An antimicrobial metabolite n- hexadecenoic acid from marine sponge-associated bacteria Bacillus subtilis effectively inhibited biofilm forming multidrug-resistant P. aeruginosa.

Effective drug candidates to obstruct the emergence of multidrug-resistant pathogens have become a major concern. A potent antimicrobial producer was isolated from a marine sponge designated as MSI38 and was identified as Bacillus subtilis by 16SrDNA sequencing. The active antimicrobial fraction was purified, and the metabolite was identified as n-hexadecanoic acid by spectroscopic analysis. The fish-borne pathogen Pseudomonas aeruginosa FP012 was found to be multidrug-resistant and poses a risk of disease to food handlers and consumers in general. The compound showed a potent bactericidal effect against P. aeruginosa FP012 with a MIC of 31.33 ± 5.67 mg L-1 and MBC of 36.66 ± 5.17 mg L-1. The time-based biofilm inhibitory potential of MSI38 and ciprofloxacin was analyzed by confocal laser scanning microscopy. A synergistic effect of MSI38 and ciprofloxacin on biofilm showed 85% inhibition.

A novel anti-infective molecule nesfactin identified from sponge associated bacteria Nesterenkonia sp. MSA31 against multidrug resistant Pseudomonas aeruginosa.

Overuse of antibiotics coupled with biofilm-forming ability has led to the emergence of multi-drug P. aeruginosa strains worldwide. Quorum sensing is a bacterial cell-cell communication system that regulates the expression of genes, including virulence factors, through production of acyl-homoserine lactones (AHLs) in Pseudomonas aeruginosa. The phenotypic expression of virulence factors in P. aeruginosa is mediated by quorum sensing systems (las and rhl). In this study an anti-infective molecule produced by a marine actinomycetes Nesterenkonia sp. MSA31 was elucidated as lipopeptide by NMR and LC-MS/MS analysis. The new lipopeptide molecule was named Nesfactin. This molecule effectively inhibited virulence phenotypes including production of hemolysin, protease, lipase, phospholipase, esterase, elastase, rhamnolipid, alginate, and pyocyanin, as well as motility and biofilm formation in P. aeruginosa. The high-performance thin layer chromatography (HPTLC) analysis revealed that the lipopeptide (50 µg/mL) inhibited production of the AHLs produced by the las and rhl quorum sensing systems (3-oxo-C12-HSL and C4-HSL, respectively). Docking analysis showed the binding affinity of the ligand towards the quorum sensing receptor molecules. The confocal laser scanning microscopy images showed the anti-biofilm effect of lipopeptide against P. aeruginosa. Nesfactin based hydrogel showed a significant antibiofilm effect on the catheter. This study suggests that the lipopeptide may be an effective anti-virulence treatment for Pseudomonas aeruginosa infections.

Anti-diabetic Potential of a Stigmasterol From the Seaweed Gelidium spinosum and Its Application in the Formulation of Nanoemulsion Conjugate for the Development of Functional Biscuits.

The seaweed Gelidium spinosum was selected for the extraction of phytosterol by the Soxhlet method. The extracted phytosterol was chemically characterized as stigmasterol using Fourier-transform infrared spectrometry and gas chromatography-mass spectrometry analysis. The antioxidant and α-amylase inhibitory activity of stigmasterol has been confirmed by in vitro assays. The in vivo studies demonstrated an anti-diabetic effect in streptozotocin (STZ)-induced hyperglycemic rats. Biochemical analysis showed administration of stigmasterol reduced the blood sugar, urea, and creatinine level. The stigmasterol was then nano-emulsified and incorporated into dough for biscuit formulation. The stigmasterol incorporated biscuit showed higher proximate values, low moisture content, lighter color and the textural property revealed lower hardness. Sensorial results showed acceptability when compared to the control. This study demonstrated the stigmasterol reduced hyperglycemic effects and therefore could be used as a supplement in diets for diabetic patients.

Revealing antibiotic resistance in therapeutic and dietary probiotic supplements.

