Newcastle Disease Virus in Madagascar: Identification of an Original Genotype Possibly Deriving from a Died Out Ancestor of Genotype IV

In Madagascar, Newcastle disease (ND) has become enzootic after the first documented epizootics in 1946, with recurrent annual outbreaks causing mortality up to 40%. Four ND viruses recently isolated in Madagascar were genotypically and pathotypically characterised. By phylogenetic inference based on the F and HN genes, and also full-genome sequence analyses, the NDV Malagasy isolates form a cluster distant enough to constitute a new genotype hereby proposed as genotype XI. This new genotype is presumably deriving from an ancestor close to genotype IV introduced in the island probably more than 50 years ago. Our data show also that all the previously described neutralising epitopes are conserved between Malagasy and vaccine strains. However, the potential implication in vaccination failures of specific amino acid substitutions predominantly found on surface-exposed epitopes of F and HN proteins is discussed

Pseudomonas aeruginosa biofilm formation and persistence, along with the production of quorum sensing-dependent virulence factors, are disrupted by a triterpenoid coumarate ester isolated from dalbergia trichocarpa, a tropical legume

Recently, extracts of Dalbergia trichocarpabark have been shown to disrupt P. aeruginosa- PAO1 quorum sensing (QS) mechanisms, which are key regulators of virulence factor expression and implicated in biofilm formation. One of the active compounds has been isolated and identified as oleanolic aldehyde coumarate (OALC), a novel bioactive compound that inhibits the formation of P. aeruginosa PAO1 biofilm and its maintenance as well as the expression of the las and rhl QS systems. Consequently, the production of QS-controlled virulence factors including, rhamnolipids, pyocyanin, elastase and extracellular polysaccharides as well as twitching and swarming motilities is reduced. Native acylhomoserine lactones (AHLs) production is inhibited by OALC but exogenous supply of AHLs does not restore the production of virulence factors by OALC-treated cultures, indicating that OALC exerts its effect beyond AHLs synthesis in the QS pathways. Further experiments provided a significant inhibition of the global virulence factor activator gacA by OALC. OALC disorganizes established biofilm structure and improves the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Finally, a significant reduction of Caenorhabditis elegans paralysis was recorded when the worms were infected with OALC-pre-treated P. aeruginosa. Taken together, these results show that triterpenoid coumarate esters are suitable chemical backbones to target P. aeruginosa virulence mechanisms.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Endemic Chromoblastomycosis Caused Predominantly by Fonsecaea nubica , Madagascar1

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PAO1 biofilm phenotypes as affected by DMSO, naringin, naringenin or OALC.

<p>(A) Fluorescence microscopy images of PAO1 cells incubated statically at 37°C for 24 hours. Cells were visualized after staining with SYTO-9 (green fluorescence for living bacteria) and propidium iodide (red fluorescence for dead bacteria) furnished in the LIVE/DEAD <i>Bac</i>Light kit. (B) Fluorescence microscopic images of biofilm formation by PAO1 cells incubated for 24 hours and then treated for 24 hours with DMSO, naringin, naringenin or OALC. Fluorescence microscopy was achieved by using a Leica DM IRE2 inverted fluorescence microscope using a 40x objective lens and images were false-colored and assembled using Adobe Photoshop.</p

Synergistic activity of OALC with tobramycin against biofilm-encapsulated <i>P</i>. <i>aeruginosa</i> PAO1.

<p>PAO1 cells were incubated statically for 24 hours and then treated for 24 hours with tobramycin (100 μg mL^-1) and DMSO, naringin (4 mM), naringenin (4 mM) or OALC (200 μM final concentration). (A) OALC + tobramycin, (B) tobramycin, (C) naringenin + tobramycin, (D) DMSO + tobramycin, (E) naringin + tobramycin. Assessment of bacterial viability and microscopy were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132791#pone.0132791.g006" target="_blank">Fig 6</a>. (F) Quantification of bacterial viability. Error bars represent the standard errors of the means; all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s <i>t</i>-tests; <i>P</i> ≤ 0.01).</p

Effect of OALC on extracellular polysaccharides production by <i>P</i>. <i>aeruginosa</i> PAO1.

<p>(A) Quantification of total extracellular polysaccharides. The cell density of the bacteria was assessed at 600nm and extracellular polysaccharides production was measured using Phenol-Sulfuric Acid (PSA) method and expressed in μg mL^-1 with glucose as standard. (B) Quantification of alginate. The cell density of the bacteria was assessed at 600nm and alginate production was measured using carbazole method and expressed in μg mL^-1 with sodium alginate as standard. Error bars represent the standard errors of the means; all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s <i>t</i>-tests; <i>P</i> ≤ 0.01). Naringenin (Nar) and naringin (Nin) both at 4 mM final concentration were used as quorum sensing inhibitor positive and negative controls, respectively.</p

Effect of OALC on virulence factors and acylhomoserine lactones production in <i>P</i>. <i>aeruginosa</i> PAO1.

<p>(A) rhamnolipids production, (B) pyocyanin production, (C) Elastase production. The cell density of the bacteria was assessed at 600 nm and elastase production was assessed via an elastolysis assay and calculated as the ratio between <i>A</i>₄₉₅ and <i>A</i>₆₀₀. The rhamnolipids production was measured using methylene-blue-based method and expressed in μg mL^-1. Pyocyanin was extracted, quantified by absorbance measurements at 380 nm and calculated as the ratio between <i>A</i>₃₈₀ and <i>A</i>₆₀₀. (D) Quantification of <i>N</i>-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL; grey bar) and <i>N</i>-butanoyl-L-homoserine lactone (C4-HSL; clear bar) produced by PAO1 cells. Acylhomoserine lactones were extracted and quantified by mass spectrometry. Error bars represent the standard errors of the means and all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO controls (Student’s <i>t</i>-tests; <i>P</i> ≤ 0.01). Naringenin (Nar) and naringin (Nin) were used as a quorum sensing inhibitor control and negative control, respectively.</p

Chemical structure of the bioactive compound assigned as 3β-hydroxyolean-12-en-28-al 3-<i>p</i>-coumarate (oleanolic aldehyde coumarate, OALC).

<p>Chemical structure of the bioactive compound assigned as 3β-hydroxyolean-12-en-28-al 3-<i>p</i>-coumarate (oleanolic aldehyde coumarate, OALC).</p