Contribution of Microbiota to Bioactivity Exerted by Bee Bread.

Bee-collected pollen (BCP) and bee bread (BB) are honey bee products known for their beneficial biological properties. The main goal of this study was to investigate BB microbiota and its contribution to bioactivity exerted by BB. The microbiota of BB samples collected at different maturation stages was investigated via culture-independent (Next Generation Sequencing, NGS) and culture-dependent methods. Microbial communities dynamically fluctuate during BB maturation, ending in a stable microbial community structure in mature BB. Bee bread bacterial isolates were tested for phenotypes and genes implicated in the production and secretion of enzymes as well as antibacterial activity. Out of 309 bacterial isolates, 41 secreted hemicellulases, 13 cellulases, 39 amylases, 132 proteinases, 85 Coomassie brilliant blue G or R dye-degrading enzymes and 72 Malachite Green dye-degrading enzymes. Furthermore, out of 309 bacterial isolates, 42 exhibited antibacterial activity against Staphylococcus aureus, 34 against Pseudomonas aeruginosa, 47 against Salmonella enterica ser. Typhimurium and 43 against Klebsiella pneumoniae. Artificially fermented samples exerted higher antibacterial activity compared to fresh BCP, strongly indicating that BB microbiota contribute to BB antibacterial activity. Our findings suggest that BB microbiota is an underexplored source of novel antimicrobial agents and enzymes that could lead to new applications in medicine and the food industry.

HPLC- and NMR-Based Chemical Profiling, Wound-Healing Potential, Anti-Inflammatory and Antibacterial Activities of Satureja pilosa (Lamiaceae), a Neglected Medicinal-Aromatic Herb.

Satureja pilosa Velen. (Lamiaceae) is a perennial and melliferous aromatic-medicinal subshrub which is range-restricted in adjacent parts of Greece and Bulgaria and locally in Italy, known in Northern Greece as wild oregano ("agriorigani") and traditionally collected from the wild for culinary purposes. Since the ethnopharmacological data and modern biological activities of Satureja spp. suggest promising applications in skin conditions, the present study aimed to investigate the hitherto unknown phenolic content of cultivated S. pilosa and its potential biological activities, focusing mainly on wound-healing and anti-inflammatory effects. An HPLC-PDA-MS-targeted phytochemical investigation, along with NMR, allowed for the isolation and characterization of the main constituents, resulting in 18 compounds. Representative extracts and purified compounds were tested for wound-healing activity on NIH/3T3 fibroblasts. The butanol extract exhibited a significantly higher cell migration rate (73.4%) compared to aqueous (50.6%) and methanolic (49.6%) ones, enhancing the cell migration more rapidly at both concentration levels, whilst rosmarinic acid was the most potent among the isolated compounds, with a migration rate of 64.0% at the concentration level of 10-5 mg/mL, followed by 3,4-dihydrophenyllactic acid (54.7%). Moreover, potential effects on endothelial activation processes were explored, including the leukocyte-endothelial cell interaction during inflammatory processes and the migratory capacity during angiogenic actions, since these processes are commonly associated with skin diseases. Finally, extracts and purified compounds demonstrated weak antibacterial potential against two important pathogens (Staphylococcus aureus and Pseudomonas aeruginosa), suggesting that further investigation is warrented.

Antioxidant and Antibacterial Properties of a Functional Sports Beverage Formulation.

Athletes often consume functional beverages in order to improve performance and reduce oxidative stress caused by high-intensity exercise. The present study aimed to evaluate the antioxidant and antibacterial properties of a functional sports beverage formulation. The beverage's antioxidant effects were assessed on human mesenchymal stem cells (MSCs) by determining thiobarbituric acid reactive substances (TBARS; TBARS levels decreased significantly by 52.67% at 2.0 mg/mL), total antioxidant capacity (TAC; TAC levels increased significantly by 80.82% at 2.0 mg/mL) and reduced glutathione (GSH; GSH levels increased significantly by 24.13% at 2.0 mg/mL) levels. Furthermore, the beverage underwent simulated digestion following the INFOGEST protocol to assess its oxidative stability. The analysis of the total phenolic content (TPC) using the Folin-Ciocalteu assay revealed that the beverage contained a TPC of 7.58 ± 0.066 mg GAE/mL, while the phenolics identified by HPLC were catechin (2.149 mg/mL), epicatechin (0.024 mg/mL), protocatechuic acid (0.012 mg/mL), luteolin 7-glucoside (0.001 mg/mL), and kaempferol-3-O-ß-rutinoside (0.001 mg/mL). The beverage's TPC was strongly correlated with TAC (R2 = 896). Moreover, the beverage showcased inhibitory and bacteriostatic effects against Staphylococcus aureus and Pseudomonas aeruginosa. Lastly, the sensory acceptance test demonstrated that the functional sports beverage was well accepted by the assessors.