OBJECTIVES: The aim of this study was to evaluate antibiotic susceptibility patterns in commercially available dietary and probiotic supplements. METHODS: Probiotic strains were isolated from the dietary supplements (designated as sample B, D and V), and multidrug resistance profiles were tested using the Kirby-Bauer test. Minimum inhibitory concentrations and double-disk synergy tests were performed to detect the mechanism of action of the resistance, and the presence of extended spectrum ß-lactamase activity (ESBL) was confirmed. RESULTS: The isolates Streptococcus faecalis and Bacillus mesentericus (both from sample B) were found to be resistant to penicillin G, Lactobacillus acidophilus (sample D) was resistant to ampicillin, and all the isolates from samples B, D and V were resistant to ceftazidime. The isolates Lactobacillus sporogenes, S. faecalis, B. mesentericus from sample B, Lactobacillus. rhamnosus, Saccharomyces boulardii from sample D, and L. sporogenes (sample V) were resistant to erythromycin. CONCLUSIONS: The findings showed the presence of antibiotic resistance in probiotic bacteria isolated from commercially available dietary supplements. Because multidrug resistance is a serious emerging issue, and the risk of drug-resistant gene transfer to commensals or pathogens of the gut is inevitable, the safety of probiotics has become a major criterion of interest. The findings of this study would serve as a platform for further screening and characterization of the determinants of antibiotic resistance and the genetic mechanisms of resistance.

Revealing the Efficacy of Thermostable Biosurfactant in Heavy Metal Bioremediation and Surface Treatment in Vegetables.

Biosurfactants are amphiphilic molecules which showed application in the food, medical, and cosmetics industries and in bioremediation. In this study, a marine sponge-associated bacteria (MSI 54) was identified as a biosurfactant producer which showed high emulsification and surface tension-reducing property. The isolate MSI 54 was identified as Bacillus sp. and the biosurfactant was chemically characterized as a lipopeptide analog based on the spectral data including Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The MSI 54 lipopeptide biosurfactant was an anionic molecule which showed high affinity toward cationic heavy metals including Pb, Hg, Mn, and Cd. The heavy metal bioremediation efficacy of the biosurfactant was evaluated using atomic absorption spectroscopy, scanning electron microscopy/energy-dispersive X-ray spectroscopy, and high-resolution transmission electron microscopy analysis. When MSI 54 lipopeptide biosurfactant was added to heavy metals, this resulted in a white co-precipitate of the metal-biosurfactant complex. The heavy metal remediation efficacy of the biosurfactant at a 2.0 × critical micelle concentration (CMC) showed removal of 75.5% Hg, 97.73% Pb, 89.5% Mn, and 99.93% Cd, respectively, in 1,000 ppm of the respective metal solution. The surface treatment of farm fresh cabbage, carrot, and lettuce with 2.0 × CMC of the lipopeptide showed effective removal of the surface heavy metal contaminants.

Photoprotective effect of nanomelanin-seaweed concentrate in formulated cosmetic cream: With improved antioxidant and wound healing properties.

A melanin producer bacteria Halomonas venusta was isolated from a marine sponge Callyspongia sp. and optimized for melanin production. The optimized fermented media supplemented with 1% tyrosine yielded 4.92 mg/ml of melanin. The melanin incorporated cream was formulated and fortified with concentrates of seaweed Gelidium spinosum. Melanin and seaweed concentrate were found to be rich in antioxidant activity and were effectively inhibited the growth of S. aureus (MTCC 96) and S. pyogenes (MTCC 442). Various combinations of the cream were optimized and selected a formula containing 0.25% of melanin and 0.75% of seaweed concentrate which showed improved texture quality of cream. The formulated cream showed a pH of 5.52, spreadability 23 mm, and smooth and homogeneous texture. On application over skin provides a cooling effect and immediate disappearance without formation of white or oily film. Texture analysis of newly formulated cream showed similar results with that of control cream in terms of firmness, cohesiveness, index of viscosity and consistency. The formulated cream showed significant reduction of reactive oxygen species generated on exposure to direct sunlight. The cream showed protective effect on photohemolysis thus protecting the skin from lysis of red blood cells. The sun protection factor of the formulated cream F3 was found to be 18.373 ± 1.45. The combined antimicrobial and antioxidant effect of melanin and seaweed concentrate increased the shelf life of cream over the control. This study was the first report on photoprotective cream formulation using melanin and seaweed concentrate, which improved antioxidant and wound healing properties. The antimicrobial effect of the formulated natural cream could reduce the emergence of drug resistant bacteria and side effects of synthetic creams.

An antibiotic agent pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro isolated from a marine bacteria Bacillus tequilensis MSI45 effectively controls multi-drug resistant Staphylococcus aureus.

