Acinetobacter baumannii is a serious nosocomial pathogen with multiple drug resistance (MDR), the control of which has become challenging due to the currently used antibiotics. Our main objective in this study is to determine the antibacterial and antibiofilm activities of the antimicrobial peptide, Octominin, against MDR A. baumannii and derive its possible modes of actions. Octominin showed significant bactericidal effects at a low minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of 5 and 10 µg/mL, respectively. Time-kill kinetic analysis and bacterial viability tests revealed that Octominin showed a concentration-dependent antibacterial activity. Field-emission scanning electron microscopy (FE-SEM) analysis revealed that Octominin treatment altered the morphology and membrane structure of A. baumannii. Propidium iodide (PI) and reactive oxygen species (ROS) generation assays showed that Octominin increased the membrane permeability and ROS generation in A. baumannii, thereby causing bacterial cell death. Further, a lipopolysaccharides (LPS) binding assay showed an Octominin concentration-dependent LPS neutralization ability. Biofilm formation inhibition and eradication assays further revealed that Octominin inhibited biofilm formation and showed a high biofilm eradication activity against A. baumannii. Furthermore, up to a concentration of 100 µg/mL, Octominin caused no hemolysis and cell viability changes in mammalian cells. An in vivo study in zebrafish showed that the Octominin-treated group had a significantly higher relative percentage survival (54.1%) than the untreated group (16.6%). Additionally, a reduced bacterial load and fewer alterations in histological analysis confirmed the successful control of A. baumannii by Octominin in vivo. Collectively, these data suggest that Octominin exhibits significant antibacterial and antibiofilm activities against the multidrug-resistant A. baumannii, and this AMP can be developed further as a potent AMP for the control of antibiotic resistance.

Antimicrobial peptides (AMPs) have become a key solution for controlling multi-drug-resistant (MDR) pathogens, and the nanoencapsulation of AMPs has been used as a strategy to overcome challenges, such as poor stability, adverse interactions, and toxicity. In previous studies, we have shown the potent antimicrobial activity of Octominin against Candida albicans and Acinetobacter baumannii. This study is focused on the nanoencapsulation of Octominin with chitosan (CS) and carboxymethyl chitosan (CMC) as a drug delivery system using the ionotropic gelation technique. Octominin-encapsulated CS nanoparticles (Octominin-CNPs) had an average diameter and zeta potential of 372.80 ± 2.31 nm and +51.23 ± 0.38 mV, respectively, while encapsulation efficiency and loading capacity were 96.49 and 40.20%, respectively. Furthermore, Octominin-CNPs showed an initial rapid and later sustained biphasic release profile, and up to 88.26 ± 3.26% of the total Octominin release until 96 h. Transmission electron microscopy data showed the irregular shape of the Octominin-CNPs with aggregations. In vitro and in vivo toxicity of Octominin-CNPs was significantly lower than the Octominin at higher concentrations. The antifungal and antibacterial activities of Octominin-CNPs were slightly higher than those of Octominin in both the time-kill kinetic and microbial viability assays against C. albicans and A. baumannii, respectively. Mode of action assessments of Octominin-CNPs revealed that morphological alterations, cell membrane permeability alterations, and reactive oxygen species generation were slightly higher than those of Octominin at the tested concentrations against both C. albicans and A. baumannii. In antibiofilm activity assays, Octominin-CNPs showed slightly higher biofilm inhibition and biofilm eradication activities compared to that of Octominin. In conclusion, Octominin was successfully encapsulated into CS, and Octominin-CNPs showed lower toxicity and greater antimicrobial activity against C. albicans and A. baumannii compared to Octominin.

This study evaluated the modulation of gut microbiota, immune responses, and gut morphometry in C57BL/6 mice, upon oral administration of S. maxima-derived modified pectin (SmP, 7.5 mg/mL) and pectin nanoparticles (SmPNPs; 7.5 mg/mL). Metagenomics analysis was conducted using fecal samples, and mice duodenum and jejunum were used for analyzing the immune response and gut morphometry, respectively. The results of metagenomics analysis revealed that the abundance of Bacteroidetes in the gut increased in response to both modified SmP and SmPNPs (75%) as compared with that in the control group (66%), while that of Firmicutes decreased in (20%) as compared with that in the control group (30%). The mRNA levels of mucin, antimicrobial peptide, and antiviral and gut permeability-related genes in the duodenum were significantly (p < 0.05) upregulated (> 2-fold) upon modified SmP and SmPNPs feeding. Protein level of intestinal alkaline phosphatase was increased (1.9-fold) in the duodenum of modified SmPNPs feeding, evidenced by significantly increased goblet cell density (0.5 ± 0.03 cells/1000 µm²) and villi height (352 ± 10 µm). Our results suggest that both modified SmP and SmPNPs have the potential to modulate gut microbial community, enhance the expression of immune related genes, and improve gut morphology.

Exosomes are important mediators of intercellular communication and modulate many physiological and pathological processes. Knowledge of secretion, content, and biological functions of fish exosomes during pathological infection is still scarce due to lack of suitable standardized isolation techniques. In this study, we aimed to isolate exosomes from the plasma of marine fish, rock bream (Oplegnathus fasciatus), by two isolation methods: differential ultracentrifugation (UC) and a commercial membrane affinity spin column technique (kit). Morphological and physicochemical characteristics of the isolated exosomes were determined by these two methods, and the efficiencies of the two methods were compared. Exosomes isolated by both methods were in the expected size range (30–200 nm) and had a characteristic cup-shape in transmission electron microscopy observation. Moreover, more intact exosomes were identified using the kit-based method than UC. Nanoparticle tracking analysis demonstrated a heterogeneous population of exosomes with a mean particle diameter of 114.6 ± 4.6 and 111.2 ± 2.2 nm by UC and a kit-based method, respectively. The particle concentration obtained by the kit method (1.05 × 10¹¹ ± 1.23 × 10¹⁰ particles/mL) was 10-fold higher than that obtained by UC (4.90 × 10¹⁰ ± 2.91 × 10⁹ particles/mL). The kit method had a comparatively higher total protein yield (1.86 mg) and exosome protein recovery (0.55 mg/mL plasma). Immunoblotting analysis showed the presence of exosome marker proteins (CD81, CD63, and HSP90) in the exosomes isolated by both methods and suggests the existence of exosomes. However, the absence of cytotoxicity or adverse immune responses to fish and mammalian cells by the exosomes isolated by the UC procedure indicates its suitability for functional studies in vitro. Overall, our basic characterization results indicate that the kit-based method is more suitable for isolating high-purity exosomes from fish plasma, whereas UC has higher safety in terms of yielding exosomes with low toxicity. This study provides evidence for the existence of typical exosomes in rock beam plasma and facilitates the selection of an efficient exosome isolation procedure for future applications in disease diagnosis and exosome therapy as fish medicine.