Extensive analysis of the cultivated medicinal herbal drug Origanum dictamnus L. and antimicrobial activity of its constituents.

Phytochemical investigations of the methanol extract from Origanum dictamnus L. (Lamiaceae) resulted in the isolation of forty compounds belonging to the classes of terpenes, resorcinol derivatives, flavonoids, depsides, neolignans and jasmonates. Chromatographic isolations were targeted by using two analytical platforms, NMR and HPLC-PDA-MS. In parallel, HPLC-PDA-MS of individual fractions enabled the unambiguous identification of additionally eight components. In total 48 constituents were isolated/identified. Among the isolated constituents are four undescribed compounds, one resorcinol derivative, one monoterpene, one diterpene and one acylated flavonoid glycoside. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses, including 1D and 2D NMR, and HPLC-ESI-MS and HRMS experiments. Representative compounds were tested for their antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. I4-II7-dicarvacrol was the most potent constituent.

Physicochemical Characterization and Biological Properties of Pine Honey Produced across Greece.

Pine honey is a honeydew honey produced in the East Mediterranean region (Greece and Turkey) from the secretions of the plant sucking insect Marchalina hellenica (Gennadius) (Coccoidea: Marchalini-dae) feeding on living parts of Pinus species. Nowadays, honeydew honey has attracted great attention due to its biological activities. The aim of this study was to study unifloral pine honey samples produced in Greece regarding their physicochemical parameters and antioxidant and antibacterial activity against five nosocomial and foodborne pathogens. These honeys showed physicochemical and microscopic characteristics within the legal limits, except for diastase activity, a parameter known to be highly variable, depending on various factors. Substantially higher levels of H2O2 were estimated compared to other types of honeydew honey, whereas protein content was similar. The total phenolic content was 451.38 ± 120.38 mg GAE/kg and antiradical activity ranged from 42.43 to 79.33%, while FRAP values (1.87 to 9.43 mmol Fe+2/kg) were in general higher than those reported in the literature. Various correlations could be identified among these parameters. This is the first attempt to investigate in depth the antibacterial activity of pine honey from Greece and correlate it with honey quality parameters. All tested honeys exerted variable but significant antibacterial activity, expressed as MIC and MBC values, comparable or even superior to manuka honey for some tested samples. Although honey antibacterial activity is mainly attributed to hydrogen peroxide and proteins in some cases (demonstrated by elevated MICs after catalase and Proteinase K treatment, respectively), no strong correlation between the antibacterial activity and hydrogen peroxide concentration or total protein content was demonstrated in this study. However, there was a statistically significant correlation of moisture, antioxidant and antibacterial activity against Klebsiella pneuomoniae, as well as antioxidant and antibacterial activity against Salmonella ser. Typhimurium. Interestingly, a statistically significant negative correlation has been observed between diastase activity and Staphylococcus aureus antibacterial activity. Overall, our data indicate multiple mechanisms of antibacterial activity exerted by pine honey.

Transcriptomic Analysis of Pseudomonas aeruginosa Response to Pine Honey via RNA Sequencing Indicates Multiple Mechanisms of Antibacterial Activity.

