Extreme marine environments, such as the submarine shallow vents of the Eolian Islands (Italy), offer an almost unexplored source of microorganisms producing unexploited and promising biomolecules for pharmaceutical applications. Thermophilic and thermotolerant bacilli isolated from Eolian vents are able to produce exopolysaccharides (EPSs) with antiviral and immunomodulatory effects against Herpes simplex virus type 2 (HSV-2). HSV-2 is responsible for the most common and continuously increasing viral infections in humans. Due to the appearance of resistance to the available treatments, new biomolecules exhibiting different mechanisms of action could provide novel agents for treating viral infections. The EPSs hinder the HSV-2 replication in human peripheral blood mononuclear cells (PBMC) but not in WISH (Wistar Institute Susan Hayflic) cells line, indicating that cell-mediated immunity was involved in the antiviral activity. High levels of Th1-type cytokines were detected in PBMC treated with all EPSs, while Th2-type cytokines were not induced. These EPSs are water soluble exopolymers able to stimulate the immune response and thus contribute to the antiviral immune defense, acting as immunomodulators. As stimulants of Th1 cell-mediated immunity, they could lead to the development of novel drugs as alternative in the treatment of herpes virus infections, as well as in immunocompromised host.

The eradication of bacterial biofilm represents a crucial strategy to prevent a clinical problem associated with microbial persistent infection. In this study we evaluated the ability of the exopolysaccharide (EPS) B3-15, produced by the marine Bacillus licheniformis B3-15, to prevent the adhesion and biofilm formation of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 on polystyrene and polyvinyl chloride surfaces. The EPS was added at different times (0, 2, 4 and 8 h), corresponding to the initial, reversible and irreversible attachment, and after the biofilm development (24 or 48 h). The EPS (300 µg/mL) impaired the initial phase, preventing bacterial adhesion even when added after 2 h of incubation, but had no effects on mature biofilms. Without exerting any antibiotic activity, the antibiofilm mechanisms of the EPS were related to the modification of the (i) abiotic surface properties, (ii) cell-surface charges and hydrophobicity, and iii) cell-to-cell aggregation. The addition of EPS downregulated the expression of genes (lecA and pslA of P. aeruginosa and clfA of S. aureus) involved in the bacterial adhesion. Moreover, the EPS reduced the adhesion of P. aeruginosa (five logs-scale) and S. aureus (one log) on human nasal epithelial cells. The EPS could represent a promising tool for the prevention of biofilm-related infections.

Bioprospecting in unusual marine environments provides an innovative approach to search novel biomolecules with antibiofilm activity. Antarctic sponge-associated bacteria belonging to Colwellia, Pseudoalteromonas, Shewanella and Winogradskyella genera were evaluated for their ability to contrast the biofilm formation by Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213, as model organisms. All strains were able to produce biofilm at both 4 and 25 °C, with the highest production being for Colwellia, Shewanella and Winogradskyella strains at 4 °C after 24 h. Antibiofilm activity of cell-free supernatants (CFSs) differed among strains and on the basis of their incubation temperature (CFSs_4°C and CFSs_25°C). The major activity was observed by CFSs_4°C against S. aureus and CFSs_25°C against P. aeruginosa, without demonstrating a bactericidal effect on their growth. Furthermore, the antibiofilm activity of crude extracts from Colwellia sp. GW185, Shewanella sp. CAL606, and Winogradskyella sp. CAL396 was also evaluated and visualized by confocal laser scanning microscopic images. Results based on the surface-coating assay and surface tension measurements suggest that CFSs and the crude extracts may act as biosurfactants inhibiting the first adhesion of P. aeruginosa and S. aureus. The CFSs and the novel biopolymers may be useful in applicative perspectives for pharmaceutical and environmental purposes.

