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Sample records for aeruginosa extracellular products

  1. Pseudomonas aeruginosa extracellular products inhibit staphylococcal growth, and disrupt established biofilms produced by Staphylococcus epidermidis

    DEFF Research Database (Denmark)

    Qin, Zhiqiang; Yang, Liang; Qu, Di

    2009-01-01

    Multiple bacterial species often coexist as communities, and compete for environmental resources. Here, we describe how an opportunistic pathogen, Pseudomonas aeruginosa, uses extracellular products to interact with the nosocomial pathogen Staphylococcus epidermidis. S. epidermidis biofilms and p...... of a novel strategy for controlling S. epidermidis biofilms....

  2. Effects of lead(II) on the extracellular polysaccharide (EPS) production and colony formation of cultured Microcystis aeruginosa.

    Science.gov (United States)

    Bi, Xiang-dong; Zhang, Shu-lin; Dai, Wei; Xing, Ke-zhing; Yang, Fan

    2013-01-01

    To investigate the effects of lead(II) on the production of extracellular polysaccharides (EPS), including bound extracellular polysaccharides (bEPS) and soluble extracellular polysaccharides (sEPS), and the colony formation of Microcystis aeruginosa, cultures of M. aeruginosa were exposed to four concentrations (5.0, 10.0, 20.0 and 40.0 mg/L) of lead(II) for 10 d under controlled laboratory conditions. The results showed that 5.0 and 10.0 mg/L lead(II) stimulated M. aeruginosa growth throughout the experiment while 20.0 and 40.0 mg/L lead(II) inhibited M. aeruginosa growth in the first 2 d exposure and then stimulated it. As compared to the control group, significant increases in the bEPS and sEPS production were observed in 20.0 and 40.0 mg/L lead(II) treatments (P bEPS production, which conversely promoted colony formation, suggesting that heavy metals might be contributing to the bloom-forming of M. aeruginosa in natural conditions.

  3. Abiotic factors in colony formation: effects of nutrition and light on extracellular polysaccharide production and cell aggregates of Microcystis aeruginosa

    Science.gov (United States)

    Yang, Zhen; Kong, Fanxiang

    2013-07-01

    Colony morphology is important for Microcystis to sustain a competitive advantage in eutrophic lakes. The mechanism of colony formation in Microcystis is currently unclear. Extracellular polysaccharide (EPS) has been reported to play an important role in cell aggregate formation of some phytoplankton. Microcystis aeruginosa was cultivated under varied abiotic conditions, including different nutrient, light, and temperature conditions, to investigate their effects on EPS production and morphological change. The results show that nutrient concentration and light intensity have great effects on EPS productionin M. aeruginosa. There was a considerable increase in EPS production after M. aeruginosa was cultivated in adjusted culture conditions similar to those present in the field (28.9 mg C/L, 1.98 mg N/L, 0.65 mg P/L, light intensity: 100 μmol/(m2 · s)). These results indicate that abiotic factors might be one of the triggers for colony formation in Microcystis.

  4. Extracellular toxins of pseudomonas aeruginosa. Pt. 4

    International Nuclear Information System (INIS)

    Obernesser, H.J.; Doering, G.

    1982-01-01

    A sensitive and specific solid phase radioimmunoassay (RIA) for detection of the elastase (Ela) of Pseudomonas aeruginosa (PA) was developed and the RIA was used to assay 10 PA strains of various origin and serotype. A great strain variability of Ela production was found which different from 94.1 to 0.1 μg per ml of culture supernatant fluid (CSF). The Ela and alkaline protease (AP) concentrations were converted to proteolytic activity and combined. The sum of the calculated enzymatic values of Ela and AP correlated well with the experimentally determined values of total proteolytic activity of the CSF. (orig.) [de

  5. Biofilm formation by Staphylococcus epidermidis on peritoneal dialysis catheters and the effects of extracellular products from Pseudomonas aeruginosa.

