No abstract

clinicaltrials.gov Identifier: NCT02034513.

Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta. Antifungal activity was extractable from spent growth media, and fractionation by semi‐preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H‐, 13C‐NMR and mass spectrometry. The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2). No viscosin production was observed, however, when Ps. fluorescens DR54 was cultured in media enriched with glutamate. In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P. ultimum and R. solani. Viscosinamide production by Ps. fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value. In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase. Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps. fluorescens DR54. The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps. fluorescens DR54 in biological control.

Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLPproducing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.Cyclic lipopeptides (CLPs) are produced by distinctively different groups of bacteria, both gram-positive (20) and gramnegative (28). The high diversity of CLP-producing microorganisms (28) and differences in chemical structure suggest that the CLP compounds may serve different, and possibly multiple, purposes. This may explain why the specific role of CLP production is often unclear (28,40). For a limited number of CLPs (28), the reported functions include promotion of bacterial swarming (12, 26) and biosurfactant properties (19,24,41). In many cases, CLP compounds are also known to exert a role in antagonistic interactions with other organisms (28), e.g., plant pathogenicity (5) and antifungal (19,30,31,38,44), antibacterial (11), antiviral (49), or cytotoxic (16) activity.Synthesis of CLPs is nonribosomal and catalyzed by large peptide synthetase complexes (27). Various environmental stimuli may affect CLP production, i.e., carbon substrate (36), limitation by C, N, or P (15, 37), Fe limitation (15), growth phase conditions (15), and interaction with interfaces (32). Little information is available on production rates and regulating factors for the compounds in natural environments. Asaka and Shoda (2) detected surfactin and iturine production by Bacillus subtilis RB14 in a sterilized vermiculite-soil system, and Nakayama et al. (31) detected xanthobaccin A production by a Stenotrophomonas sp. strain, SB-K88, in a hydroponic sugar beet rhizosphere system, but documentation for in situ production of CLPs...

Aim: To study the antagonistic activity by Pseudomonas¯uorescens strain 96.578 on the plant pathogenic fungus Rhizoctonia solani. Methods and Results: Strain 96.578 produced a new cyclic lipopeptide, tensin. High tensin production per cell was detected in liquid media with glucose, mannitol or glutamate as growth substrate while fructose, sucrose and asparagine supported low production. Tensin production was nearly constant in media with different initial C levels, while low initial N contents reduced production. When applied to sugar beet seeds, strain 96.578 produced tensin during seed germination. When challenged with strain 96.578 or puri®ed tensin, Rhizoctonia solani reduced radial mycelium extension but increased branching and rosette formation. Conclusion: The antagonistic activity of strain 96.578 towards Rhizoctonia solani was caused by tensin. Signi®cance and Impact of the Study: When coated onto sugar beet seeds, tensin production by strain 96.578 could be of signi®cant importance for inhibition of mycelial growth and seed infection by Rhizoctonia solani.

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 g g ؊1 ) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ϳ2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.

have been investigators for and have received lecture honoraria from LEO Pharma. P.E. has been a study investigator for, has received research funding (departmental) and has received speaker honoraria from LEO Pharma. V.M. has been a study investigator for LEO Pharma. S.M. has been a study investigator and has received speaker honoraria from LEO Pharma. T.S.S.N. is an employee of LEO Pharma.The present address for V.M. is SummaryBackground Management of chronic hand eczema (CHE) remains a challenge; effective topical treatment is limited to corticosteroids. Objectives To assess the efficacy and safety of a novel, pan-Janus kinase inhibitor (delgocitinib) in patients with CHE. Methods In this randomized, double-blind, phase IIa study, patients with CHE received delgocitinib ointment 30 mg g À1 or vehicle ointment for 8 weeks. The primary end point was the proportion of patients achieving treatment success ['clear'/ 'almost clear' skin with ≥ 2-point improvement in the Physician's Global Assessment of disease severity (PGA)] at week 8. Secondary end points included Hand Eczema Severity Index (HECSI) score changes and the proportion of patients achieving treatment success on the Patient's Global Assessment of disease severity (PaGA). Results Ninety-one patients were randomized. More patients receiving delgocitinib (46%) than vehicle (15%) [odds ratio 4Á89, 95% confidence interval (CI) 1Á49-16Á09; P = 0Á009] achieved treatment success (PGA). Adjusted mean HECSI score at week 8 was lower with delgocitinib (13Á0) than with vehicle (25Á8) (adjusted mean difference À12Á88, 95% CI À21Á47 to À4Á30; P = 0Á003). More patients receiving delgocitinib than vehicle achieved treatment success by PaGA, but this did not reach statistical significance. The incidence of adverse events was similar with delgocitinib and vehicle; none led to discontinuation of delgocitinib. Conclusions Delgocitinib ointment was efficacious and well tolerated. As a plateau of efficacy was not observed, a longer treatment period may lead to increased efficacy. Further clinical studies are warranted to confirm these findings in patients with CHE.What's already known about this topic?• Chronic hand eczema (CHE) has a significant burden. • Few randomized controlled studies have evaluated current treatments for CHE; only limited data are available to inform and guide clinical practice decisions.• There is currently an unmet need for efficacious and well-tolerated topical treatment options for patients with CHE.What does this study add?• Delgocitinib is a novel, pan-Janus kinase (JAK) inhibitor specific for JAK1, JAK2, JAK3 and tyrosine kinase 2.1104 Topical delgocitinib in patients with chronic hand eczema, M. Worm et al.

Pseudomonas sp. strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi. By Tn5::luxAB mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8. The gene interrupted by the transposon in strain DSS73-15C2 (amsY) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase. DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS. Growth of strain DSS73-15C2 (amsY) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds. This growth phenotype could be complemented by purified amphisin. Seed exudate further induced expression of bioluminescence from the amsY::luxAB reporter during the transition to stationary phase. This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase. Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional gacS locus. Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.