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.

Low complexity regions in the proteins of prokaryotes perform important functional roles and are highly conserved.

We provide the first high-throughput analysis of the properties and functional role of Low Complexity Regions (LCRs) in more than 1500 prokaryotic and phage proteomes. We observe that, contrary to a widespread belief based on older and sparse data, LCRs actually have a significant, persistent and highly conserved presence and role in many and diverse prokaryotes. Their specific amino acid content is linked to proteins with certain molecular functions, such as the binding of RNA, DNA, metal-ions and polysaccharides. In addition, LCRs have been repeatedly identified in very ancient, and usually highly expressed proteins of the translation machinery. At last, based on the amino acid content enriched in certain categories, we have developed a neural network web server to identify LCRs and accurately predict whether they can bind nucleic acids, metal-ions or are involved in chaperone functions. An evaluation of the tool showed that it is highly accurate for eukaryotic proteins as well.

The complex evolutionary history of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (AARSs) are a superfamily of enzymes responsible for the faithful translation of the genetic code and have lately become a prominent target for synthetic biologists. Our large-scale analysis of >2500 prokaryotic genomes reveals the complex evolutionary history of these enzymes and their paralogs, in which horizontal gene transfer played an important role. These results show that a widespread belief in the evolutionary stability of this superfamily is misconceived. Although AlaRS, GlyRS, LeuRS, IleRS, ValRS are the most stable members of the family, GluRS, LysRS and CysRS often have paralogs, whereas AsnRS, GlnRS, PylRS and SepRS are often absent from many genomes. In the course of this analysis, highly conserved protein motifs and domains within each of the AARS loci were identified and used to build a web-based computational tool for the genome-wide detection of AARS coding sequences. This is based on hidden Markov models (HMMs) and is available together with a cognate database that may be used for specific analyses. The bioinformatics tools that we have developed may also help to identify new antibiotic agents and targets using these essential enzymes. These tools also may help to identify organisms with alternative pathways that are involved in maintaining the fidelity of the genetic code.

Intra- and inter-serotypic recombinations in the 5΄ UTR-VP4 region of Echovirus 30 strains.

Recombination has been recognized as a major mechanism of evolution in enteroviruses. The Echovirus 30 (E-30) strain Gior was sequenced and phylogenetically compared to all available E-30 sequences to detect recombination events between the 5΄UTR and VP1 genomic regions. The comparison of phylogenetic trees of the 5΄UTR and VP1 revealed incongruences concerning strains, lineages and sub-lineages. Comparative analysis of 62 E-30 sub-genomic sequences revealed six different recombination events that almost all occurred in the same region, having a start point in the 3΄end of the 5΄ UTR and end point in VP4. The only exception was the sub-lineage of Gior for which both borders of recombination were located in the 5΄UTR. These results describe for the first time recombination events in this region in circulating EV-B strains, revealing the exact points of these recombination events, highlighting the impact of such events on the evolution and epidemiology of enteroviruses.

Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes.

Considering that 70% of our planet's surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds.

Cutibacterium acnes induces acne-like lesions in hairless mice models - A comparative study.

Acne vulgaris, a chronic inflammatory skin disease with a high prevalence worldwide, necessitates reliable preclinical models for both understanding its pathogenesis and evaluating potential anti-acne therapies. This study aims to establish a robust mouse model using intracutaneous injection of Cutibacterium acnes bacterial suspension. Three hairless mouse strains (SKH-hr1, SKH-hr2 brown, and SKH-hr2 + ApoE) were systematically compared to ascertain the stains most closely resembling acne in humans. Various assessments, including photo documentation, biophysical evaluation, blood analysis, and histopathology, were conducted. Despite all strains exhibiting acne-like lesions, SKH-hr1 mice emerged as the most suitable model, demonstrating the most satisfactory results across multiple criteria. This research underscores the significance of employing hairless mice strains as models in acne studies to enhance and facilitate the development of effective therapeutic interventions.