    Science.gov (United States)

    Pihl, Maria; Arvidsson, Anna; Skepö, Marie; Nilsson, Martin; Givskov, Michael; Tolker-Nielsen, Tim; Svensäter, Gunnel; Davies, Julia R

    2013-04-01

    Biofilm formation by Staphylococcus epidermidis is a cause of infections related to peritoneal dialysis (PD). We have used a PD catheter flow-cell model in combination with confocal scanning laser microscopy and atomic force microscopy to study biofilm formation by S. epidermidis. Adherence to serum-coated catheters was four times greater than to uncoated ones, suggesting that S. epidermidis binds to serum proteins on the catheter surface. Pseudomonas aeruginosa biofilm supernatant interfered with the formation of a serum protein coat thereby reducing the capacity for biofilm formation in S. epidermidis. Supernatants from ΔpelA, ΔpslBCD and ΔrhlAB strains of P. aeruginosa showed no differences from the wild-type supernatant indicating that the effect on serum coat formation was not due to rhamnolipids or the PelA and PslBCD polysaccharides. Supernatant from P. aeruginosa also dispersed established S. epidermidis biofilms. Supernatants lacking PelA or PslBCD showed no differences from the wild type but that from a ΔrhlAB strain, showed reduced, but not abolished, capacity for dispersal. This suggests that rhamnolipids are involved but not wholly responsible for the effect. Thus, supernatants from P. aeruginosa contain promising substances for the prevention and treatment of biofilm infections, although further work is required to identity more active components. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. Biofilm formation by Staphylococcus epidermidis on peritoneal dialysis catheters and the effects of extracellular products from Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Pihl, Maria; Arvidsson, Anna; Skepö, Marie

    2013-01-01

    Biofilm formation by Staphylococcus epidermidis is a cause of infections related to peritoneal dialysis (PD). We have used a PD catheter flow-cell model in combination with confocal scanning laser microscopy and atomic force microscopy to study biofilm formation by S. epidermidis. Adherence....... aeruginosa contain promising substances for the prevention and treatment of biofilm infections, although further work is required to identity more active components....

  7. Extracellular DNA Shields against Aminoglycosides in Pseudomonas aeruginosa Biofilms

    DEFF Research Database (Denmark)

    Chiang, Wen-Chi; Nilsson, Martin; Jensen, Peter Østrup

    2013-01-01

    Within recent years, it has been established that extracellular DNA is a key constituent of the matrix of microbial biofilms. In addition, it has recently been demonstrated that DNA binds positively charged antimicrobials such as aminoglycosides and antimicrobial peptides. In the present study, we...... provide evidence that extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. We show that exogenously supplemented DNA integrates into P. aeruginosa biofilms and increases their tolerance toward aminoglycosides. We provide evidence that biofilms formed by a DNA release......-deficient P. aeruginosa quorum-sensing mutant are more susceptible to aminoglycoside treatment than wild-type biofilms but become rescued from the detrimental action of aminoglycosides upon supplementation with exogenous DNA. Furthermore, we demonstrate that exposure to lysed polymorphonuclear leukocytes...

  8. Swimming Motility Mediates the Formation of Neutrophil Extracellular Traps Induced by Flagellated Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Madison Floyd

    2016-11-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections often characterized by robust neutrophilic infiltration. Neutrophils provide the first line of defense against P. aeruginosa. Aside from their defense conferred by phagocytic activity, neutrophils also release neutrophil extracellular traps (NETs to immobilize bacteria. Although NET formation is an important antimicrobial process, the details of its mechanism are largely unknown. The identity of the main components of P. aeruginosa responsible for triggering NET formation is unclear. In this study, our focus was to identify the main bacterial factors mediating NET formation and to gain insight into the underlying mechanism. We found that P. aeruginosa in its exponential growth phase promoted strong NET formation in human neutrophils while its NET-inducing ability dramatically decreased at later stages of bacterial growth. We identified the flagellum as the primary component of P. aeruginosa responsible for inducing NET extrusion as flagellum-deficient bacteria remained seriously impaired in triggering NET formation. Purified P. aeruginosa flagellin, the monomeric component of the flagellum, does not stimulate NET formation in human neutrophils. P. aeruginosa-induced NET formation is independent of the flagellum-sensing receptors TLR5 and NLRC4 in both human and mouse neutrophils. Interestingly, we found that flagellar motility, not flagellum binding to neutrophils per se, mediates NET release induced by flagellated bacteria. Immotile, flagellar motor-deficient bacterial strains producing paralyzed flagella did not induce NET formation. Forced contact between immotile P. aeruginosa and neutrophils restored their NET-inducing ability. Both the motAB and motCD genetic loci encoding flagellar motor genes contribute to maximal NET release; however the motCD genes play a more important role. Phagocytosis of P. aeruginosa and superoxide production by neutrophils were also

  9. Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development

    DEFF Research Database (Denmark)

