Hypersaline solar saltworks represent unique ecological niches that harbor extremophilic microalgae with considerable biotechnological potential. Within these environments, members of the genus Dunaliella are particularly noteworthy due to their remarkable metabolic plasticity and ability to accumulate high-value biomolecules. In the present study, we investigated the biodiversity of Dunaliella in hypersaline saltworks by isolating and identifying autochthonous strains and assessing their growth kinetics and biomass biochemical composition in the context of potential biotechnological applications. Specifically, sixteen strains of Dunaliella were isolated from evaporation and crystallizer ponds of the Kalloni saltworks in Lesvos, Greece, and subjected to an integrative characterization combining morphological observations, molecular phylogenetics, growth kinetics, and biochemical profiling. Phylogenetic analyses based on four genetic markers (18S, ITS, rbcL, tufA) consistently resolved the isolates into three distinct clades: one corresponding to Dunaliella salina/D. minutissima, one to D. parva, and a third representing a clearly divergent lineage. Growth assays revealed marked variability in cell density, biomass productivity and specific growth rate, with certain strains exhibiting enhanced proliferation under controlled conditions. Biochemical analyses demonstrated distinct allocation patterns, with evaporation pond isolates comparatively enriched in proteins (up to 60.8% DW), whereas crystallizer pond isolates accumulated higher levels of carbohydrates (up to 19.0% DW), carotenoids (up to 7.34% mg g-1 DW) and phenolic compounds (up to 8.68% mg GAE g-1 DW). Antioxidant assays (FRAP, TEAC) further indicated significantly elevated reducing and radical scavenging activities among crystallizer isolates. These findings expand current knowledge on the biodiversity of autochthonous Dunaliella strains and support their potential as sustainable sources of bioactive compounds for applications in the agri-food, nutraceutical, pharmaceutical, and cosmeutical sectors.

Hydroponic systems are gaining prominence in sustainable agriculture, yet their nutrient-rich effluents remain an underexplored source of microbial biodiversity with potential biotechnological interest. In this study, shotgun metagenomic sequencing was employed to profile, with a high taxonomic resolution, the photosynthetic microbial community in hydroponic effluent before and after a natural algal bloom, revealing pronounced shifts in microbial composition. Notably, relative abundance increased sixfold for Chlamydomonas reinhardtii and tenfold for Bigelowiella natans. Four dominant microalgal strains (PR1-PR4) were subsequently isolated and characterized through integrative morphological and molecular taxonomy, with phylogenetic analyses based on four genetic markers (18S rRNA, ITS, rbcL and tufA) confirming that each isolate represents a distinct lineage within Chlorophyceae families, including Chlorella sp., Chlamydomonas sp., and Scenedesmus sp. Growth kinetics under three temperature regimes, typical of Greek environmental conditions from spring to autumn (15 °C, 23 °C, 32 °C), demonstrated broad ecological plasticity and rapid biomass production, highlighting strains with strong adaptive resilience. Biochemical profiling of the isolates revealed significant inter-strain differences in primary and secondary metabolite content, including proteins (up to 43% DW), lipids (up to 31% DW), carbohydrates (up to 44% DW), photosynthetic pigments, phenolics, flavonoids, and antioxidant activity. The observed metabolic diversity of autochthonous microalgal strains from hydroponic environments, combined with their high growth rates, underscores their potential for applications in bioremediation, bioenergy, and the development of value-added products within a circular bioeconomy framework.

