Search Results (114)

Background: Live biotherapeutic products (LBPs) require robust genetic stability and effective biocontainment to support safe clinical translation and regulatory acceptance. Aim: This study presents a single-step chromosomal engineering strategy that integrates auxotrophy-mediated biocontainment with therapeutic gene insertion to support regulatory-oriented live biotherapeutic chassis design. Methods: A no-SCAR genome-editing approach combining CRISPR/Cas9 and λ-Red recombineering was used to generate an Escherichia coli MG1655 ΔilvC::hlyA strain by replacing ilvC with the hlyA gene encoding listeriolysin O. Chromosomal and episomal expression systems were compared for auxotrophy, growth, haemolytic activity, plasmid stability, and intracellular DNA delivery to RAW 264.7 macrophages. Results: Auxotrophy was successfully established and restored by branched-chain amino acid supplementation. Chromosomal integration preserved haemolytic activity and bacterial growth while improving long-term genetic stability and plasmid maintenance compared with episomal expression. Both systems supported intracellular DNA delivery, whereas the chromosomal construct showed improved host-cell preservation under higher bacterial challenge. Conclusions: This proof-of-concept study supports the feasibility of using a single-step chromosomal engineering strategy to combine intrinsic biocontainment with therapeutic-gene integration in an engineered bacterial chassis. Full article

Background/Objectives: Therapeutic plasma exchange (TPE) is an extracorporeal treatment used in thrombotic microangiopathies (TMAs) and various autoimmune and neurological disorders. However, data regarding its use during early pregnancy remain limited. This study aimed to evaluate maternal laboratory response and perinatal outcomes in pregnant women who underwent TPE before 26 weeks of gestation. Methods: This retrospective case series included 10 pregnant women diagnosed before 26 weeks of gestation who underwent TPE between 2010 and 2023. Clinical and laboratory parameters before and after TPE were compared. Results: Indications for TPE included HELLP syndrome (n = 4), thrombotic thrombocytopenic purpura (n = 3), presumed atypical haemolytic uremic syndrome (n = 1), neuromyelitis optica (n = 1), and Guillain–Barré syndrome (n = 1). The mean gestational age at diagnosis was 22.1 ± 3.1 weeks, and the mean gestational age at delivery was 27.1 ± 6.9 weeks. Five fetuses (50%) died and five (50%) survived to discharge. In patients with TMAs, TPE was associated with significant decreases in LDH, INR, APTT, ALT, AST, and total bilirubin levels, along with a significant increase in platelet count and ADAMTS13 activity (p < 0.01). No maternal complications occurred in neurological cases, all of which resulted in term deliveries with healthy neonates. Conclusions: In this uncontrolled case series, TPE was associated with rapid maternal clinical and laboratory improvement in selected pregnant women with TMAs, although a causal effect cannot be established from these data. However, perinatal outcomes were primarily determined by gestational age at delivery: all fetal losses occurred before 26 weeks, whereas all infants survived when delivery occurred after 26 weeks. Larger studies are needed to confirm these findings. Full article

Iron is essential for life, but its safe use by the body depends on it being kept within tightly controlled compartments. When this compartmentalisation is disrupted—through haemolysis, saturation of scavenger proteins, or dysregulation of the hepcidin–ferroportin axis—damaging iron species accumulate in the circulation and within vascular cells, with potentially serious consequences for endothelial function. This review explores the mechanisms by which iron dysregulation compromises vascular endothelial cell biology across a range of disease states, including haemolytic anaemias, atherosclerosis, cerebrovascular disease, extracorporeal circulatory support, and iatrogenic iron loading. Common pathological themes emerge: depletion of nitric oxide bioavailability, oxidative stress, endothelial activation, and in chronic settings, vascular remodelling. The review subsequently focuses in depth on the pulmonary vasculature, where dysregulated iron compartmentalisation has emerged as a key contributor to the pathogenesis of pulmonary hypertension. Here, iron-driven mitochondrial dysfunction, smooth muscle cell proliferation, and iron-dependent lipid peroxidation via ferroptosis are discussed as mechanistic drivers of pulmonary vascular remodelling. The therapeutic implications of targeting iron handling in pulmonary hypertension are considered, including modulation of the hepcidin–ferroportin axis. Together, the evidence presented highlights disordered iron compartmentalisation as a unifying pathological thread across vascular disease and a compelling target for intervention. Full article

