Search

Gaetana Pezzino,1 Alessia Calabrò,1 Fabiana Drommi,1 Stefania Campana,1 Riccardo Cavaliere,1,2 Irene Bonaccorsi,1 Paolo Carrega,1 Vincenzo Zammuto,3– 5 Maria Giovanna Rizzo,3 Concetta Gugliandolo,3,4 Guido Ferlazzo,6,7 Claudia De Pasquale1 1Laboratory of Immunology and Biotherapy, Department Human Pathology ”G. Barresi”, University of Messina, Messina, Italy; 2Division of Clinical Pathology, University Hospital Policlinico G. Martino, Messina, Italy; 3Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; 4Research Centre for Extreme Environments and Extremophiles, Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; 5ATHENA Green Solutions S.r.l., Messina, Italy; 6Department of Experimental Medicine (DIMES), University of Genoa, Genova, Italy; 7Unit of Experimental Pathology and Immunology, IRCCS Ospedale Policlinico San Martino, Genova, ItalyCorrespondence: Guido Ferlazzo, Unit of Experimental Pathology and Immunology, IRCCS Ospedale Policlinico San Martino, Genoa, Italy, Email guido.ferlazzo@unige.itBackground: Respiratory viral infections are a leading cause of severe diseases and mortality; therefore, novel treatments effective for their prevention are highly requested. Here, we identified a broad-spectrum antiviral activity of a natural exopolysaccharide, EPS T14, purified from a marine thermotolerant strain of Bacillus licheniformis strain T14.Methods: The effects on human normal nasal epithelial cells (HNEpCs) following treatment with EPS T14 was evaluated at different time points and with increasing concentration of compound. To assess the antiviral properties, viability of HNEpCs treated with EPS T14 was analysed following infection with different respiratory viruses.Results: Neither toxicity nor pro-inflammatory properties were observed in vitro on HNEpCs treated with EPS T14 up to high concentrations, thus ensuring its safety. Cell culture-based assays revealed that treatment of HNEpCs with EPS T14 (used at 400ug/mL) results in efficient prevention of cell infection by different respiratory viruses through physically hindering the entry of the viruses via cell surface receptors. Interestingly, in addition to this prophylactic antiviral activity, EPS T14 also shows a long-lasting efficacy by inhibiting viral spread in the cell culture. Finally, combination of EPS T14 with a hypertonic saline solution shows a synergistic antiviral activity.Conclusion: EPS T14 can exert both prophylactic and therapeutic antiviral activity by blocking viral attachment to cellular receptors and could therefore represent a promising antiviral agent for preventing infections by different respiratory viruses. Keywords: exopolysaccharides, infection prophylaxis, nasal epithelium, respiratory virus, viral infection, EPSs

The mannose-rich exopolysaccharide EPS B3-15, produced by the thermophilic Bacillus licheniformis B3-15, was previously reported to possess promising potentialities as antiviral and immunomodulatory agent, and in preventing the adhesion of Pseudomonas aeruginosa and Staphylococcus aureus . In this study, EPS B3-15 was evaluated for its anti-inflammatory activity in LPS-induced macrophages and the ability to contrast the adhesion of Klebsiella pneumoniae and Streptococcus pneumoniae as pathogenic bacteria of the respiratory tract. Without affecting the macrophages viability, the EPS at low concentration (300 μg/mL) significantly downregulated the gene expression of iNOS and the consequent NO generation, and it also decreased the production of pro-inflammatory cytokines. Moreover, the EPS reduced the adhesion of Str. pneumoniae (47 %) more efficiently than K. pneumoniae (38 %), due to its ability to modify the abiotic surfaces properties and alter the charges of bacterial-cell surface of Gram-positive more than Gram-negative. As able to reduce the inflammatory responses in macrophage cells and simultaneously prevent biofilm-related to the respiratory tract infections, EPS B3-15 could have potential use as nasal spray with anti-inflammatory action and surface-coating agent for medical devices., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

We report the ability of the crude biosurfactant (BS B3-15), produced by the marine, thermotolerant Bacillus licheniformis B3-15, to hinder the adhesion and biofilm formation of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 to polystyrene and human cells. First, we attempted to increase the BS yield, optimizing the culture conditions, and evaluated the surface-active properties of cell-free supernatants. Under phosphate deprivation (0.06 mM) and 5% saccharose, the yield of BS (1.5 g/L) increased by 37%, which could be explained by the earlier (12 h) increase in lch AA expression compared to the non-optimized condition (48 h). Without exerting any anti-bacterial activity, BS (300 µg/mL) prevented the adhesion of P. aeruginosa and S. aureus to polystyrene (47% and 36%, respectively) and disrupted the preformed biofilms, being more efficient against S. aureus (47%) than P. aeruginosa (26%). When added to human cells, the BS reduced the adhesion of P. aeruginosa and S. aureus (10× and 100,000× CFU/mL, respectively) without altering the epithelial cells' viability. As it is not cytotoxic, BS B3-15 could be useful to prevent or remove bacterial biofilms in several medical and non-medical applications.