Background: A Tetraselmis chui-derived ingredient exhibiting a high superoxide dismutase (SOD) activity (TSOD) has previously been shown to act as a cellular health promoter, activating cellular antioxidant and anti-inflammatory mechanisms, and protecting DNA from damage, among other bioactivities. To date, the potential of TSOD to protect telomeric DNA from shortening has not been studied. Aim: In this study, potential protective effects of TSOD on telomere length were evaluated in neonatal human fibroblasts both under standard and oxidative conditions induced by H2O2. Methods: Based on cytotoxicity tests, three different dilutions of a phosphate-buffered saline extract of TSOD (TSOD_A: 1/160, TSOD_B: 1/320, TSOD_C: 1/640) were assessed after four and eight weeks of treatment using the proprietary telomere analysis technology. Results: Under standard culture conditions, no significant differences in telomere-related variables were observed between control and treated cells after 4 weeks. However, by week 8, cells treated with TSOD_A exhibited a higher 20th percentile compared to the untreated control. Under oxidative stress conditions, both TSOD_A and TSOD_C significantly increased both median telomere length and 20th percentile, and reduced critically short telomeres (< 3 kbp) and the telomere shortening rate. Notably, TSOD_A maintained these significant differences also after 8 weeks of exposure. Conclusion: Taken together, these results demonstrated a protective effect of TSOD on telomere length in human cells, supporting the potential of TSOD to combat the deleterious effects of telomere shortening, thus promoting healthy ageing and longevity.

The application of microalgal biostimulants has emerged as a sustainable strategy to enhance crop performance, improve stress tolerance, and reduce the environmental footprint of agricultural practices. The present study investigates the biostimulant potential of Chlorella vulgaris extract on lettuce (Lactuca sativa L.) grown hydroponically under both normal and salinity stress conditions. Two extract concentrations (CV10 and CV50) were foliarly applied, and their effects on plant growth, nutrient balance, stress tolerance, and metabolomic responses were assessed. Both concentrations significantly promoted lettuce growth under normal conditions and effectively mitigated the negative impacts of salinity stress. Application of C. vulgaris extract improved nitrate balance and reduced nitrate accumulation in both normal and stressed plants, thereby enhancing nutritional quality. The extract also alleviated oxidative stress induced by salinity, boosting antioxidant capacity by increasing phenolic and flavonoid content. Metabolomic analysis revealed that C. vulgaris triggered distinct metabolic reprogramming, particularly at the higher concentration (CV50), influencing processes related to osmotic adjustment, energy metabolism, and secondary metabolite biosynthesis. Salinity-stressed plants treated with CV50 exhibited significant accumulation of key sugars, amino acids, organic acids, and phenylpropanoids associated with stress resilience. These findings underscore the multifaceted role of C. vulgaris extract in improving lettuce yield, physiological performance, stress tolerance, and nutritional quality, while also reducing nitrate content.

Microalgae have gained significant attention as sustainable sources of high value compounds, such as bioactive polysaccharides that are usually rich in sulfated groups and exhibit antioxidant properties. Here, 14 new microalgae strains of the genera Tetraselmis, Dunaliella, and Nannochloropsis, isolated from Greek coastal lagoons were analyzed to quantify and characterize their polysaccharide content. Heterogeneity was observed regarding the content of their total sugars (5.5-40.9 g/100 g dry biomass). The strains with a total sugar content above 20% were analyzed concerning the content of total, α- and β-glucans. Tetraselmis verrucosa f. rubens PLA1-2 and T. suecica T3-1 were rich in β-glucans (11%, and 8.1%, respectively). The polysaccharides of the two Tetraselmis strains were isolated and they were mainly composed of glucose and galactose. The isolated polysaccharides were fractionated using ion-exchange-chromatography. The anionic fraction from T. verrucosa f. rubens PLA1-2 was rich in sulfated polysaccharides, had antioxidant capacity, and exhibited healing properties. The anionic polysaccharides from the two Tetraselmis strains did not negatively influence the viability of human cells, while exhibiting antiviral properties against the replication of Hepatitis C Virus (HCV), with median efficient concentrations (EC50) at a range of 210-258 μg/mL.