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.

Detection of a phylogenetically distinct IMP-type metallo-ß-lactamase, IMP-35, in a CC235 Pseudomonas aeruginosa from the Dutch-German border region (Euregio).

OBJECTIVES: To characterize a highly divergent IMP-type metallo-ß-lactamase (MBL) variant detected in a multidrug-resistant Pseudomonas aeruginosa clinical isolate. METHODS: P. aeruginosa isolate 1876 was recovered from an anal swab of an inpatient at a German hospital in the Dutch-German border region (Euregio), where cross-border patient healthcare occurs. MICs were determined by agar dilution and phenotypic screening for MBL production by Etest MBL. Typing was performed by multilocus sequence typing (MLST). PCR assays and nucleotide sequencing were employed for identification of bla gene types. The class 1 integron carrying the blaIMP-type gene was characterized by PCR mapping and sequencing using a set of specific primers. A phylogenetic tree was constructed for the new blaIMP variant. RESULTS: Isolate 1876 was phenotypically positive for MBL production, exhibited resistance to carbapenems and harboured a new blaIMP-type gene, blaIMP-35. MLST showed that the allelic profile corresponded to ST622, which belongs to the prevalent international clonal complex CC235. The blaIMP-35 gene was located in a class 1 integron as the first gene cassette, followed by blaOXA-35, aacA6, qacEΔ1 and sul1, suggesting its recent integration. IMP-35 was highly divergent, possessing 33/246 (13.4%) different amino acid residues from its closest IMP variants (IMP-8 and IMP-12) and was phylogenetically distinct, representing a separate group in the phylogenetic tree of IMP proteins. CONCLUSIONS: The identification of this phylogenetically distinct IMP-type variant in a CC235 P. aeruginosa suggests the ongoing spread of new IMP-type carbapenemases as well as the potential of the blaIMP-35 gene to evolve in the hospital environment.

Posttranslational regulation impacts the fate of duplicated genes.

Gene and genome duplications create novel genetic material on which evolution can work and have therefore been recognized as a major source of innovation for many eukaryotic lineages. Following duplication, the most likely fate is gene loss; however, a considerable fraction of duplicated genes survive. Not all genes have the same probability of survival, but it is not fully understood what evolutionary forces determine the pattern of gene retention. Here, we use genome sequence data as well as large-scale phosphoproteomics data from the baker's yeast Saccharomyces cerevisiae, which underwent a whole-genome duplication approximately 100 mya, and show that the number of phosphorylation sites on the proteins they encode is a major determinant of gene retention. Protein phosphorylation motifs are short amino acid sequences that are usually embedded within unstructured and rapidly evolving protein regions. Reciprocal loss of those ancestral sites and the gain of new ones are major drivers in the retention of the two surviving duplicates and in their acquisition of distinct functions. This way, small changes in the sequences of unstructured regions in proteins can contribute to the rapid rewiring and adaptation of regulatory networks.

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.

Impact of Mt. Olympus Honeys on Virulence Factors Implicated in Pathogenesis Exerted by Pseudomonas aeruginosa.

The aim of this study was to examine the impact of twenty honey samples, harvested in Mt. Olympus (Greece), on the virulence factors implicated in P. aeruginosa pathogenesis. Six key virulence factors (protease and elastase activity, pyocyanin and pyoverdine concentration, biofilm formation, and swimming motility) were selected in order to assess the effect of the tested honeys compared with Manuka honey. All tested honeys demonstrated a significant inhibition of protease and elastase activity compared with the control. Six and thirteen honeys exerted superior protease (no inhibition zone) and elastase (values lower than 55%) activity, respectively, compared with Manuka honey. Seventeen tested honeys exhibited reduced pyoverdine production compared with the control; all tested honeys, except for one, showed an inhibitory effect on pyocyanin production compared with the control. Regarding swimming motility, nine tested honeys demonstrated significantly higher inhibition compared with Manuka honey. Honey concentrations (6% v/v and 8% v/v) had the most profound impact, as they reduced biofilm formation to less than 20% compared with the control. Overall, our data demonstrate a significant inhibition of the virulence factors in the tested Mt. Olympus honeys, highlighting the strong antimicrobial activity against P. aeruginosa, an antibiotic-resistant pathogen of growing concern, which is implicated in severe nosocomial infections globally.