    Yang, Liang; Hu, Yifan; Liu, Yang

    2011-01-01

    Bacteria form surface attached biofilm communities as one of the most important survival strategies in nature. Biofilms consist of water, bacterial cells and a wide range of self‐generated extracellular polymeric substances (EPS). Biofilm formation is a dynamic self‐assembly process and several d...... polysaccharide is more important than Pel polysaccharide in P. aeruginosa PAO1 biofilm formation and antibiotic resistance. Our study thus suggests that different EPS materials play distinct roles during bacterial biofilm formation.......Bacteria form surface attached biofilm communities as one of the most important survival strategies in nature. Biofilms consist of water, bacterial cells and a wide range of self‐generated extracellular polymeric substances (EPS). Biofilm formation is a dynamic self‐assembly process and several...... distinguishable stages are observed during bacterial biofilm development. Biofilm formation is shown to be coordinated by EPS production, cell migration, subpopulation differentiation and interactions. However, the ways these different factors affect each other and contribute to community structural...

  10. Reexamining intra and extracellular metabolites produced by Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Maria Shuja

    2016-02-01

    Full Text Available Objective: To isolate, screen and analyze bacteria from different areas of Pakistan for the production of antimicrobial compounds, zinc solubilization and bioplastic production. Methods: Isolation and purification was proceeding with streak plate method. Antagonistic assay was completed with well diffusion and thin-layer chromatography. In vivo analysis of bioplastic was analyzed with Nile blue fluorescence under UV and Sudan staining. Results: A total of 18 bacterial strains purified from soil samples while 148 strains form stock cultures were used. Out of 166 only 94 showed antimicrobial activity against each of Grampositive and Gram-negative; cocci and rods. In case of heavy metal (ZnO and Zn3(PO42.4H2O solubilization, 54 strains solubilized ZnO and 23 strains solubilized Zn3(PO42.4H2O, while 127 strains grown on polyhydroxyalkanoate detection meedia supplemented with Nile blue medium showed bioplastic production by producing fluorescence under UV light. Four bacterial strains (coded as 100, 101, 104 and 111 were selected for further characterization. Induction time assay showed that strains 101, 104, and 111 showed inhibitory activity after 4 h of incubation while strain 100 showed after 8 h. All four strains were tolerable to the maximum concentration of ZnO. Amplified products of both 16S rRNA and PhaC gene fragments of strain 111 were sequenced and submitted to GenBank as accession numbers EU781525 and EU781526. Conclusions: Bacterial strain Pseudomonas aeruginosa-111 has potential to utilize as biofertilize and bioplastic producer.

  11. Capsule production by Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Lynn, A.R.

    1984-01-01

    Mucoid strains of Pseudomonas aeruginosa, associated almost exclusively with chronic respiratory infections in patients with cystic fibrosis, possess a capsule composed of alginic acid similar to one produced by Azotobacter vinelandii. Recent reports have provided evidence that the biosynthetic pathway for alginate in P. aeruginosa may differ from the pathway proposed for A. vinelandii in that synthesis in P. aeruginosa may occur by way of the Entner-Doudoroff pathway. Incorporation of isotope from (6-/sup 14/C)glucose into alginate by both P. aueroginosa and A. vinelandii was 10-fold greater than that from either (1-/sup 14/C)/sup -/ or (2-/sup 14/C)glucose, indicating preferential utilization of the bottom half of the glucose molecule for alginate biosynthesis. These data strongly suggest that the Entner-Doudoroff pathway plays a major role in alginate synthesis in both P. aeruginosa and A. vinelandii. The enzymes of carbohydrate metabolism in mucoid strains of P. aeruginosa appear to be unchanged whether alignate is actively produced or not and activities do not differ significantly from nonmucoid strain PAO.

  12. Influence of extracellular polymeric substances on deposition and redeposition of Pseudomonas aeruginosa to surfaces

    NARCIS (Netherlands)

    Gomez-Suarez, C; Pasma, J; van der Borden, AJ; Wingender, J; Flemming, HC; Busscher, HJ; van der Mei, HC

    In this study, the role of extracellular polymeric substances (EPS) in the initial adhesion of EPS-producing Pseudomonas aeruginosa SG91 and SG81R1, a non-EPS-producing strain, to substrata with different hydrophobicity was investigated. The release of EPS by SG81 was concurrent with a decrease in

  13. Extracellular matrix metalloproteinase inducer enhances host resistance against pseudomonas aeruginosa infection through MAPK signaling pathway

    OpenAIRE

    Li, Yongwei; Chen, Lu; Wang, Chunxia; Chen, Jianshe; Zhang, Xiaoqian; Hu, Yue; Niu, Xiaobin; Pei, Dongxu; He, Zhiqiang; Bi, Yongyi

    2016-01-01

    This study aims to explore the role of extra-cellular matrix metalloproteinase inducer (EMMPRIN) in the drug resistance of the pseudomonas aeruginosa (PA). The BALB/c mice were transfected with PA, then the mice were infected with the siRNA of EMMPRIN to silence the EMMPRIN gene. The EMMPRIN mRNA and protein were detected by using RT-PCR and western blot, respectively. In order to examine the function of EMMPRIN in drug resistance of PA, the BALB/c and C57BL/6 mice were treated with EMMPRIN s...