Background/Objectives: The aquaculture industry represents a fundamental food sector. One of the main limiting factors for this sector is related to bacterial diseases, for which antibiotics have been widely used worldwide for decades. In recent years, a more conscious approach to the use of antimicrobials within the framework of the One Health approach has increased the need for alternatives capable of helping with disease management while avoiding the onset of antimicrobial resistance phenomena. Antimicrobial peptides, which have a broad spectrum of action against pathogens, are a promising solution. Methods: In this work, we investigated the capability of three peptides (Trematocine, Chionodracine, and Cnd-m3) isolated from Antarctic fish to target bacterial and viral pathogens affecting aquaculture. Successively, we investigated their cytotoxicity versus a continuous embryonic cell line (DLEC) derived from European sea bass and their haemolytic activity against fish erythrocytes. Moreover, we evaluated their immunomodulatory effect. Results: Regarding antibacterial properties, Cnd-m3 was identified as the best peptide, demonstrating good bactericidal and bacteriostatic activity against various bacterial strains, including Lactococcus garvieae. Concerning this bacterium, ANS permeability assays showed that the Cnd-m3 peptide has a great ability to interact with its outer membrane, while TEM analysis revealed that the peptide, after destabilization of the cell membrane, interacts with nucleic structures. Considering the antiviral activity, Trematocine was effective against two tested pathogenic enveloped viruses. Moreover, the toxicity of Trematocine and Cnd-m3 was evaluated by investigating their cytotoxicity against a cell line derived from Dicentrarchus labrax and haemolysis against sea bass erythrocytes. Both revealed good selectivity towards pathogens at the lowest concentration. Finally, Cnd-m3 manifested light in vitro immunomodulatory properties. Conclusions: Overall, these data provide a solid basis for future studies assessing the potential applications of two of the tested peptides in aquaculture. Full article

Background/Objectives: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) necessitates innovative strategies to overcome conventional resistance mechanisms. This study investigated the potential of the natural flavonolignan silibinin (SIL) as an antivirulence agent against S. aureus, with a particular emphasis on its putative multi-target antibacterial activity and its capacity to potentiate the effects of ciprofloxacin (CIP). Methods: The antibacterial and antivirulence properties of SIL were assessed using both in vitro and in silico approaches. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined, and its synergistic interaction with CIP was evaluated using checkerboard assays. Inhibition of biofilm formation, as well as disruption of established biofilms, was assessed using an MTT-based viability assay. Staphyloxanthin (STX) inhibition was examined through pigment extraction and spectrophotometric quantification of pathway intermediates. Molecular docking studies were conducted to predict the binding affinities of the compounds to key bacterial targets, while safety was evaluated through haemolytic and cytotoxicity assays. Results: SIL exhibited weak to moderate direct antibacterial activity (MICs of 256–512 µg/mL), which is characteristic of many natural product scaffolds. Notably, SIL potentiated the activity of CIP, reducing its MIC by up to fourfold against selected resistant strains of S. aureus. SIL significantly inhibited biofilm formation and disrupted established mature biofilms in a strain-dependent manner. In vitro metabolic profiling and in silico analyses provided mechanistic insights into the effects of SIL on STX biosynthesis. Precursor accumulation data suggest inhibition at the diapophytoene desaturase (CrtN) catalytic step, representing a potential mechanism not previously reported for flavonolignans. Molecular docking studies further predicted favourable binding affinities for CrtM and other key targets. Importantly, SIL exhibited no haemolytic activity and low cytotoxicity in macrophages at synergistic concentrations. Conclusions: This study provides evidence that SIL functions as a dual-action agent, potentiating ciprofloxacin efficacy while reducing STX production and inhibiting biofilm formation, thereby impairing key virulence mechanisms of S. aureus. These findings, together with its favourable safety profile, provide a strong rationale for the development of silibinin-based topical adjuvants to combat drug-resistant Staphylococcus infections in humans. Full article

Staphylococcus aureus is a major cause of skin and soft tissue infections, necessitating the development of new topical agents with rapid bactericidal activity and low resistance potential. Here, we evaluated the antibacterial activity of a pyrazolone copper complex (P-FAH-Cu-phen) against S. aureus, investigated its in vitro mode of action, and its assessed therapeutic efficacy in a murine model of S. aureus-infected skin trauma. P-FAH-Cu-phen exhibited potent bactericidal activity (minimum inhibitory concentration [MIC] 1.4 μg/mL; minimum bactericidal concentration [MBC] 2.8 μg/mL) and rapid killing (>91% eradication within 2.5 min), with no detectable MIC increase under the tested serial passaging conditions. Cell-envelope dysfunction was evidenced by increased supernatant alkaline phosphatase activity, elevated leakage of nucleic acids and proteins, and reduced membrane-associated Na⁺/K⁺- and Ca²⁺/Mg²⁺-ATPase activities. At sub-inhibitory concentrations, P-FAH-Cu-phen reduced haemolytic and coagulase activities, modulated virulence gene expression (sea, hla, agrA), and inhibited biofilm formation and biofilm-associated metabolic activity. In vivo, topical treatment accelerated wound closure and histopathological repair, increased hydroxyproline content, reduced bacterial burden, and lowered TNF-α and IL-10 levels in wound tissues. Collectively, P-FAH-Cu-phen shows multi-faceted anti-infective activity and exhibits further development as a topical candidate for S. aureus-infected skin wounds. Full article

