Biofilm inhibition and anti-quorum sensing activity of phytosynthesized silver nanoparticles against the nosocomial pathogen Pseudomonas aeruginosa

Quorum sensing (QS), the communication signaling network, regulates biofilm formation and several virulence factors in Pseudomonas aeruginosa PAO1, a nosocomial opportunistic pathogen. QS is considered to be a challenging target for compounds antagonistic to virulent factors. Biologically synthesized silver nanoparticles (AgNPs) are reported as anti-QS and anti-biofilm drugs against bacterial infections. The present study reports on the synthesis and characterization of Piper betle (Pb) mediated AgNPs (Pb-AgNPs). The anti-QS activity of Pb-AgNPs against Chromobacterium violaceum and the potential effect of Pb-AgNPs on QS-regulated phenotypes in PAO1 were studied. FTIR analysis exhibited that Pb-AgNPs had been capped by phytochemical constituents of Pb. Eugenol is one of the active phenolic phytochemicals in Pb leaves, therefore molecular docking of eugenol-conjugated AgNPs on QS regulator proteins (LasR, LasI and MvfR) was performed. Eugenol-conjugated AgNPs showed considerable binding interactions with QS-associated proteins. These results provide novel insights into the development of phytochemically conjugated nanoparticles as promising anti-infective candidates.     

Identification and genetic diversity analysis of strain Pseudomonas syringae S5: A pathogen responsible for bacteriosis in Avena sativa plants

The genus Pseudomonas includes pathogenic species P. syringae, which can be found in various agricultural environments and which can affect a wide variety of plants, causing significant economic losses when the environmental conditions for its proliferation are optimal. Comprehensive characterizations of phytopathogenic bacteria belonging to the genus Pseudomonas are scarce in Argentina. In this work, the tabtoxin‐producing strain Pseudomonas S5, isolated from oat, was identi?ed as a P. syringae through biochemical tests such as the LOPAT test, and genetic tests such as the analysis of the small subunit ribosomal RNA gene (16S rRNA) sequence and repetitive elements, using BOX and ERIC primers. It was also determined that this phytopathogen is potentially capable of infecting other crops of agricultural importance for our region, such as soybean. This ability to infect different hosts gives it an adaptive advantage that allows it to endure seasonal changes in the environment where it lives. Our work contributes to the physiological classification of the phytopathogen P. syringae S5 isolated from our region, as well as to the knowledge about its range of potential hosts.     

Enumeration, isolation and identification of diazotrophs from Korean wetland rice varieties grown with long-term application of N and compost and their short-term inoculation effect on rice plants

AIM: This study has been aimed (i) to isolate and identify diazotrophs from Korean rice varieties; (ii) to examine the long-term effect of N and compost on the population dynamics of diazotrophs and (iii) to realize the shot-term inoculation effect of these diazotrophs on rice seedlings. METHODS AND RESULTS: Diazotrophic and heterotrophic bacterial numbers were enumerated by most probable number method and the isolates were identified based on morphological, physiological, biochemical and 16s rDNA sequence analysis. Long-term application of fertilizer N with compost enhanced both these numbers in rice plants and its environment. Bacteria were high in numbers when malate and azelaic acids were used as carbon source, but less when sucrose was used as a carbon substrate. The combined application promoted the association of diazotrophic bacteria like Azospirillum spp., Herbaspirillum spp., Burkholderia spp., Gluconacetobacter diazotrophicus and Pseudomonas spp. in wetland rice plants. Detection of nifD genes from different diazotrophic isolates indicated their nitrogen fixing ability. Inoculation of a representative isolate from each group onto rice seedlings of the variety IR 36 grown in test tubes indicated the positive effect of these diazotrophs on the growth of rice seedlings though the percentage of N present in the plants did not differ much. CONCLUSIONS: Application of compost with fertilizer N promoted the diazotrophic and heterotrophic bacterial numbers and their association with wetland rice and its environment. Compost application in high N fertilized fields would avert the reduction of N(2)-fixing bacterial numbers and their association was beneficial to the growth of rice plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of high N fertilization on diazotrophic bacterial numbers could be reduced by the application of compost and this observation would encourage more usage of organic manure. This study has also thrown light on the wider geographic distribution of G. diazotrophicus with wetland rice in temperate region where sugarcane (from which this bacterium was first reported to be associating and thereon from other plant species) is not cultivated.     

Effect of antibiotic-induced morphological changes on surface properties, motility and adhesion of nosocomial Pseudomonas aeruginosa strains under different physiological states

AIM: To investigate the influence of antibiotic-induced morphological changes on adhesion and motility abilities and surface properties of nosocomial Pseudomonas aeruginosa under different physiological states. METHODS AND RESULTS: The effects of subinhibitory concentration (sub-MIC) of Piperacillin/Tazobactam (P/T) and Imipenem (IMP) were studied on P. aeruginosa adhesion (1 h) using a modified microtitre-plate assay, on their ability to swim and to twitch, on surface hydrophobicity and on acid-base interactions of P. aeruginosa strains by measuring their ability to adhere to n-hexadecane, chloroform and ethyl acetate, respectively. Our results show that antibiotic-induced morphological changes and bacterial physiological state can affect differently surface properties, motility and adhesion abilities of P. aeruginosa. CONCLUSIONS: Under different physiological states P/T induced morphological changes, reduced motility abilities, decreased adhesion to polystyrene and cell surface hydrophobicity (CSH). Moreover, P/T and IMP led to similar changes in exponential population adhesion to chloroform and ethyl acetate. Additionally, IMP induced morphological changes and showed no differences on CSH, adhesion and motility abilities in both growth phases. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhesion is an attractive target for new antibacterial strategies, namely by using sub-MIC antibiotics that induce morphological, motility and surface properties changes, which are dependent of P. aeruginosa phenotype and physiological state.     

Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability

Exotoxin Y (ExoY) is a type III secretion system effector found in ~ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases.