Biosynthesis enhancement of tropodithietic acid (TDA) antibacterial compound through biofilm formation by marine bacteria Phaeobacter inhibens on micro-structured polymer surfaces.

Although aquaculture is a major player in current and future food production, the routine use of antibiotics provides ample ground for development of antibiotic resistance. An alternative route to disease control is the use of probiotic bacteria such as the marine bacteria Phaeobacter inhibens which produces tropodithietic acid (TDA) that inhibit pathogens without affecting the fish. Improving conditions for the formation of biofilm and TDA-synthesis is a promising avenue for biocontrol in aquaculture. In this study, the biosynthesis of TDA by Phaeobacter inhibens grown on micro-structured polymeric surfaces in micro-fluidic flow-cells is investigated. The formation of biofilms on three surface topographies; hexagonal micro-pit-arrays, hexagonal micro-pillar-arrays, and planar references is investigated. The biomass on these surfaces is measured by a non-invasive confocal microscopy 3D imaging technique, and the corresponding TDA production is monitored by liquid chromatography mass spectrometry (LC-MS) in samples collected from the outlets of the microfluidic channels. Although all surfaces support growth of P. inhibens, biomass appears to be decoupled from total TDA biosynthesis as the micro-pit-arrays generate the largest biomass while the micro-pillar-arrays produce significantly higher amounts of TDA. The findings highlight the potential for optimized micro-structured surfaces to maintain biofilms of probiotic bacteria for sustainable aquacultures.

A reliable quantitative method for determining CBD content and release from transdermal patches in Franz cells.

INTRODUCTION: There are several cannabidiol (CBD) transdermal patches available on the market. However, none are FDA-approved. Furthermore, not much evidence has been published about CBD release and skin permeation from such patches, so the effectiveness and reliability remain unclear. OBJECTIVES: We aimed to develop a method to determine the in vitro release and skin permeation of CBD from transdermal patches using Franz cell diffusion in combination with quantitative 1 H-NMR (qNMR). MATERIALS AND METHODS: The study was conducted on CBD patches with known CBD content and six different commercially available or market-ready CBD patches using a Franz cell with a Strat-M™ membrane and with samples taken directly from the transdermal patch for qNMR analysis. RESULTS: The use of qNMR yielded an average recovery of 100% ± 7% when samples with known CBD content were tested. Results from the testing of six commercially available patches indicated that five out of six patches did not contain the CBD amount stated by the manufacturer according to a ± 10% variance margin, of which four patches were under-labeled and one was over-labeled. The release rate of patches was determined, and significant differences between the patches were shown. Maximum release of CBD was calculated to occur after 39 to 70 h. CONCLUSION: The established method was proven to be a reliable means of determining the quantity and release of CBD from transdermal patches and can be used to verify CBD content and release rate in transdermal patches.

Alkali Metal- and Acid-Catalyzed Interconversion of Goniodomin A with Congeners B and C.

Goniodomin A (GDA, 1) is a phycotoxin produced by at least four species of Alexandrium dinoflagellates that are found globally in brackish estuaries and lagoons. It is a linear polyketide with six oxygen heterocyclic rings that is cyclized into a macrocyclic structure via lactone formation. Two of the oxygen heterocycles in 1 comprise a spiro-bis-pyran, whereas goniodomin B (GDB) contains a 2,7-dioxabicyclo[3.3.1]nonane ring system fused to a pyran. When H2O is present, 1 undergoes facile conversion to isomer GDB and to an α,ß-unsaturated ketone, goniodomin C (GDC, 7). GDB and GDC can be formed from GDA by cleavage of the spiro-bis-pyran ring system. GDA, but not GDB or GDC, forms a crown ether-type complex with K+. Equilibration of GDA with GDB and GDC is observed in the presence of H+ and of Na+, but the equilibrated mixtures revert to GDA upon addition of K+. Structural differences have been found between the K+ and Na+ complexes. The association of GDA with K+ is strong, while that with Na+ is weak. The K+ complex has a compact, well-defined structure, whereas Na+ complexes are an ill-defined mixture of species. Analyses of in vitro A. monilatum and A. hiranoi cultures indicate that only GDA is present in the cells; GDB and GDC appear to be postharvest transformation products.

Phages carry interbacterial weapons encoded by biosynthetic gene clusters.