Antiviral Activity of Beebread, Bee-Collected Pollen and Artificially Fermented Follen against Influenza A Virus.

Bee-collected pollen (BCP) and the naturally fermented BCP product known as bee bread (BB) are functional foods renowned for their nutritious, antioxidant, antibacterial and other therapeutic properties. This is the first study employed to assess the antiviral activity of BCP and BB against influenza A virus (IAV) H1N1 along with their proteinaceous, aqueous and n-butanol fractions. Additionally, artificially fermented BCP has been evaluated against IAV (H1N1). Antiviral activity was assessed in vitro by comparative real-time PCR assay. IC50 values ranged from 0.022 to 10.04 mg/mL, and Selectivity Index (SI) values ranged from 1.06 to 338.64. Artificially fermented BCP samples AF5 and AF17 demonstrated higher SI values than unfermented BCP, and proteinaceous fractions demonstrated the highest SI values. The chemical profile of BCP and BB samples, analyzed using NMR and LC-MS, revealed the presence of specialized metabolites that may contribute toward the antiviral activity. Overall, the significant anti-IAV activity of BB and BCP harvested in Thessaly (Greece) could be attributed to chemical composition (especially undiscovered yet proteinaceous compounds) and possibly to microbiome metabolism. Further research regarding the antiviral properties of BCP and BB will elucidate the mode of action and could lead to new treatments against IAV or other viral diseases.

A panoramic view of the genomic landscape of the genus Streptomyces.

We delineate the evolutionary plasticity of the ecologically and biotechnologically important genus Streptomyces, by analysing the genomes of 213 species. Streptomycetes genomes demonstrate high levels of internal homology, whereas the genome of their last common ancestor was already complex. Importantly, we identify the species-specific fingerprint proteins that characterize each species. Even among closely related species, we observed high interspecies variability of chromosomal protein-coding genes, species-level core genes, accessory genes and fingerprints. Notably, secondary metabolite biosynthetic gene clusters (smBGCs), carbohydrate-active enzymes (CAZymes) and protein-coding genes bearing the rare TTA codon demonstrate high intraspecies and interspecies variability, which emphasizes the need for strain-specific genomic mining. Highly conserved genes, such as those specifying genus-level core proteins, tend to occur in the central region of the chromosome, whereas those encoding proteins with evolutionarily volatile species-level fingerprints, smBGCs, CAZymes and TTA-codon-bearing genes are often found towards the ends of the linear chromosome. Thus, the chromosomal arms emerge as the part of the genome that is mainly responsible for rapid adaptation at the species and strain level. Finally, we observed a moderate, but statistically significant, correlation between the total number of CAZymes and three categories of smBGCs (siderophores, e-Polylysin and type III lanthipeptides) that are related to competition among bacteria.

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.

Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics.

Non-ribosomal peptide synthetases (NRPS) and type-I polyketide synthases (PKS-I) are multimodular enzymes involved in biosynthesis of oligopeptide and polyketide secondary metabolites produced by microorganisms such as bacteria and fungi. New findings regarding the mechanisms underlying NRPS and PKS-I evolution illustrate how microorganisms expand their metabolic potential. During the last decade rapid development of bioinformatics tools as well as improved sequencing and annotation of microbial genomes led to discovery of novel bioactive compounds synthesized by NRPS and PKS-I through genome-mining. Taking advantage of these technological developments metagenomics is a fast growing research field which directly studies microbial genomes or specific gene groups and their products. Discovery of novel bioactive compounds synthesized by NRPS and PKS-I will certainly be accelerated through metagenomics, allowing the exploitation of so far untapped microbial resources in biotechnology and medicine.