  14. Aspergillus triggers phenazine production in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Jensen, Britt Guillaume; Jelsbak, Lars; Søndergaard, Ib

    in the contact area of A. niger, A. flavus, A. oryzae, but not A. fumigatus. In addition, other metabolites with UV chromophores similar to the phenazines were only found in the contact zone between Aspergillus and Pseudomonas. No change in secondary metabolite profiles were seen for the Aspergilli, when......Objectives: Pseudomonas aeruginosa is an opportunistic human pathogen, commonly infecting cystic fibrosis (CF) patients. Aspergilli, especially Aspergillus fumigatus, are also frequently isolated from CF patients. Our aim was to examine the possible interaction between P. aeruginosa and different...... Aspergillus species. Methods: A suspension of fungal spores was streaked onto WATM agar plates. After 24 hours incubation at 37 °C, a P. aeruginosa overnight culture was streaked out perpendicular to the fungal streak. The plates were incubated at 37 °C for five days, examined and plugs were extracted...

  15. Induction of beta-lactamase production in Pseudomonas aeruginosa biofilm

    DEFF Research Database (Denmark)

    Giwercman, B; Jensen, E T; Høiby, N

    1991-01-01

    Imipenem induced high levels of beta-lactamase production in Pseudomonas aeruginosa biofilms. Piperacillin also induced beta-lactamase production in these biofilms but to a lesser degree. The combination of beta-lactamase production with other protective properties of the biofilm mode of growth c...... could be a major reason for the persistence of this sessile bacterium in chronic infections....

  16. Production of extracellular nucleic acids by genetically altered bacteria in aquatic-environment microcosms

    International Nuclear Information System (INIS)

    Paul, J.H.; David, A.W.

    1989-01-01

    The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [ 3 H]thymidine or [ 3 H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degree C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physicochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms

  17. Chemical Analysis of Cellular and Extracellular Carbohydrates of a Biofilm-Forming Strain Pseudomonas aeruginosa PA14

    Science.gov (United States)

    Coulon, Charlène; Vinogradov, Evgeny; Filloux, Alain; Sadovskaya, Irina

    2010-01-01

    Background Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A–L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the “scaffolding” polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A–L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated. Principal Findings In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1–3)-α-D-QuiNAc-(1–3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic β-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ∼20% of dry weight) of LPS-like material. Conclusions We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is

  18. Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa.

    Science.gov (United States)

    Galdino, Anna Clara M; Viganor, Lívia; Ziccardi, Mariangela; Nunes, Ana Paula F; Dos Santos, Kátia R N; Branquinha, Marta H; Santos, André L S

    2017-12-01

    Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites. Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR. Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility. The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  19. Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.

    Science.gov (United States)

    Jennings, Laura K; Storek, Kelly M; Ledvina, Hannah E; Coulon, Charlène; Marmont, Lindsey S; Sadovskaya, Irina; Secor, Patrick R; Tseng, Boo Shan; Scian, Michele; Filloux, Alain; Wozniak, Daniel J; Howell, P Lynne; Parsek, Matthew R

    2015-09-08

    Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.

  20. Production of extracellular aspartic protease in submerged ...

    African Journals Online (AJOL)

    Fungal milk-clotting enzymes have gained value as bovine Chymosin substitutes in the cheese industry. In this work, the effects of culture conditions on the production of extracellular milk clotting enzymes from Mucor mucedo DSM 809 in submerged fermentation were studied. The maximum activity was observed after 48 h ...

  1. Effects of Dihydroartemisinin and Artemether on the Growth, Chlorophyll Fluorescence, and Extracellular Alkaline Phosphatase Activity of the Cyanobacterium Microcystis aeruginosa.

