13/71 day-30 isolates, but marked genotype–phenotype discordance was observed, including matrix-dependent expression patterns for tetM, ermB, and blaTEM. Collectively, these findings show that environmentally exposed artisanal dairy fermentations can enrich resistance phenotypes and may serve as sensitive sentinels of AMR dynamics at the human–animal–environment interface.

Background/Objectives: Staphylococcus aureus (S. aureus) intramammary infection remains a major global dairy problem due to its contagious nature, its ability to persist and colonize teat/skin and mucosal niches, and the often-limited bacteriological cure achieved with antimicrobial therapy. Beyond udder health, it is relevant to public health because it can enter raw milk chains and serve as a reservoir for antimicrobial resistance determinants that may circulate between dairy animals and humans. Methods: We assessed S. aureus’ ecology in raw ewe milk from 75 sheep farms in Epirus (Greece) by sampling clinically healthy controls (group A) and clinical mastitis cases pre-treatment (group B), followed by resampling at the first post-withdrawal milking after penicillin/streptomycin treatment (group C1—therapeutic protocol 1), oxytetracycline treatment (group C2—therapeutic protocol 2), or enrofloxacin treatment (group C3—therapeutic protocol 3). Results: S. aureus detection was high and comparable across groups (A 23.0%, B 22.0–30.0%, C 20.0–22.0%), and paired analyses showed no significant pre–post shifts in detection/burden within therapeutic protocols (all p > 0.05). Nevertheless, persistence remained evident. The chromosomal gene mecA was detected in S. aureus strains in all groups, ranging from 13.6% in controls to 54.5% post-withdrawal in group C1, and was also present in the pre-treatment group. In paired sampling animals, mecA was mostly stable, with rare emergence or loss. Across antibiotic classes, within-animal resistance transitions were generally uncommon and non-significant (p > 0.05); β-lactam resistance was fully stable (p = 1.00). Descriptively, resistance to protein synthesis inhibitors tended to decline after therapy in protocol 1 and protocol 3, while protocol 3 showed post-treatment gains in fluoroquinolone resistance. By contrast, virulence-associated phenotype traits shifted after therapy: enterotoxigenicity increased post-withdrawal (especially in the C3 group), Staphylococcal Enterotoxin A (SEA) and Staphylococcal Enterotoxin B (SEB) appeared only post-therapy, Staphylococcal Enterotoxin D (SED) increased significantly in paired isolates (p = 0.002), and strong biofilm adherence increased (in C3, p = 1.5 × 10⁻⁵). Conclusions: The detection of S. aureus after therapy suggests that one possibility is that antimicrobial exposure may select for, or otherwise reshape, the residual intramammary population, rather than reliably eliminating it—an outcome that remains clinically relevant for udder health. Moreover, the persistence of mecA/methicillin-resistant Staphylococcus aureus (MRSA)-compatible profiles indicates that milk released to the food chain after withdrawal compliance may still harbor S. aureus with enhanced preservation capacity and significant food safety relevance.