In this work, two biosurfactant-producing strains, Bacillus subtilis and Bacillus licheniformis, have been characterized. Both strains were able to grow at high salinity conditions and produce biosurfactants up to 10% NaCl. Both extracted-enriched biosurfactants showed good surface tension reduction of water, from 72 to 26-30 mN/m, low critical micelle concentration, and high resistance to pH and salinity. The potential of the two lipopeptide biosurfactants at inhibiting biofilm adhesion of pathogenic bacteria was demonstrated by using the MBEC device. The two biosurfactants showed interesting specific anti-adhesion activity being able to inhibit selectively biofilm formation of two pathogenic strains. In particular, Escherichia coli CFT073 and Staphylococcus aureus ATCC 29213 biofilm formation was decreased of 97% and 90%, respectively. The V9T14 biosurfactant active on the Gram-negative strain was ineffective against the Gram-positive and the opposite for the V19T21. This activity was observed either by coating the polystyrene surface or by adding the biosurfactant to the inoculum. Two fractions from each purified biosurfactant, obtained by flash chromatography, fractions (I) and (II), showed that fraction (II), belonging to fengycin-like family, was responsible for the anti-adhesion activity against biofilm of both strains.

Lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain showed an interesting anti-adhesion activity against biofilm formation of human pathogenic bacterial strains. The chemical characterisation of the crude extract of V9T14 strain was first developed through electrospray ionisation mass spectrometry (ESI-MS) and ESI-MS/MS direct infusions: two sets of molecular ion species belonging to the fengycin and surfactin families were revealed and their structures defined, interpreting their product ion spectra. The LC/ESI-MS analysis of the crude extract allowed to separate in different chromatogram ranges the homologues and the isoforms of the two lipopeptide families. The extract was then fractionated by silica gel chromatography in two main fractions, I and II. The purified biosurfactants were analysed through a new, rapid and suitable LC/ESI-MS/MS method, which allowed characterising the composition and the structures of the produced lipopeptides. LC/ESI-MS/MS analysis of fraction I showed the presence of C(13), C(14) and C(15) surfactin homologues, whose structures were confirmed by the product ion spectra of the sodiated molecules [M + Na](+) at m/z 1030, 1044 and 1058. LC/ESI-MS/MS analysis of fraction II confirmed the presence of two main fengycin isoforms, with the protonated molecules [M + H](+) at m/z 1478 and 1506 corresponding to C(17) fengycin A and C(17) fengycin B, respectively. Other homologues (C(14) to C(16)) were revealed and confirmed as belonging to fengycin A or B according to the retention times and the product ions generated, although with the same nominal mass. Finally, a relative percentage content of each homologue for both lipopeptides families in the whole extract was proposed.

Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its nicotinamide dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN (NAD nucleotidase), which plays a central role by degrading NAD into adenosine and NR (nicotinamide riboside), the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN which determined that the enzyme is a Zn2+-dependent 5'-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3 Å resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate specificity, recognizing either mono- or di-nucleotide nicotinamides and different adenosine phosphates with a maximal activity on 5'-adenosine monophosphate. Sequence and structural analysis of H. influenzae NadN led us to discover that human CD73 is capable of processing both NAD and NMN, therefore disclosing a possible novel function of human CD73 in systemic NAD metabolism. Our data may prove to be useful for inhibitor design and disclosed unanticipated fascinating evolutionary relationships.

Staphylococcus aureus and Staphylococcus epidermidis are considered two of the most important pathogens, and their biofilms frequently cause device-associated infections. Microbial biosurfactants recently emerged as a new generation of anti-adhesive and anti-biofilm agents for coating implantable devices to preserve biocompatibility. In this study, R89 biosurfactant (R89BS) was evaluated as an anti-biofilm coating on medical-grade silicone. R89BS is composed of homologues of the mono- (75%) and di-rhamnolipid (25%) families, as evidenced by mass spectrometry analysis. The antimicrobial activity against Staphylococcus spp. planktonic and sessile cells was evaluated by microdilution and metabolic activity assays. R89BS inhibited S. aureus and S. epidermidis growth with minimal inhibitory concentrations (MIC99) of 0.06 and 0.12 mg/mL, respectively and dispersed their pre-formed biofilms up to 93%. Silicone elastomeric discs (SEDs) coated by R89BS simple adsorption significantly counteracted Staphylococcus spp. biofilm formation, in terms of both built-up biomass (up to 60% inhibition at 72 h) and cell metabolic activity (up to 68% inhibition at 72 h). SEM analysis revealed significant inhibition of the amount of biofilm-covered surface. No cytotoxic effect on eukaryotic cells was detected at concentrations up to 0.2 mg/mL. R89BS-coated SEDs satisfy biocompatibility requirements for leaching products. Results indicate that rhamnolipid coatings are effective anti-biofilm treatments and represent a promising strategy for the prevention of infection associated with implantable devices.

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Fresh juices of four Italian cultivars of lemons (Citrus limon Burm) have been analyzed by headspace solid phase microextraction coupled to gas chromatography and gas chromatography-mass spectrometry (GC-MS). The best results have been obtained with the 2 cm 50/30 microm divinylbenzene/carboxen on poly(dimethylsiloxane) fiber, using a homogenization time of 1 h at 40 degrees C and a sampling period of 30 min. A total of 35 volatile compounds have been identified by GC-MS, and their relative amounts have been calculated by adding internal standard to the samples. Differences in composition of lemon juices volatile components have been observed. Verdello Siracusano lemon juice has the highest amount of volatile compounds (50.28 mg/L), followed by Interdonato (8.39 mg/L), Primo Fiore Capo d'Orlando (5.75 mg/L), and Femminello Siracusano (2.62 mg/L) juices. Volatile compounds mainly consist of mono- and sesquiterpene hydrocarbons and oxygenated molecules (aldheydes, monoterpene alcohols, and monoterpene esters). Headspace solid phase microextraction coupled to a gas chromatograph equipped with a specific sulfur detector, a sulfur chemiluminescence detector, let us detect and quantify dimethyl sulfide compound at the microgram/liter level in lemon juices.

Candida albicans is the major fungus that colonises medical implants, causing device-associated infections with high mortality. Antagonistic bacterial products with interesting biological properties, such as biosurfactants, have recently been considered for biofilm prevention. This study investigated the activity of lipopeptide biosurfactant produced by Bacillus subtilis AC7 (AC7 BS) against adhesion and biofilm formation of C. albicans on medical-grade silicone elastomeric disks (SEDs). Chemical analysis, stability, surface activities of AC7 BS crude extract and physicochemical characterisation of the coated silicone disk surfaces were also carried out. AC7 BS showed a good reduction of water surface tension, low critical micelle concentration, good emulsification activity, thermal resistance and pH stability. Co-incubation with 2 mg ml(-1) AC7 BS significantly reduced adhesion and biofilm formation of three C. albicans strains on SEDs in a range of 67-69 % and of 56-57 %, respectively. On pre-coated SEDs, fungal adhesion and biofilm formation were reduced by 57-62 % and 46-47 %, respectively. Additionally, AC7 BS did not inhibit viability of C. albicans strains in both planktonic and sessile form. Chemical analysis of the crude extract revealed the presence of two families of lipopeptides, principally surfactin and a lower percentage of fengycin. The evaluation of surface wettability indicated that AC7 BS coating of SEDs surface was successful although uneven. AC7 BS significantly prohibits the initial deposition of C. albicans and slows biofilm growth, suggesting a potential role of biosurfactant coatings for preventing fungal infection associated with silicone medical devices.

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