Microbial bioconversion of agro-industrial by-products into high-value-added metabolites such as polysaccharides or lipids serves a dual purpose: mitigating environmental pollution through waste reduction and supporting the development of novel bioproducts. In this study, a non-conventional, poorly studied Cryptococcus albidus strain was initially assessed for its ability to grow on semi-defined media containing lactose, glycerol, or glucose under three distinct nitrogen availability conditions at C/N equal to 20, 80, and 160 mol/mol in shake flask cultures. The goal was to evaluate biomass production and synthesis of valuable metabolites under these conditions. C. albidus demonstrated robust growth on all commercial carbon sources, particularly under nitrogen-rich conditions, producing more than 25.0 g/L of microbial biomass with a high intracellular polysaccharide content (>45%, w/w). Additionally, mannitol production was detected in cultures with glycerol and glucose (9.1 and 13.1 g/L, respectively), especially after nitrogen depletion. Subsequently, C. albidus and a Cutaneotrichosporon curvatus strain were batch-cultivated using pretreated secondary cheese whey (SCW) as a carbon-rich waste substrate. When cultivated on SCW, both yeast strains partially metabolized lactose and produced polysaccharide-rich biomass, dominated by β-glucans (>29% of total biomass), compounds known for their functional and bioactive properties. The cellular polysaccharides (cPS extracted from C. albidus exhibited cytotoxic effects against cancer cells, suggesting their potential use as biological response modifiers. In contrast, the cPS from C. curvatus did not affect cell viability, indicating their promise as ingredients for applications in the food, feed, pharmaceutical, or cosmetic sectors.

Therapeutic options against metallo-β-lactamase producing P. aeruginosa (MBL-PA) are limited due to multi-drug resistance. A jumbo phage isolated from wastewater in Greece was characterized microbiologically and genetically and evaluated for its potential as a therapeutic agent alone or in combination with antibiotics in an experimental thigh infection mouse model. The host range of the jumbo phage vB_PaerM_AttikonH10 (AttikonH10) against 20 MBL-PA clinical isolates and 10 susceptible strains, one-step phage growth and growth curves of mid-exponential phase bacteria upon addition of the phage were analyzed. Whole-genome sequencing was performed and the de novo assembled complete phage genome was compared with other jumbo phages. In vivo pharmacokinetics in different tissues as well as the efficacy of two dosing regimens 109 and 106 PFU/mouse administered intraperitoneally alone and in combination with amikacin (384 mg/kg/day) was tested against an MBL-PA clinical isolate in murine thigh infection model. The phage formed small plaques in double-layer agar and demonstrated clear or semi-clear lysis in 83.3

Smart packaging is a continuously evolving sector that may provide solutions to several cold chain management challenges and shows great potential toward food waste reduction. Fish is a highly nutritious yet perishable commodity with increasing statistics of annual loss and waste. This study investigates the application of pH-sensitive indicators on fish products, aiming to analyze and highlight the importance of comprehensive monitoring of the physicochemical alterations and their interactions with the headspace composition during refrigerated storage of fresh fish. Insights into this process can contribute to developing more practical spoilage indicators and dynamic shelf life prediction methods. A brief case study is presented on the development and application of a smart indicator prototype. The freshness pH indicator was prepared by incorporating methyl red into a starch and cellulose matrix using the dip coating method and was tested for its sensitivity to pH, ammonia vapor, and the detection of gilthead sea bream spoilage. The deterioration in the quality of sea bream fillets, as indicated by the observed color change of the indicator, was confirmed through microbiological and chemical analyses of the fish flesh. The color response of the pH indicator (red–pink turned pale yellow) was found to correlate with fish alteration patterns and reflected the headspace gas composition thus enabling “real-time” monitoring of fish spoilage.