Antiviral Activity of Bee Products.

Honey bees provide many products exerting a wide range of benefits to humans. Honey, propolis, royal jelly, beeswax, bee venom, bee pollen and bee bread have been used as natural medicines since ancient times because of their therapeutic effects. These products have demonstrated healing properties against wounds, diabetes, gastrointestinal diseases, cancer, asthma, neurological diseases, bacterial and viral infections. The antibacterial and antibiofilm activity of honey bee products is widely studied, and a huge body of evidence supports it. On the other hand, their antiviral effect has not been extensively studied. However, recent research has demonstrated their potential against various viral infections including SARS-CoV-2. Hence, honey bee products could be alternatives to treat viral diseases, especially when there is no effective treatment available. This narrative review aims to present up-to-date data (including ongoing clinical trials) regarding the antiviral activity of honey bee products, aiming to elucidate how honey bee product supplementation contributes to antiviral treatment.

Investigation of Serotype Prevalence of Escherichia coli Strains Isolated from Layer Poultry in Greece and Interactions with Other Infectious Agents.

Colibacillosis is the most common bacterial disease in poultry and it is caused by avian pathogenic Escherichia coli (APEC), which is assigned to various O-serogroups. Previous studies have shown that APEC strains are more often related to certain O-serogroups such asO78, O2 and O1. E. coli has been reported to act either as a primary or secondary agent in complicating other infections. The aim of this study was to investigate the occurrence of and characterize the O-serogroups of E. coli strains isolated from commercial layer and layer breeder flocks showing macroscopic lesions of colibacillosis and increased or normal mortality in Greece. Furthermore, we attempted to assess the interaction between infectious agents such as Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), infectious bronchitis (IBV) and infectious laryngotracheitis (ILT) with E. coli infections in layer flocks with increased mortality. Our study revealed that in addition to the common serogroups (O78, O2), many other, and less common serogroups were identified, including O111. The O78, O111 and O2 serogroups were frequently detected in flocks with lesions of colibacillosis and increased mortality whereas O2, O88 and O8 were reported more commonly in birds with colibacillosis lesions but normal mortality rates. These data provide important information for colibacillosis monitoring and define preventative measures, especially by using effective vaccination programs because E. coli vaccines are reported to mainly offer homologous protection. Finally, concerning the association of the four tested infectious agents with E. coli mortality, our study did not reveal a statistically significant effect of the above infectious agents tested with E. coli infection mortality.

A Comparative Analysis of the Core Proteomes within and among the Bacillus subtilis and Bacillus cereus Evolutionary Groups Reveals the Patterns of Lineage- and Species-Specific Adaptations.

By integrating phylogenomic and comparative analyses of 1104 high-quality genome sequences, we identify the core proteins and the lineage-specific fingerprint proteins of the various evolutionary clusters (clades/groups/species) of the Bacillus genus. As fingerprints, we denote those core proteins of a certain lineage that are present only in that particular lineage and absent in any other Bacillus lineage. Thus, these lineage-specific fingerprints are expected to be involved in particular adaptations of that lineage. Intriguingly, with a few notable exceptions, the majority of the Bacillus species demonstrate a rather low number of species-specific fingerprints, with the majority of them being of unknown function. Therefore, species-specific adaptations are mostly attributed to highly unstable (in evolutionary terms) accessory proteomes and possibly to changes at the gene regulation level. A series of comparative analyses consistently demonstrated that the progenitor of the Cereus Clade underwent an extensive genomic expansion of chromosomal protein-coding genes. In addition, the majority (76-82%) of the B. subtilis proteins that are essential or play a significant role in sporulation have close homologs in most species of both the Subtilis and the Cereus Clades. Finally, the identification of lineage-specific fingerprints by this study may allow for the future development of highly specific vaccines, therapeutic molecules, or rapid and low-cost molecular tests for species identification.

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.