The decline of honey bee health has intensified interest in microbiome-based strategies to support colony resilience and reduce reliance on chemical interventions. In this study, we performed an in vitro probiotic screening of five lactic acid bacteria (LAB) strains of honey bee origin and two multi-strain consortia for prospective application in apiculture. Two formulations were evaluated: LAB Mix 1 (Apilactobacillus kunkeei and Lactobacillus apis) and LAB Mix 2 (Lactiplantibacillus plantarum, Fructobacillus fructosus, and A. kunkeei). Functional and safety-related traits were investigated, including auto-aggregation, cell-surface hydrophobicity, inter-strain compatibility, organic acid production, oxidative detoxification capacity, antibiotic susceptibility, haemolytic activity, and growth dynamics in sugar-based feeding syrups. All strains exhibited time-dependent increases in aggregation and hydrophobicity, with A. kunkeei and F. fructosus showing particularly strong surface-associated properties. No mutual antagonism or haemolytic activity was observed. Organic acid profiling revealed strain-specific metabolic signatures, with high lactic and citric acid production by L. plantarum and LAB consortia. Several strains displayed peroxidase activity, suggesting a role in oxidative stress mitigation. Growth assays demonstrated that high sugar concentrations severely limited bacterial growth, whereas moderate dilution significantly improved growth. Under osmotic stress conditions, mixed cultures generally achieved higher optical density values than individual strains. Collectively, these findings support bee-associated LAB and multi-strain formulations as promising candidates for further probiotic development. Full article

Amazonian communities traditionally use plant leaves to wrap food; however, there is little information available on the species and their health benefits. This study aimed to characterise the physicochemical properties of the samples, including pH, total soluble solids, total titratable acidity, moisture content, ash, and mineral composition determined by atomic absorption spectroscopy. Major bioactive compounds, including vitamin C, organic acids, carotenoids, chlorophylls and derivatives, and phenolic compounds, were determined by liquid chromatography. The antioxidant potential was examined using ABTS and DPPH, antimicrobials (bacteria and fungi), biofilm inhibition (bacteria), and the haemolytic activity of Calathea lutea and Calathea inocephala leaves was evaluated. C. lutea showed high iron (2930.0 mg/100 g DW), vitamin C (4.6 mg/100 g DW), and tartaric acid (722.3 mg/100 g DW). C. inocephala showed high lutein (83.5 mg/100 g DW) and pheophytin b (177.5 mg/100 g DW). Major phenolics included caffeic acid (16,996.3 mg/100 g DW). Extracts at 1 mg/mL inhibited multidrug resistance in Enterococcus faecalis and Enterococcus faecium and showed strong antibiofilm activity against Listeria monocytogenes. The antioxidant activity was 4.6 mmol TE/100 g DW in the DPPH method, and the compound was haemocompatible at concentrations below 600 µg/mL. These findings highlight its biotechnological potential and importance for sustainable community use. Full article

Background: It is well known that host factors are capable of regulating microbial behaviours such as growth, metabolic pathways, virulence properties, and antimicrobial susceptibilities. In this respect, the present study aimed to investigate the relationship between insulin and various virulence properties of Pseudomonas aeruginosa ATCC 27853. Methods: Growth alterations, biofilm formation, motility, haemolytic activity, and pigment production (pyocyanin and pyoverdine) were determined in the presence/absence of three different insulin concentrations (10 µU/mL –20 µU/mL –200 µU/mL) under in vitro conditions. In addition, changes in bacterial virulence were evaluated in an in vivo animal (Caenorhabditis elegans) model. Alterations in growth, haemolytic activity, and pigment production were investigated spectrophotometrically. Biofilm formation was examined using the crystal violet well-plate assay. A soft agar plate method was used to detect swimming motility. Results: According to the results, all three insulin concentrations enhanced the bacterial growth. On the other hand, biofilm production, swimming motility, and haemolytic activity decreased in the presence of all insulin concentrations. Pyocyanin production was shown to be increased in the presence of only 10 µU/mL of insulin, but pyoverdine production did not change. In vivo animal survival rates showed that 200 µU/mL of insulin decreased bacterial virulence. Conclusions: This research demonstrates that P. aeruginosa can sense and respond to mammalian hormones (insulin), which can modulate microbial virulence through diverse mechanisms, providing new insights that may be relevant to infection dynamics. Full article