The connection between wine microbiota and terroir has become increasingly significant in the wine industry in recent years. Indigenous yeasts have emerged as a valuable tool for imparting unique qualities to wine, enhancing the aroma characteristics specific to a given wine-producing region. This study aimed to isolate indigenous Saccharomyces cerevisiae strains from spontaneous fermentations and assess their impact on the fermentative performance, chemical composition, and wine aroma profiles, using two commercial strains serving as controls. Fermentation kinetics, organic acids, ethanol, and glycerol content were monitored daily using HPLC. The produced wines were evaluated for their organoleptic properties and underwent volatile compound profiling using GC-MS. Additionally, the gene transcription patterns of the isolated yeasts and their connection to the resulting oenological traits were further explored, employing RNAseq during fermentation. Our study revealed that native strains supported volatilome, promoted ester and terpene formation, enhanced fruity, floral, and sweet attributes, and contributed to a distinct wine aroma compared to the control ones. Of particular interest were the differences in organic acid metabolism and glycerol formation. Transcriptomes of the indigenous yeasts identified different genomic responses and explained the variations in metabolite production between strains. In conclusion, the data obtained highlights the different transcriptomic and metabolic profiles of the indigenous yeasts isolated during this study. Besides, the importance of understanding yeast genomics and metabolism to achieve promising sensory characteristics and unique wine styles was emphasized, and these insights could contribute to the development of new products while preserving the identity of a region.

Soil salinization and its accompanying soil degradation pose a major threat to plant growth and the sustainability of terrestrial ecosystems worldwide. Therefore, exploring methods to improve the efficiency of phytoremediation of saline soils has been the focus of current research. The symbiosis between biological nitrogen-fixing (BNF) plants and rhizosphere microorganisms plays a crucial role in improving plant resilience and to cope with salt stress. Especially the interaction between nitrogen (N2)-fixing bacteria and mycorrhizal fungi can mitigate the negative effects of salt stress on N2-fixing plants. However, a comprehensive review on the mechanisms of interaction between N2-fixing bacteria and mycorrhizal fungi that confer salt tolerance to N2-fixing plants is still lacking. In this review, we summarize the effects of salt stress on N2-fixing plants and their root colonizing microorganisms with a focus on mechanisms of interaction between N2-fixing bacteria and mycorrhizal fungi under salt stress. These interactions enhance host plant resilience through nutrient complementation, hormonal regulation, and improved antioxidant capacity, but may also be antagonistic through nutrient competition. Finally, we identified research gaps of the analyses of the tripartite symbioses of N2-fixing plants, N2-fixing bacteria, and mycorrhizal fungi to elucidate future research directions.

Mutualistic symbioses between plants and microorganisms have served as a cornerstone for terrestrial ecosystem establishment since the colonization of land by plants ca. 470 million years ago (Ma). These mutualisms diversified in symbiont partners and ecological functions in response to dynamic environmental shifts, with root-based architectures emerging later as a key adaptive innovation. Phylogenomic analyses reveal a conserved common symbiotic signalling pathway (CSSP) through the mycorrhizal-actinorhizal-rhizobial (MAR) evolutionary trajectory, underscoring convergent evolutionary mechanisms that facilitated the repeated emergence of mutualistic root-microbe interactions. Despite this shared foundation, recent studies highlight lineage-specific adaptations in symbiont recognition, immune evasion, and nutrient exchange, reflecting divergent evolutionary pressures and ecological niches. For instance, actinorhizal symbioses, although understudied compared to legume-rhizobia systems, exhibit unique adaptations in host specificity and nitrogen-fixation efficiency, offering untapped potential for sustainable agriculture and reforestation. This review synthesizes information from different disciplines to elucidate the origin and diversification of root symbioses, emphasizing molecular innovations and ecological drivers that shaped their evolution. We further explore the role of environmental pressures, such as resource availability and climate change, in driving the adaptive diversification of these symbiotic relationships. By integrating evolutionary, molecular, and ecological perspectives, this work advances our understanding of root symbioses as dynamic systems shaped by both conserved mechanisms and context-dependent adaptations.

