This study utilized phytochemical screening to conduct the qualitative analysis of plant extracts, aiming to identify various classes of secondary metabolites. Moreover, the antibacterial activity of different types of Oregano vulgare and Salvia triloba extracts was determined. To achieve the aim of this study, aqueous, ethanolic, and enzymatic extracts were prepared and screened for phytochemical capacity and antioxidant activities. The determination of the antibacterial activity included phenotypic screening of antibiotic susceptibility pattern of oral and food pathogenic bacterial strains, determination of the minimum inhibitory concentration and minimum bactericidal concentration—via microdilution broth test and in vitro valuation of antibacterial efficacies—of the anti-biofilm properties of the studied herbal extractions. Results: Our study evaluated the phytochemical composition and the antioxidant, antibacterial, and anti-biofilm properties of O. vulgare and S. triloba extracts. The analyzed samples contained bioactive compounds, such as phenolics and flavonoids, contributing to the observed strong antioxidant effect. Furthermore, they exhibited notable activity against oral biofilm formation and demonstrated significant antibacterial efficacy against dental caries’ microorganisms as well as food pathogens. Despite methodological variations, all extracts showed significant antioxidant capacity and promising antibacterial activity against various pathogens, including resistant strains, while also inhibiting biofilm formation. Although limited to two plant species and facing methodological constraints, this study lays the groundwork for future research, indicating the therapeutic potential of O. vulgare and S. triloba extracts. Further exploration is needed to report on underlying mechanisms and validate efficacy through clinical trials.

This study utilized phytochemical screening to conduct qualitative analysis of plant extracts, aiming to identify various classes of secondary metabolites. Moreover, the antibacterial activity of different types of O. vulgare and S. triloba extracts was determined. To achieve the aim of the study, aqueous, ethanolic, and enzymatic extracts were prepared and screened for phytochemical capacity and antioxidant activities. Determination of the antibacterial activity included phenotypic screening of antibiotic susceptibility pattern of oral and food pathogenic bacterial strains, determination of Minimum inhibitory concentration and minimum bactericidal concentration - Microdilution broth test and in vitro valuation of antibacterial efficacies - anti-biofilm properties of the studied herbal extractions. Results: Our study evaluated the phytochemical composition and the antioxidant, antibacterial, and anti-biofilm properties of O. vulgare and S. triloba extracts. The analyzed samples contained bioactive compounds, such as phenolics and flavonoids, contributing to the observed strong antioxidant effect. Furthermore, they exhibited notable activity against oral biofilm formation and demonstrated significant antibacterial efficacy against dental caries microorganisms as well as food pathogens. Despite methodological variations, all extracts showed significant antioxidant capacity and promising antibacterial activity against various pathogens, including resistant strains, while also inhibiting biofilm formation. Although limited to two plant species and facing methodological constraints, the study lays a groundwork for future research, indicating the therapeutic potential of O. vulgare and S. triloba extracts. Further exploration is needed to report on underlying mechanisms and validate efficacy through clinical trials.

Dairy products from ovine milk are very popular in the Mediterranean countries and are gaining a large portion of the market in EU countries and worldwide. EU legislation permits the dairy processing of raw ovine milk under certain conditions. To study the ecology and prevalence of E. coli in raw ewes’ milk and assess thus the public health risk, samples of milk were taken from 75 different sheep farms in the rural area of Epirus, Greece. The initial sampling was conducted in clinically healthy animals which were noted as controls (group A). From the same farms, samples were taken from animals with clinical mastitis and before treatment (group B). For therapeutic purposes, to some animals, a combination of penicillin and streptomycin was administrated (group C1), or tetracycline (group C2), or enrofloxacin (group C3). Finally, samples of raw milk were taken from the C groups, on the first day after the withdrawal period of the antibiotics used, when the milk is permitted to enter the food chain. In total, 97 isolates of Escherichia coli were recovered from all groups. Analysis revealed an impressive increase in E. coli strains in the milk of group B (39.33%) with respect to group A (5%). Even after treatment, although the prevalence was decreased, it was still found to be higher in the C groups than group A. E. coli O157:H7 strains absent from group A were detected in all other groups in relatively low occurrence rates with respect to other “O” serotypes but non-O157:H7 strains. Virulence factors such as the production of toxins (32.60% for serotoxin 1 and 18.47% for serotoxin 2) and hemolysin (42.39%) as well as biofilm formation capacity (52.17% of the total) and ESDL production (43.47% of the total) were also studied. All strains were also tested for susceptibility against 12 antibiotics by the MIC method and the results showed a high prevalence of resistance and multi-resistance. The presence of various resistant strains to antibiotics and pathogenic “O” serotype strains in the milk when it can enter the food chain again is an alarming conclusion.

Rural households all over the world rear backyard chicken mainly for their own consumption and, to a lesser extent, for barter trade. These chickens represent a staple dish with numerous culinary variations and a cheap source of protein. Although some Campylobacter species, and particularly Campylobacter jejuni and Campylobacter coli, have been associated with industrial poultry carcasses, studies concerning the ecology of this genus in rural households do not exist. To assess the prevalence of Campylobacter species in the tissues of backyard chickens, samples were collected from birds Gallus domesticus bred in households in the rural area of Epirus (Greece), and Campylobacter strains were isolated by quantitative methods at 37 °C and 42 °C. In total, 256 strains were identified, belonging to 17 Campylobacter species, with C. jejuni and C. coli being the most prevalent. From the four ecological parameters studied (size of the flock, presence of small ruminants in the same household, presence of other poultry species in the same household, and feeding leftovers of the household), the size of the flock and the presence of small ruminants and/or pigs in the same household mostly affected the distribution of these strains. To study the phenotypical resistance against 14 antibiotics, 215 strains were selected. The results showed a high prevalence of multidrug-resistance (MDR) strains extending to all classes of antibiotics. Further genome analysis revealed the presence of genes coding resistance (bla_OxA-61, tet(O), tet(A) cmeA, cmeB, cmeC, and gyrA (Thr-86-Ile mutation)), with the efflux pump CmeABC being the most prevalent. All antimicrobial resistance-encoded genes co-circulated, except for bla_OXA-61, which moved independently. The minimum inhibitory concentration (MIC) values of two out of three antibiotics (representing different classes) were reduced when the strains tested were exposed to carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a known efflux pump inhibitor. The same result was obtained with the addition of CCCP to the MIC values of bile salts. These results lead to the conclusion that Campylobacter species are present in an impressive diversity in backyard chicken tissues and that they exert a significant resistance to antibiotics, raising a potential danger for public health.

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.