Sponge associated bacteria are a rich source of bioactive secondary metabolites. This study aims to isolate bacteria producing antimicrobial agents from a marine sponge, Callyspongia diffusa. A total of fifty-six bacteria were isolated and screened for antibacterial activity against multidrug resistant S. aureus. Based on the 16S rRNA sequence and phylogenetic analysis the antimicrobial producer strain MSI45 was identified as a novel Bacillus tequilensis. The culture conditions of strain MSI45 were optimized to enhance the yield of the antimicrobial compound. The antimicrobial compound was purified using a silica gel column chromatography and high performance liquid chromatography. On the basis of spectroscopic analysis such as FT-IR, NMR and GC-MS, the bioactive metabolite was identified as pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro. The extracted compound MSI45 showed a potent inhibitory effect on multidrug resistant S. aureus with an MIC of 15 ± 0.172 mg L-1 and MBC of 20 ± 0.072 mg L-1. The compound was non-hemolytic and showed high antioxidant activity. The antioxidant activity may increase the efficacy and safety of the molecule in drug development. Hence, this compound produced by Bacillus tequilensis MSI45 could have potent antimicrobial and antioxidant activity against S. aureus infection.

Revealing the ability of a novel polysaccharide bioflocculant in bioremediation of heavy metals sensed in a Vibrio bioluminescence reporter assay.

A bioflocculant-producing bacterial strain, designated MSI021, was isolated from the marine sponge Dendrilla nigra and demonstrated 94% flocculation activity in a kaolin clay suspension. MSI021 was identified as Bacillus cereus based on phylogenetic affiliation and biochemical characteristics. The purified extra-cellular bioflocculant was chemically elucidated as a polysaccharide molecule. The polysaccharide bioflocculant was stable under both acidic and alkaline conditions (pH 2.0-10.0) and temperatures up to 100 °C. The purified bioflocculant efficiently nucleated the formation of silver nanoparticles which showed broad spectrum antibacterial activity. The ability of the bioflocculant to remediate heavy metal toxicity was evaluated by measuring the inhibition of bioluminescence expression in Vibrio harveyi. Enrichment of heavy metals such as zinc, mercury and copper at concentrations of 1, 2 and 3 mM in culture media showed significant reduction of bioluminescence in Vibrio, whereas media enriched with heavy metals and bioflocculant showed dose dependent improvement in the expression of bioluminescence. The assay results demonstrated that the polysaccharide bioflocculant effectively mitigates heavy metal toxicity, thereby improving the expression of bioluminescence in Vibrio. This bioluminescence reporter assay can be developed into a high-throughput format to monitor and evaluate of heavy metal toxicity. The findings of this study revealed that a novel polysaccharide bioflocculant produced by a marine B. cereus demonstrated strong flocculating performance and was effective in nucleating the formation antibacterial silver nanoparticles and removing heavy metals. These results suggest that the MSI021 polysaccharide bioflocculant can be used to develop greener waste water treatment systems.

Production of Lipopeptide Biosurfactant by a Marine Nesterenkonia sp. and Its Application in Food Industry.

Biosurfactants are smart biomolecules which have wide spread application in medicines, processed foods, cosmetics as well as in bioremediation. In food industry, biosurfactants are used as emulsion stabilizing agents, antiadhesives, and antimicrobial/antibiofilm agents. Nowadays biosurfactant demands in industries has increased tremendously and therefore new bacterial strains are being explored for large scale production of biosurfactants. In this study, an actinobacterial strain MSA31 was isolated from a marine sponge Fasciospongia cavernosa which showed high activity in biosurfactant screening assays such as drop collapsing, oil displacement, lipase and emulsification. Lipopeptide produced by MSA31 was found to be thermostable which was evident in differential scanning calorimetry analysis. The spectral data obtained in the Fourier transform infrared spectroscopy showed the presence of aliphatic groups combined with peptide moiety which is a characteristic feature of lipopeptides. The stability index of lipopeptide MSA31 revealed "halo-alkali and thermal tolerant biosurfactant" which can be used in the food industry. Microtiter plate assay showed 125 µg/ml of lipopeptide was effective in reducing the biofilm formation activity of pathogenic multidrug resistant Staphylococcus aureus. The confocal laser scanning microscopic images provided further evidences that lipopeptide MSA31 was an effective antibiofilm agent. The antioxidant activity of lipopeptide MSA31 may be due to the presence of unsaturated fatty acid present in the molecule. The brine shrimp cytotoxicity assay showed lipopeptide MSA31 was non-toxic and can be used as food additives. Incorporation of lipopeptide MSA31 in muffin showed improved organoleptic qualities compared to positive and negative control. This study provides a valuable input for this lipopeptide to be used in food industry as an effective emulsifier, with good antioxidant activity and as a protective agent against S. aureus.