Exopolysaccharide (EPS) production represents an adaptive strategy developed by extremophiles to cope with environmental stresses. The EPS-producing Bacillus licheniformis B3-15, of shallow marine vent origin (Vulcano Island, Italy), was previously reported as tolerant to arsenate (As^V). In this study, we evaluated: (i) the increasing production of EPS by Bacillus licheniformis B3-15 in the novel SG17 medium; (ii) the arsenic absorption capacity of the EPS by mass spectroscopy; (iii) the functional groups of EPS interacting with As by ATR-FTIR spectroscopy; and (iv) the ability of EPS to prevent arsenic toxicity by the bioluminescent assay. The EPS yield (240 mg L⁻¹) was 45% higher than previously reported. The EPS was mainly constituted of disaccharide repeating units with a manno-pyranosidic configuration and low protein content, attributed to the poly-gamma glutamic acid component as evidenced by NMR analysis. ATR-FTIR spectra indicated that the functional groups of the EPS (O–H, C=O, C–O and C=C and N–O) were involved in the adsorption of the arsenic cations, with greater interactions between EPS and arsenate (As^V) than arsenite (As^III). Consequently, the EPS at increasing concentration (100 and 300 µg mL⁻¹) adsorbed As^V more efficiently (20.5% and 34.5%) than As^III (0.7% and 1.8%). The bioluminescence assay showed that the EPS was not toxic, and its addition reduced the toxicity of both As forms by more than twofold. The crude EPS B3-15 could be used in arsenic bioremediation as a possible eco-friendly alternative to other physical or chemical methods.

The surfactin-like lipopeptide (BS-SBP3) and the exopolysaccharide (EPS-SBP3) produced by the polyextremophilic Bacillus horneckiae SBP3 (DSM 103063) have been recently described as valuable biopolymers useful in biotechnological applications. To investigate the hydrating capabilities of BS-SBP3 and EPS-SBP3, here we evaluated (i) their wetting properties, measuring the contact angle; (ii) their moisture uptake abilities using the gravimetric method; and (iii) their hydrating states (from 0 to 160% w/w of water content) using ATR-FTIR spectroscopy. BS-SBP3 reduced the water contact angle on a hydrophobic surface from 81.7° to 51.3°, whereas the contact angle in the presence of EPS-SBP3 was 72.9°, indicating that BS-SBP3 improved the wettability of the hydrophobic surface. In the moisture uptake tests, EPS-SBP3 absorbed more water than BS-SBP3, increasing its weight from 10 mg to 30.1 mg after 36 h of 100% humidity exposure. Spectral distance and cross-correlation analyses were used to evaluate the molecular changes of the two biopolymers during the hydration process. As the water concentration increased, BS-SBP3 spectra changed in intensity in the two contributions of the OH-stretching band named “closed” and “open” (3247 and 3336 cm⁻¹, respectively). Differently, the spectra of EPS-SBP3 exhibited a broader peak (3257 cm⁻¹), which shifted at higher water concentrations. As evaluated by the spectral distance and the wavelet cross-correlation analysis, the OH-stretching bands of the BS-SBP3 and EPS-SBP3 changed as a function of water content, with two different sigmoidal trends having the inflection points at 80% and 48%, respectively, indicating peculiar water-properties of each biopolymer. As wetting agents, these biopolymers might replace industrially manufactured additives in agriculture and the food and cosmetic industries.

The exploitation of thermophilic hydrocarbon-utilizing bacilli could provide novel environmentally friendly surfactants. In this work, 80 thermophilic bacilli isolated from shallow hydrothermal vents of the Eolian Islands (Italy) were screened for their ability to utilize hydrocarbons and produce biosurfactants (BSs). Among them, 15 strains grew with kerosene or gasoline (2% v/v) as the only carbon and energy source, and most of them were positive to the methylene blue agar as prescreening assay for BSs production and displayed emulsifying activity. The cell-free supernatants (CFSs) from two selected strains, Bacillus licheniformis B3-15 and Bacillus horneckiae SBP3, were both surface active and able to emulsify different hydrocarbons and vegetable oils. BSs from B3-15 (910 mg L⁻¹) and SBP3 (950 mg L⁻¹) were chemically different surfactin-like lipopeptides, with specific mineral-, castor- and crude oil removal ability from the cotton matrix. CFSs from the 15 thermophilic strains, which harbor both lipolytic and surfactant abilities, could be suitable for industrial-based applications and environmental issues, such as oil recovery and removal from polluted areas or surfaces, (e.g., oil pipelines, bilge tankers, or industrial silos), whereas the crude BSs, as high-value compounds, may be used in different fields of application, as detergent, cosmeceutical, and pharmaceutical industries.