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 ( lec A and pslA of P. aeruginosa and clf A 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.

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.

Aims: This study was to analyse the biomass production and fatty acids (FAs) profiles in a newly isolated chlorophyte, namely Coccomyxa AP01, under nutritionally balanced (NB) conditions (comparing nitrate and urea as nitrogen sources) and nitrogen or phosphate deprivation., Methods and Results: Lipid yields was about 30%-40% of dried biomasses in all examined nutritional conditions. Under NB conditions, lipids were principally constituted by monounsaturated FAs, mainly represented by oleic acid, and saturated and polyunsaturated FAs at similar concentrations. Nutrients deprivation induced remarkable changes in FAs profiles, with the highest amounts of saturated (42%-46%), followed by similar amounts of monounsaturated and polyunsaturated, and the emergence of rare long-chain FAs. Under phosphate deprivation, biomass yield was similar to NB conditions, with the highest yield of saturated (mainly palmitic acid) and of polyunsaturated FAs (33%) (mainly linoleic and linolenic acids)., Conclusions: Balanced or deprived nutritional conditions in Coccomyxa AP01 induced a selective production and composition of FAs. The phosphate-deprivation condition concomitantly provided high biomass yield and the production of high value saturated and polyunsaturated FAs with industrial interest., Significance and Impact of the Study: Coccomyxa AP01 could be considered a promising source of different FAs, including also docosapentaenoic acid, for several commercial purposes spanning from biodiesel production, pharmaceutical and cosmetic applications to innovative aquaculture fish feeds., (© 2021 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

The conformational variation of the viral capsid structure plays an essential role both for the environmental resistance and acid nuclear release during cellular infection. The aim of this study was to evaluate how capsid rearrangement in engineered phages of M13 protects viral DNA and peptide bonds from damage induced by UV-C radiation. From in silico 3D modelling analysis, two M13 engineered phage clones, namely P9b and 12III1, were chosen for (i) chemical features of amino acids sequences, (ii) rearrangements in the secondary structure of their pVIII proteins and (iii) in turn the interactions involved in phage capsid. Then, their resistance to UV-C radiation and hydrogen peroxide (H 2 O 2 ) was compared to M13 wild-type vector (pC89) without peptide insert. Results showed that both the phage clones acquired an advantage against direct radiation damage, due to a reorganization of interactions in the capsid for an increase of H-bond and steric interactions. However, only P9b had an increase in resistance against H 2 O 2 . These results could help to understand the molecular mechanisms involved in the stability of new virus variants, also providing quick and necessary information to develop effective protocols in the virus inactivation for human activities, such as safety foods and animal-derived materials.

Extremophiles are optimal models in experimentally addressing questions about the effects of cosmic radiation on biological systems. The resistance to high charge energy (HZE) particles, and helium (He) ions and iron (Fe) ions (LET at 2.2 and 200 keV/µm, respectively, until 1000 Gy), of spores from two thermophiles, Bacillus horneckiae SBP3 and Bacillus licheniformis T14, and two psychrotolerants, Bacillus sp. A34 and A43, was investigated. Spores survived He irradiation better, whereas they were more sensitive to Fe irradiation (until 500 Gy), with spores from thermophiles being more resistant to irradiations than psychrotolerants. The survived spores showed different germination kinetics, depending on the type/dose of irradiation and the germinant used. After exposure to He 1000 Gy, D-glucose increased the lag time of thermophilic spores and induced germination of psychrotolerants, whereas L-alanine and L-valine increased the germination efficiency, except alanine for A43. FTIR spectra showed important modifications to the structural components of spores after Fe irradiation at 250 Gy, which could explain the block in spore germination, whereas minor changes were observed after He radiation that could be related to the increased permeability of the inner membranes and alterations of receptor complex structures. Our results give new insights on HZE resistance of extremophiles that are useful in different contexts, including astrobiology.