Pine honey is a unique type of honeydew honey produced exclusively in Eastern Mediterranean countries like Greece and Turkey. Although the antioxidant and anti-inflammatory properties of pine honey are well documented, few studies have investigated so far its antibacterial activity. This study investigates the antibacterial effects of pine honey against P. aeruginosa PA14 at the molecular level using a global transcriptome approach via RNA-sequencing. Pine honey treatment was applied at sub-inhibitory concentration and short exposure time (0.5× of minimum inhibitory concentration -MIC- for 45 min). Pine honey induced the differential expression (>two-fold change and p ≤ 0.05) of 463 genes, with 274 of them being down-regulated and 189 being up-regulated. Gene ontology (GO) analysis revealed that pine honey affected a wide range of biological processes (BP). The most affected down-regulated BP GO terms were oxidation-reduction process, transmembrane transport, proteolysis, signal transduction, biosynthetic process, phenazine biosynthetic process, bacterial chemotaxis, and antibiotic biosynthetic process. The up-regulated BP terms, affected by pine honey treatment, were those related to the regulation of DNA-templated transcription, siderophore transport, and phosphorylation. Pathway analysis revealed that pine honey treatment significantly affected two-component regulatory systems, ABC transporter systems, quorum sensing, bacterial chemotaxis, biofilm formation and SOS response. These data collectively indicate that multiple mechanisms of action are implicated in antibacterial activity exerted by pine honey against P. aeruginosa.

Antibiotic Resistance Patterns and mcr-1 Detection in Avian Pathogenic Escherichia coli Isolates from Commercial Layer and Layer Breeder Flocks Demonstrating Colibacillosis in Greece.

Objectives: The aim of this study was to investigate the antimicrobial resistance (AMR) patterns of Escherichia coli strains isolated from poultry flocks suffering from colibacillosis in Greece and to detect the presence of the mcr-1 gene in isolates being phenotypically resistant to colistin. Results: A total of 150 E. coli strains were isolated from commercial layers and layer breeder flocks in Greece and tested for antimicrobial susceptibility. A high level of susceptibility was revealed for cephalosporins, neomycin, and colistin. Susceptibility varied for other antimicrobials (tetracycline, doxycycline, lincospectin, trimethoprim/sulfamethoxazole, enrofloxacin, amoxicillin), whereas no susceptibility was reported for macrolides, tiamulin, lincomycin, oxacillin. Concerning colistin resistance, 20 E. coli strains were found to be phenotypically resistant (13 strains showed intermediate resistance pattern and 7 strains fully resistance trait). Further investigation was performed by PCR, which has revealed the presence of the mcr-1 gene in one phenotypically colistin-resistant isolate. Conclusion: AMR is prevalent in layer poultry production, including resistance against colistin confirmed by the presence of the mcr-1 gene.

Antibacterial and antioxidant activity of different types of honey derived from Mount Olympus in Greece.

The aim of the present study was to examine the antioxidant and antibacterial activity of 21 types of honey derived from Mount Olympus (Mt. Olympus), a region with great plant biodiversity. The antibacterial activity was examined against the growth of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) by the agar well diffusion assay and the determination of the minimum inhibitory concentration (MIC). The antioxidant activity was assessed by using the 2,2­diphenyl­1­picrylhydrazyl (DPPH) and 2,2'­azino­bis(3­ethylbenzothiazoline­6­sulphonic acid (ABTS•+) free radical scavenging assays. These activities were compared to Manuka honey which is used as an alternative medicine. The results revealed that all tested honey types exhibited antibacterial activity against S. aureus and P. aeruginosa. The MIC of the tested honey types against S. aureus ranged from 3.125 to 12.5% (v/v), while MIC of Manuka honey was determined to be 6.25% (v/v). The MIC values of the tested honey types against P. aeruginosa ranged from 6.25 to 12.5% (v/v) and the MIC of Manuka honey was determined at 12.5% (v/v). Moreover, the results suggested that the presence of hydrogen peroxide and proteinaceous compounds in the honey types accounted, at least in part, for the antibacterial activity. In addition, the total polyphenolic content (TPC) of the honey types seemed to contribute to the antibacterial activity against P. aeruginosa. Furthermore, some of the tested honey types exhibited potent free radical scavenging activity against DPPH and ABTS•+ radicals, which was greater than that of Manuka honey. The results indicated that not only the quantity, but also the quality of the polyphenols were responsible for the antioxidant activity. Moreover, four honey types exhibiting great antioxidant activity were converted to powder using a freeze drying method. The results indicated that following conversion to powder all honey types, apart from one, retained their antioxidant activity, although their TPC was reduced. On the whole, and at least to the best of our knowledge, the present study is the first that extensively examined the bioactivities of different types of honey derived from Mt. Olympus.