Drug-resistant bacteria cause millions of global infections each year, and the development of alternative antimicrobial drugs has become a serious undertaking. Currently, peptides with antimicrobial activity represent potential candidates for new antibiotic discovery as they are less likely to cause drug resistance in bacteria. In this study, bifunctional peptides with potent trypsin-inhibitory activity and antimicrobial activity were obtained by rational computation-based structural modifications to a novel Bowman–Birk-type inhibitor (BBI) peptide. The analogues not only displayed potent bacterial killing ability against two drug-resistant bacteria strains of E. coli but also an excellent safety profile, as assessed by low haemolytic activity and low anti-proliferation activity on HaCaT cells. Throughout the molecular dynamics simulations, the peptides exhibited stable adsorption onto the mixed POPE/POPG membrane; most amino acid residues of the AMPs remained bound to the membrane surface, with a few amino acid residues partially penetrating the membrane interior. This showed that the electrostatic interactions were the dominant driving force mediating the peptide–membrane associations. In addition, the tested peptides displayed a degree of stability in the presence of salt ions, serum, and trypsin. These modified peptides thus possess potential as clinical antibacterial agents, and the strategies used in structural modification may also provide a different path to developing new antimicrobial peptides. Full article

Background: The golden era of antibiotics has long passed, and the clinical failures caused by emerging drug-resistant bacteria have intensified the demand for novel antimicrobial agents. Antimicrobial peptides have attracted significant attention as promising candidates for next-generation antibiotics. Methods: In this study, we identified a novel antimicrobial peptide, Caerin 1.1-LC, from the skin secretion of the Australian green tree frog, Litoria caerulea. Subsequent structure–activity relationship studies led us to design a series of analogues and revealed the critical role of the peptide’s intrinsic hinge structure in shaping its biological activity. Results: Incorporation of D-isomers at the valine residues within the hinge preserved overall helical content but altered the hinge conformation, resulting in an 8-fold increase in antibacterial activity against Gram-negative bacteria. Simultaneously, haemolytic activity was markedly reduced, leading to a 56-fold improvement in therapeutic index (from 0.47 to 26.6). Structural modulation of the hinge also switched the mechanism of action from classical membrane disruption with associated permeability changes to a non-membrane-permeabilising, ‘cell-penetrating-like’ behaviour, inducing membrane potential depolarisation and ATP disruption to trigger bacterial death. In vivo studies using infected larval models, along with in vitro LPS neutralisation assays, further demonstrated the therapeutic potential of the D-analogue as a novel antibacterial agent. Conclusions: This work highlights the pivotal role of hinge structures in Caerin-family/hinge-containing AMPs, offering a strategic avenue for optimising antibacterial efficacy. Full article

Lacmellea edulis, traditionally known as chicle, is a species that has received little attention despite its nutraceutical potential. This study aimed to evaluate the physicochemical characteristics (pH, soluble solids, titratable acidity, moisture, ash, and minerals) and the content of bioactive compounds (vitamin C, organic acids, carotenoids, and phenols) of fruits at different stages of ripeness, as well as their antimicrobial (against Candida albicans, Candida tropicalis, Escherichia coli, Staphylococcus aureus, and Streptococcus mutans), antiproliferative and antihaemolytic activity. Bioactive compounds were quantified using liquid chromatography, while biological activities were assessed via spectrophotometric assays. The results revealed a high concentration of ascorbic acid in the ripe pulp (3.0 mg/100 g DW), higher levels of organic acids in the unripe pulp (3947.6 mg/100 g DW), and a high total phenol content in the ripe peel (10,890.9 mg/100 g DW). The peel exhibited the highest antioxidant activity (63.3 mmol ET/100 g DW). Regarding antimicrobial activity, the pulp exhibited the lowest MIC values against E. coli (2.7 mg/mL) and S. mutans (2.6 mg/mL), the peel against S. aureus (21.3 mg/mL) and C. tropicalis (5.3 mg/mL), and the seeds against C. albicans (20.8 mg/mL). Additionally, the peel exhibited the greatest antiproliferative efficacy against cervical (HeLa) and hepatoma (HepG2) cancer cells. None of the evaluated extracts showed significant haemolytic effects, confirming their safety. Overall, L. edulis appears to be a promising source of bioactive metabolites with potential applications in functional foods and pharmaceutical products. Full article