    Science.gov (United States)

    Wang, Shoubing; Xu, Ziran

    2016-01-01

    Increased eutrophication in the recent years has resulted in considerable research focus on identification of methods for preventing cyanobacterial blooms that are rapid and efficient. The objectives of this study were to investigate the effects of dihydroartemisinin and artemether on the growth of Microcystis aeruginosa and to elucidate its mode of action. Variations in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular alkaline phosphatase activity (APA), and chlorophyll fluorescence parameters (Fv/Fm, ΦPSII, ETR, rapid light curves, fast chlorophyll fluorescence curves on fluorescence intensity, and relative variable fluorescence) were evaluated by lab-cultured experiments. Our results demonstrated that both dihydroartemisinin and artemether inhibited the growth of M.aeruginosa by impairing the photosynthetic center in photosystem II and reducing extracellular APA, with a higher sensitivity exhibited toward artemether. The inhibitory effects of dihydroartemisinin on M.aeruginosa increased with concentration, and the maximum growth inhibitory rate was 42.17% at 24 mg·L-1 after 120h exposure, whereas it was 55.72% at 6 mg·L-1 artemetherafter 120h exposure. Moreover, the chlorophyll fluorescence was significantly inhibited (p<0.05) after 120h exposure to 12 and 24 mg·L-1 dihydroartemisinin. Furthermore, after 120h exposure to 6 mg·L-1 artemether, Fv/Fm, ΦPSII, ETR and rETRmax showed a significant decrease (p<0.01) from initial values of 0.490, 0.516, 17.333, and 104.800, respectively, to 0. One-way analysis of variance showed that 6 mg·L-1 artemether and 24 mg·L-1 dihydroartemisinin had significant inhibitory effects on extracellular APA (p<0.01). The results of this study would be useful to further studies to validate the feasibility of dihydroartemisinin and artemether treatment to inhibit overall cyanobacterial growth in water bodies, before this can be put into practice.

  2. Enhancement of Biogas Production from Bakery Waste by Pseudomonas aeruginosa

    OpenAIRE

    S. Potivichayanon; T. Sungmon; W. Chaikongmao; S. Kamvanin

    2011-01-01

    Production of biogas from bakery waste was enhanced by additional bacterial cell. This study was divided into 2 steps. First step, grease waste from bakery industry-s grease trap was initially degraded by Pseudomonas aeruginosa. The concentration of byproduct, especially glycerol, was determined and found that glycerol concentration increased from 12.83% to 48.10%. Secondary step, 3 biodigesters were set up in 3 different substrates: non-degraded waste as substrate in fir...

  3. Physiology and biochemistry of polysaccharide production by Azotobacter vinelandii and Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Annison, G.

    1985-01-01

    The extracellular production and the composition of the acetylated alginates of Azotobacter vinelandii and Pseudomonas aeruginosa were investigated. A reverse-phase HPLC method to determine D-mannuronate/L-guluronate (M/G) ratios in alginates was developed. Comparison between the HPLC procedure and a /sup 1/H-NMR method was made. Growth and alginate production by A. vinelandii in batch and continuous culture were studied. Polysaccharide production was favored in batch culture by reduced aeration rates by by intermediate aeration rates in continuous culture. The partitioning of Ca/sup 45/ in solution and associated with alginates during the growth cycle of A. vinelandii was studied. The change in the composition of polysaccharide produced during growth of A. vinelandii was related to the level of free Ca/sup + +/ which fell rapidly during initial stages of incubation. The degree of acetylation of the polyuronate was shown to be variable. Production of alginates with high O-acetate contents was observed in cultures maintained at high growth rates and high Ca/sup + +/ levels. The degree of acetylation of the polyuronate was not related to the level of epimerization in vivo although in vitro studies demonstrated that chemical acetylation of the alginate inhibited epimerization. Alginate production by clinical isolates of Pseudomonas aeruginosa was shown to be unstable and no polyguluronate was isolated from cultures. It was also noted that Ca/sup + +/ played a less important role in the composition of the polysaccharide.

  4. Genetic improvement of Rhamnolipid Production from Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Al-Gelawi, Majed Hussain; Al-Makadci, O.A.