The novel exopolysaccharide (EPS-B3-15) produced by the marine thermotolerant Bacillus licheniformis strain B3-15, constituted by mannose and glucose, has been recently reported as a valuable biopolymer in pharmaceutical applications. To dynamically characterize the thermal behavior of the whole EPS-B3-15 system, the Attenuated Total Reflectance Fourier Transform Infra-Red spectroscopy technique was used over a temperature range from ambient to 78.5 °C. The molecular changes of EPS-B3-15 during the heating process were evaluated by the spectral distance (SD) and wavelet cross-correlation (XWT) analysis. The thermal analysis revealed that the EPS-B3-15 possessed high stability until 78.5 °C. As evaluated by SD and XWT, the molecular structure of EPS-B3-15 at 45 °C was partially modified, due to -OH groups and the -COOH and -OH interactions, conferring a new conformational structure to the biopolymer. The thermal characterization provides novel information about the molecular conformations of the whole EPS-B3-15 system at different temperatures. The thermostable EPS-B3-15 can be successfully employed for biotechnological, nanotechnological and material science applications even at high temperatures., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A straightforward and green method for the synthesis of gold, silver, and silver chloride nanoparticles (Au NPs and Ag/AgCl NPs) was developed using three different microbial exopolymers (EP) as reducing and stabilizing agents. The exopolysaccharides EPS B3-15 and EPS T14 and the poly- γ -glutamic acid γ-PGA-APA were produced by thermophilic bacteria isolated from shallow hydrothermal vents off the Eolian Islands (Italy) in the Mediterranean Sea. The production of metal NPs was monitored by UV-Vis measurements by the typical plasmon resonance absorption peak and their antimicrobial activity towards Gram-positive and Gram- negative bacteria ( Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa ), as well as fungi ( Candida albicans ) was investigated. The biological evaluation showed no activity for EP-Au NPs, except against E. coli , whereas EP-Ag NPs exhibited a broad-spectrum of activity. The chemical composition, morphology, and size of EP-Ag NPs were investigated by UV-Vis, zeta potential (ζ), dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM). The best antimicrobial results were obtained for EPS B3-15-Ag NPs and EPS T14-Ag NPs (Minimum Inhibitory Concentration, MIC: 9.37-45 µg/mL; Minimum Bactericidal Concentration/Minimum Fungicidal Concentration, MBC/MFC: 11.25-75 µg/mL).

Submarine hydrothermal vents are inhabited by a variety of microorganisms capable of tolerating environmental extremes, making them ideal candidates to further expand our knowledge of the limitations for terrestrial life, including their ability to survive the exposure of spaceflight-relevant conditions. The spore resistance of two Bacillus spp. strains, APA and SBP3, isolated from two shallow vents off Panarea Island (Aeolian Islands, Italy), to artificial and environmental stressors (i.e., UVC radiation, X-rays, heat, space vacuum, hydrogen peroxide [H 2 O 2 ], and low-pressure plasma), was compared with that of two close phylogenetic relatives (Bacillus horneckiae and Bacillus oceanisediminis). Additional comparisons were made with Bacillus sp. isolated from spacecraft assembly facilities (B. horneckiae, Bacillus pumilus SAFR-032, and Bacillus nealsonii) and the biodosimetry strain and space microbiology model organism Bacillus subtilis. Overall, a high degree of spore resistance to stressors was observed for the strains isolated from spacecraft assembly facilities, with an exceptional level of resistance seen by B. pumilus SAFR-032. The environmental isolate SBP3 showed a more robust spore resistance to UVC, X-rays, H 2 O 2 , dry heat, and space vacuum than the closely related B. horneckiae. Both strains (SBP3 and APA) were more thermotolerant than their relatives, B. horneckiae and B. oceanisediminis, respectively. SBP3 may have a novel use as a bacterial model organism for future interrogations into the potential of forward contamination in extraterrestrial environments (e.g., icy moons of Jupiter or Saturn), spacecraft sterilization and, broadly, microbial responses to spaceflight-relevant environmental stressors.

To dynamically characterize the thermal properties of the fructose-rich exopolysaccharide (EPS1-T14), produced by the marine thermophilic Bacillus licheniformis T14, the Attenuated Total Reflectance Fourier Transform Infra-Red spectroscopy was coupled to variable temperature ranging from ambient to 80°C. The spectra were analyzed by the following innovative mathematical tools: i) non-ideal spectral deviation, ii) OH-stretching band frequency center shift, iii) spectral distance, and iv) wavelet cross-correlation analysis. The thermal restraint analysis revealed that the whole EPS1-T14 system possessed high stability until 80°C, and suggested that fucose was mainly involved in the EPS1-T14 thermal stability, whereas glucose was responsible for its molecular flexibility. Our results provide novel insights into the thermal stability properties of the whole EPS1-T14 and into the role of its main monosaccharidic units. As a new biopolymer, the thermostable EPS1-T14 could be used in traditional biotechnology fields and in new biomedical areas, as nanocarriers, requiring high temperature processes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Herpes simplex virus type 1 (HSV-1) is responsible of common and widespread viral infections in humans through the world, and of rare, but extremely severe, clinical syndromes in the central nervous system. The emergence of resistant strains to drugs actually in use encourages the searching for novel antiviral compounds, including those of natural origin. In this study, the recently described poly-γ-glutamic acid (γ-PGA-APA), produced by the marine thermotolerant Bacillus horneckiae strain APA, and previously shown to possess biological and antiviral activity, was evaluated for its anti-HSV-1 and immunomodulatory properties. The biopolymer hindered the HSV-1 infection in the very early phase of virus replication. In addition, the γ-PGA-APA was shown to exert low cytotoxicity and noticeable immunomodulatory activities towards TNF-α and IL-1β gene expression. Moreover, the capacity to positively modulate the transcriptional activity of the cytokine genes was paired with increased level of activation of the transcription factor NF-kB by γ-PGA-APA. Overall, as non-cytotoxic biopolymer able to contribute in the antiviral defense against HSV-1, γ-PGA-APA could lead to the development of novel natural drugs for alternative therapies.