Polysaccharides produced by Papiliotrema laurentii strains are of great interest due to their functional properties and potential applications in the food, feed, pharmaceutical, and cosmetic industries. Microbial bioconversion of agro-industrial by-products into high value-added compounds, such as polysaccharides, is of major importance within the framework of the circular economy, as it significantly reduces bioprocessing costs. The present study aimed to produce cellular (cPS) and released extracellular (EPS) polysaccharides through the biotechnological valorization of secondary cheese whey (SCW), a major by-product of cheese manufacturing. The non-extensively studied strain P. laurentii NRRL Y-2536, cultivated on pretreated SCW, produced biomass (14.8 g/L) rich in intracellular polysaccharides (>46 %, w/w), as well as released EPS (22.7 g/L – 504 mg/L/h). cPS were extracted using high-pressure homogenization and deproteinized in the presence of n-butanol–chloroform solvents. Both cPS and EPS were analyzed via FTIR, HPLC, and FRAP assay to assess their structure, composition, and antioxidant capacity. The effects of cPS and EPS on the viability of two human cancer cell lines, Caco-2 and HT29-MTX, were evaluated. The cPS of P. laurentii NRRL Y-2536 and P. laurentii NRRL YB-3594 were rich in β-glucans (>62 % of total cPS), while the EPS were composed of galacto-xylo-mannans. Notably, the cPS from P. laurentii YB-3594 exhibited the highest antioxidant activity. Both cPS isolates, along with the EPS from P. laurentii Y-2536, presented cytotoxic activity at the highest concentrations tested, suggesting their potential application as anticancer agents. In contrast, the heat-treated EPS from P. laurentii Y-2536 and the untreated EPS from P. laurentii YB-3594, which were non-cytotoxic, could be further explored as functional ingredients for food, feed, cosmetic, or biomedicine applications.

Legumes produce highly nutritious seeds, making them a valuable resource of nutrients for both humans and livestock. In addition to contributing to global food security, they can also provide an ecologically sustainable and cost-effective means of soil fertilization by initiating nutrient cycling in nutrient-limited soils through symbiotic associations with beneficial microbes such as nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF). However, climate change-induced xerothermic conditions and soil salinity may threaten these beneficial associations between symbionts. Changing environmental conditions and increasing earth’s population require the development of new sustainable products that will support not only plant nutrition but also plant growth and productivity under extreme conditions. This challenge can be addressed by isolating natural beneficial microbes from extreme environments and applying optimized combinations of selected strains that carry interesting and compatible traits to legume crops. To achieve this objective, rhizobial and AMF strains were isolated from wild legumes grown in extreme environments throughout Greece and Cyprus and are currently being evaluated for their application as microbial consortia in pasture legumes for increased rangeland production under climate change. Compatibility between symbiotic partners, functionality of symbiotic relationships and performance of symbiotic plants are investigated at the ecophysiological, molecular and microbial community levels. Furthermore, in order to overcome the lack of knowledge about the molecular mechanisms regulating the interactions between symbiotic partners and to test the efficacy of different rhizobia-AMF co-inoculation strategies, studies are also being conducted in the model legume Lotus japonicus . Roots of 45 different species of wild legumes and their rhizosphere were collected from 22 locations in Greece and Cyprus, and functional nodules were obtained from 12 species. A total of 324 pure rhizobia strains were isolated and the most valuable strains were selected based on their tolerance to abiotic stress (tested in vitro ) and their ability to nodulate the legumes Trifolium resupinatum and Medicago sativa under salinity and drought conditions. In parallel, trap cultures were established to enrich AMF spores from the sampled rhizospheres, and more than 20 AMF spore morphotypes were isolated and re-inoculated to trap cultures for a second reproduction cycle. To optimize the composition of beneficial microbial consortia, legume plants were co-inoculated with different combinations of selected rhizobia and AMF strains and subjected to salinity or drought stress with the aim of achieving compatibility between symbiotic partners and improved plant fitness. Application of microbial consortia is expected to improve the survival, productivity, and nutritive value of pasture plants and promote grazing livestock nutrition and carbon sequestration in pasture soils. This study will contribute to the development of innovative bio-based products, creating new opportunities for sustainable agriculture and investment.