The remarkable metabolic adaptability of Bacillus velezensis, including efficient nutrient use, spore formation, and the secretion of antimicrobial peptides, supports its expanding role in biotechnological applications ranging from crop protection to probiotic development. In this study, the halotolerant strain 24.5 was identified as B. velezensis through 16S rDNA and gyrA gene sequencing. PCR analyses confirmed the presence of genes responsible for polyketides, lipopeptides, and dipeptides biosynthesis. These results indicate the potential for the production of structurally diverse bioactive metabolites. Strain 24.5 demonstrated remarkable antimicrobial activity against 19 bacterial pathogens and three Candida species (p < 0.05). The study demonstrated high survival rates under simulated gastrointestinal conditions, suggesting strong adaptability for gut colonization. Antioxidant evaluation revealed DPPH radical scavenging activities of 34.68% for intact cells and 18.47% for the cell-free extract (p < 0.05). The enzymatic profile highlighted versatile metabolic functions, supporting its multifaceted probiotic potential. Auto-aggregation reached 84.42% at 24 h, and high hydrophobicity toward hexane (71.62%) supported adhesion potential. Antibiotic susceptibility profiling showed sensitivity or intermediate susceptibility to 22 of 24 tested antibiotics (p < 0.05). No haemolytic activity was detected, supporting its safety profile. Overall, these results emphasise the adaptability and multifunctional properties of Bacillus velezensis strain 24.5, highlighting its potential as a promising probiotic candidate for applications in food safety and biotechnology. Full article

The genus Miconia is used in traditional medicine, but there are few studies supporting the bioactive potential of Miconia crocea. This study aimed to evaluate the physicochemical properties, bioactive compound content, and antioxidant, antimicrobial and antihaemolytic activities at four different phenological stages of M. crocea. The pH, soluble solids, titratable acidity, moisture and ash content were determined. Mineral contents were determined by atomic absorption. Vitamin C, organic acids, carotenoids, chlorophylls and derivatives and phenols were determined by chromatography. Total anthocyanins were determined by spectrophotometry. The antioxidant capacity was evaluated using ABTS and DPPH assays, and the antimicrobial activity was tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, Candida albicans and Candida tropicalis. Potassium was the predominant mineral (>1000 mg/100 g DW), while malic acid was the predominant organic acid. Lutein was the most abundant carotenoid, as among the phenolic compounds, m-coumaric acid and chlorogenic acid were most abundant (>1000 mg/100 g DW). The optimal method for preparing the extract for antimicrobial and haemolytic activity, with a focus on phenols, involved using 50% ethanol, applying ultrasound for six minutes, and carrying out three extractions. The M0% extract exhibited the most potent antimicrobial activity against S. mutans (MIC: 7.8 mg/mL). Anti-haemolytic activity indicates biocompatibility. The results emphasise the bioactive and antimicrobial potential of M. crocea, suggesting its possible application in various industries. However, further research is needed in the form of in vivo studies. Full article

Thermo-tolerance is a virulence factor responsible for the emergence of new fungal pathogens, including Candidozyma auris (formerly classified as Candida auris, C. auris). It has been shown that in C. auris the thermo-tolerance, as well as other virulence traits, such as the ability to aggregate, to form pseudo-hyphae, or to produce melanin are strain-specific features. Here, we investigated the impact of different temperatures (25 °C, 37 °C and 42 °C) on the phenotypic and virulence profile of C. auris strain CDC B11903. The results show a positive correlation between the resistance to antifungals and increasing temperature from 25 °C to 37 °C, while no differences were observed between 37 °C and 42 °C, except for Anidulafungin. Furthermore, C. auris growth was impaired at 25 °C as compared to 37 °C and 42 °C. Except for the haemolytic activity, which increased with rising temperatures, phospholipase, lipase and biofilm production were found at all tested temperatures. Moreover, the ability to produce melanin was observed only at 37 °C and 42 °C. The capacity to grow as pseudo-hyphae or in clusters and to adhere to both biotic and abiotic surfaces were observed at all the temperatures tested, with increased propensity of C. auris to adhere to abiotic surfaces with rising temperatures. The results underline the thermo-tolerance of C. auris strain B11903 and its increased virulence profile at human body temperature both in physiological (37 °C) and febrile state (42 °C). Full article