Bacteria produce diverse specialized metabolites that mediate ecological interactions and serve as a rich source of industrially relevant natural products. Biosynthetic pathways for these metabolites are encoded by organized groups of genes called biosynthetic gene clusters (BGCs). Understanding the natural function and distribution of BGCs provides insight into the mechanisms through which microorganisms interact and compete. Further, understanding BGCs is extremely important for biocontrol and the mining of new bioactivities. Here, we investigated phage-encoded BGCs (pBGCs), challenging the relationship between phage origin and BGC structure and function. The results demonstrated that pBGCs are rare, and they predominantly reside within temperate phages infecting commensal or pathogenic bacterial hosts. Further, the vast majority of pBGCs were found to encode for bacteriocins. Using the soil- and gut-associated bacterium Bacillus subtilis, we experimentally demonstrated how a temperate phage equips a bacterium with a fully functional BGC, providing a clear competitive fitness advantage over the ancestor. Moreover, we demonstrated a similar transfer of the same phage in prophage form. Finally, using genetic and genomic comparisons, a strong association between pBGC type and phage host range was revealed. These findings suggest that bacteriocins are encoded in temperate phages of a few commensal bacterial genera. In these cases, lysogenic conversion provides an evolutionary benefit to the infected host and, hence, to the phage itself. This study is an important step toward understanding the natural role of bacterial compounds encoded by BGCs, the mechanisms driving their horizontal transfer, and the sometimes mutualistic relationship between bacteria and temperate phages.

Antimicrobial Activity of Small Synthetic Peptides Based on the Marine Peptide Turgencin A: Prediction of Antimicrobial Peptide Sequences in a Natural Peptide and Strategy for Optimization of Potency.

Turgencin A, a potent antimicrobial peptide isolated from the Arctic sea squirt Synoicum turgens, consists of 36 amino acid residues and three disulfide bridges, making it challenging to synthesize. The aim of the present study was to develop a truncated peptide with an antimicrobial drug lead potential based on turgencin A. The experiments consisted of: (1) sequence analysis and prediction of antimicrobial potential of truncated 10-mer sequences; (2) synthesis and antimicrobial screening of a lead peptide devoid of the cysteine residues; (3) optimization of in vitro antimicrobial activity of the lead peptide using an amino acid replacement strategy; and (4) screening the synthesized peptides for cytotoxic activities. In silico analysis of turgencin A using various prediction software indicated an internal, cationic 10-mer sequence to be putatively antimicrobial. The synthesized truncated lead peptide displayed weak antimicrobial activity. However, by following a systematic amino acid replacement strategy, a modified peptide was developed that retained the potency of the original peptide. The optimized peptide StAMP-9 displayed bactericidal activity, with minimal inhibitory concentrations of 7.8 µg/mL against Staphylococcus aureus and 3.9 µg/mL against Escherichia coli, and no cytotoxic effects against mammalian cells. Preliminary experiments indicate the bacterial membranes as immediate and primary targets.

Isolation and Characterization of Antimicrobial Peptides with Unusual Disulfide Connectivity from the Colonial Ascidian Synoicum turgens.

This study reports the isolation of two novel cysteine-rich antibacterial peptides, turgencin A and turgencin B, along with their oxidized derivatives, from the Arctic marine colonial ascidian Synoicum turgens. The peptides are post-translationally modified, containing six cysteines with an unusual disulfide connectivity of Cys1-Cys6, Cys2-Cys5, and Cys3-Cys4 and an amidated C-terminus. Furthermore, the peptides contain methionine residues resulting in the isolation of peptides with different degrees of oxidation. The most potent peptide, turgencin AMox1 with one oxidized methionine, displayed antimicrobial activity against both Gram-negative and Gram-positive bacteria with a minimum inhibitory concentration (MIC) as low as 0.4 µM against selected bacterial strains. In addition, the peptide inhibited the growth of the melanoma cancer cell line A2058 (IC50 = 1.4 µM) and the human fibroblast cell line MRC-5 (IC50 = 4.8 µM). The results from this study show that natural peptides isolated from marine tunicates have the potential to be promising drug leads.

Development of a LC-MS/MS method for the quantification of goniodomins A and B and its application to Alexandrium pseudogonyaulax strains and plankton field samples of Danish coastal waters.

An external standard of goniodomin A (GDA) was prepared from a strain of Alexandrium pseudogonyaulax originating from New Zealand and its chemical structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. Using the GDA standard, an ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method in selected reaction monitoring (SRM) mode was developed for separation and quantification of GDA. This method was successfully applied to planktonic field samples collected during an oceanographic expedition conducted with R/V Uthörn along the Danish west coast, Limfjord and Kattegat in June 2016. In addition, this method was used to characterize goniodomin (GD) profiles of 17 A. pseudogonyaulax strains from the coastal North Sea and from Limfjord. Highest GDA levels were found in Limfjord (up to 590 ng NT-1 m-1), but GDA was also detected in the North Sea appearing at the latitude of Sylt Island northwards and in Kattegat from the eastern mouth of Limfjord down to the Kiel Bight, but at lower abundances than within Limfjord. This is the first reported detection of GDA in planktonic field samples. Chemical analysis of 17 strains of A. pseudogonyaulax revealed that all strains were producers of GDA (5-35 pg cell-1) as well as in most cases minor amounts (0.01-0.07 pg cell-1, expressed as GDA equivalents) of goniodomin B (GDB).