The global food industry faces significant challenges related to food spoilage, particularly in aquaculture, where pathogens like Vibrio species are major disease-causing agents. This study investigated the use of bacteriophage Athena1 as a biocontrol agent in biodegradable coatings for fish contaminated with Vibrio alginolyticus V1, evaluating its stability, lytic efficacy, and performance within biopolymer-based coatings. Athena1 demonstrated stable lytic activity across various pH and temperature conditions. When directly applied to fish slices, the phage significantly inhibited V. alginolyticus V1 growth, confirming its effectiveness in complex matrices like fish flesh. Results coming from the application of Coating+Athena1 varied: chitosan exhibited strong inherent antimicrobial properties with limited added benefit from the phage, while methylcellulose managed to maintain phage stability and showed effective bacterial inhibition. Sodium alginate, however, displayed inconsistent phage activity, potentially due to interactions with other fish spoilage microbiota. Overall, these findings suggest that Vibrio-infecting bacteriophages hold potential as biocontrol agents in active biodegradable coatings and films for fish products. Further research should focus on optimizing phage release and stability across biopolymer matrices to enhance practical applications in food preservation, with emphasis on selecting tailored bacteriophages for effective biocontrol.

Symbiotic associations between plants and microorganisms are crucial to global biogeochemical cycling and ecosystem stability. Mycorrhizal fungi and nitrogen (N2)-fixing bacteria are recognized as the two main groups of microorganisms involved in such symbiotic interactions. They not only constitute the most wide-spread symbiotic microorganisms, but also ensure plants to acquire additional N resources directly from the atmosphere. Although plant-microbial interactions, for example, the performance of AM-plant and rhizobia-legume plant symbioses, have been well studied and reviewed in detail previously, still less information is known about these processes in actinorhizal symbioses. The present review is aimed to summarize current knowledge of the interaction of partners in actinorhizal root symbioses, in particular the signalling processes during establishment of BNF, and the specificity of and dependency on different symbiotic partners in this interactions, based on evolution and distribution in the plant and microbial kingdom. The features of nutrient transfer in these root symbiotic relationships and the significance of actinorhizal symbioses for the performance of plants under environmental stress are discussed and compared with AM and rhizobia-legume symbioses. In addition, research gaps in actinorhizal root symbioses research are identified and future research avenues are suggested.

Soil salinisation and concomitant soil degradation pose a major threat on plant performance and terrestrial ecosystem sustainability. Rhizobia and arbuscular mycorrhizal fungi (AMF) were reported to mitigate negative effects of adversity in their host plants. Here, we exposed two cultivars of Robinia seedlings (salt-tolerant cultivar Lu155 and salt-sensitive cultivar DB) inoculated with salt-tolerant and salt-sensitive rhizobia and/or AMF to salt treatment. The results showed that individual inoculation with salt-tolerant rhizobia greatly reduced Na+ and increased K+ contents and K+/Na+ ratios in leaves and roots upon salt exposure. Co-inoculation of salt-tolerant rhizobium and AMF greatly decreased Na+ contents in leaves and increased K+ contents and Na+/K+ ratios in roots and leaves of Robinia Lu155 more than individual inoculations. Inoculation with salt-tolerant rhizobium up-regulated the expression of genes involved in K+ and Na+ homeostasis (RpSOS1, RpHKT1, RpNHX1 and RpSKOR) in roots and leaves of the Lu155 Robinia cultivar, particularly upon co-inoculated with AMF. Apparently, the positive effects of salt-tolerant rhizobia and additive effect of co-inoculation of salt-tolerant rhizobia and AMF on K+ contents and K+/Na+ homeostasis of Robinia plants under salt exposure are mediated by upregulated expression of transporter genes in Robinia roots.

According to the Food and Agriculture Organization of the United Nations (FAO) more than 14% of the world's food production is lost every year before reaching retail, and another 17% is lost during the retail stage. The use of the expiration date as the main estimator of the life-end of food products creates unjustified food waste. Sensors capable of quantifying the effective food freshness and quality could substantially reduce food waste and enable more effective management of food chain. We propose an electrolyte-gated organic transistor (EGOT) that responds to the release of biogenic amines, like diamines and tyramine, generated by degradation of protein-rich food. The EGOT sensor features a polymeric poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) gate electrode fabricated in the shape of a miniaturized beaker containing an aqueous solution in the inner side (to be exposed to food) and capacitively coupled through a hydrogel to the transistor channel on the outside (not in contact with food). The hydrogen bonds formed by the water-dissolved amines with PEDOT:PSS modulate the EGOT channel across a wide range of amine concentrations. We demonstrate that our sensor can detect different amines by the combinatorial analysis of the response from different channel materials, PEDOT:PSS and the other DPP-DTT, with a limit of detection as low as 100 pM.

Edible raw materials have gained attention as sustainable food packaging films, which are often considered a priori safe for human consumption. However, cytotoxicity issues may arise due to the incorporation of additives or modifications of film functionality during the manufacturing process. This study introduces an integrated methodology for the evaluation of potential migration of cytotoxic substances from materials used for the development of conventional and biodegradable food packaging. Carboxymethyl cellulose (CMC) and sodium alginate (SA) were tested as raw materials of an edible (CMC-SA) film, while a low-density-polyethylene (LDPE) film was tested as a conventional material. The CMC-SA film exhibited higher water vapor transmission rate and water vapor permeability, and lower hydrophobicity compared to LDPE (WVTRCMC-SA=1457.87 vs. WVTRLDPE=3.43 g×m-2×day-1, WVPCMC-SA=43.24 vs. WVPLDPE=0.0048 g×m-2×mm×day-1×kPa-1 and CACMC-SA=52.05 vs. CALDPE=94.28°, respectively). An analytical protocol based on EU Regulation 10/2011 was introduced, to evaluate the potential migration of cytotoxic packaging substances into food simulants, using different human cells. Caco2 cells were used to simulate human intestine, whereas Huh7 and Immortalized Human Hepatocytes (IHH) cells simulated human liver. Cell viability assays and gene expression results indicated that substances migrating from the tested packaging materials neither produced cell cytotoxicity, nor induced oxidative stress to Caco2 cells.

According to the ECDC in 2023, Greece presented a high rate of carbapenem-resistant Klebsiella pneumoniae (CRKP). Samples from the largest Athens' sewage treatment plant (n = 50), rectal swabs (n = 50), and stool (n = 10) from ICU and pediatric patients were screened for phages using the double-layer agar method. Plaque morphology, genomic features, biological properties (host range, phage adsorption time and rate, latent period length and burst size) and growth inhibition kinetics were determined for all isolated phages. Phage tissue distribution and efficacy against a dominant ST11 CRKP clinical isolate were assessed in murine thigh infection model. A cocktail from different phages was designed based on biological characteristics and host range spectrum. In total 7 distinct bacteriophages were isolated from different samples 5 Drulisvirus, 1 Webevirus and 1 Przondovirus with varied lytic activity against 40 (18-50) % of all isolates in host-range studies and 51 (41-85) % growth inhibition at the lowest MOI 0.00001 in growth curve experiments. The median (range) adsorption rate, time, latent phase and burst size in one-step growth experiments were 94 (88-97) %, 6 (2-10) min, 25 (15-40) min, and 63 (47-160) PFU/infected cell, respectively. Phages were rapidly distributed in different organs with high titers up to 8h and a 4log bacterial load reduction was found with the highest dose 109 PFU/mouse. A cocktail of 5 phages was effective against 87.5 % of ESBL, NDM and VIM-producing isolates. Several phages with potent lytic activity and different host spectrum range against CRKP isolates were found. The 5-phage cocktail could be used for phage therapy of CRKP infections.