    2007-01-01

    Six bacterial isolates (isolated previously) were identified and/or ensured their identification. Results showed that these isolates belong to P. aeruginosa, and all isolates were capable of producing rhamnolipid, and best ones was P. aeruginosa RB67. In order to get rhamnolipid hyper producer mutants, mutagenesis of P. aeruginosa RB 67 using UV light and MNNG were performed. Fifty colonies from each treatment (UV and MNNG) were selected and screened for their ability to produce rhamnolipid semi-quantitatively by replica plated on blood agar and CTAB-methylene blue agar. Based on the last method, twelve colonies from each treatment (UV and MNNG) were selected and used for measuring rhamnose concentration. The results showed that these mutants varied in their ability to produce rhamnolipid and some of them showed an increase in rhamnalipid production. The highest rhamnose concentration (94 ug/mL) was achieved by the mutant (MOM12). Furthermore, FTIR spectroscopy results indicated that there were no apparent qualitative differences in rhamnolipid produced from mutants. (author)

  5. Pseudomonas aeruginosa AlgR Phosphorylation Status Differentially Regulates Pyocyanin and Pyoverdine Production

    Directory of Open Access Journals (Sweden)

    Alexander S. Little

    2018-01-01

    Full Text Available Pseudomonas aeruginosa employs numerous, complex regulatory elements to control expression of its many virulence systems. The P. aeruginosa AlgZR two-component regulatory system controls the expression of several crucial virulence phenotypes. We recently determined, through transcriptomic profiling of a PAO1 ΔalgR mutant strain compared to wild-type PAO1, that algZR and hemCD are cotranscribed and show differential iron-dependent gene expression. Previous expression profiling was performed in strains without algR and revealed that AlgR acts as either an activator or repressor, depending on the gene. Thus, examination of P. aeruginosa gene expression from cells locked into different AlgR phosphorylation states reveals greater physiological relevance. Therefore, gene expression from strains carrying algR alleles encoding a phosphomimetic (AlgR D54E or a phosphoablative (AlgR D54N form were compared by microarray to PAO1. Transcriptome analyses of these strains revealed 25 differentially expressed genes associated with iron siderophore biosynthesis or heme acquisition or production. The PAO1 algR D54N mutant produced lower levels of pyoverdine but increased expression of the small RNAs prrf1 and prrf2 compared to PAO1. In contrast, the algR D54N mutant produced more pyocyanin than wild-type PAO1. On the other hand, the PAO1 algR D54E mutant produced higher levels of pyoverdine, likely due to increased expression of an iron-regulated gene encoding the sigma factor pvdS, but it had decreased pyocyanin production. AlgR specifically bound to the prrf2 and pvdS promoters in vitro. AlgR-dependent pyoverdine production was additionally influenced by carbon source rather than the extracellular iron concentration per se. AlgR phosphorylation effects were also examined in a Drosophila melanogaster feeding, murine acute pneumonia, and punch wound infection models. Abrogation of AlgR phosphorylation attenuated P. aeruginosa virulence in these infection

  6. BIOSURFACTANTS PRODUCTION BY Pseudomonas aeruginosa USING SOYBEAN OIL AS SUBSTRATE

    Directory of Open Access Journals (Sweden)

    Venty Suryanti

    2010-06-01

    Full Text Available Optimization condition of the biosurfactants production by P. aeruginosa using soybean oil as substrate has been examined. The media containing 10% v/v of the soybean oil and 6 days of the fermentation time was the optimum condition for the biosurfactants production. The extraction technique using different solvent polarity (n-hexane, chloroform, ethyl acetate and buthanol, respectively was applied for the isolation of the biosurfactants. The biosurfactant was found in the extract chloroform of the crude biospasoy (biosurfactants obtained from soybean oil as substrate which then is called chlo-biospasoy. The chlo-biospasoy was identified as rhamnolipids which had oil in water (o/w emulsion type, had the CMC of 860 mg/L and could reduced the surface tension of the water from 72 mN/m to 52 mN/m. The chlo-biospasoy could be used as an emulsifier to form emulsion between water and hydrocarbon such as palm oil, benzene, premium or toluene with various stability. The results indicated that chlo-biospasoy could be used as an emulsifying and emulsion-stabilizing agent.     Keywords: Biosurfactants, P. aeruginosa, Soybean Oil, Emulsifier

  7. Alkaline protease contributes to pyocyanin production in Pseudomonas aeruginosa.

    Science.gov (United States)

    Iiyama, Kazuhiro; Takahashi, Eigo; Lee, Jae Man; Mon, Hiroaki; Morishita, Mai; Kusakabe, Takahiro; Yasunaga-Aoki, Chisa

    2017-04-01

    The role of the alkaline protease (AprA) in pyocyanin production in Pseudomonas aeruginosa was investigated. AprA was overproduced when a plasmid carrying the aprA gene was introduced to an aprA-deletion mutant strain, EG03; thus, aprA-complemented EG03 was used as an overproducing strain. The complemented strain produced higher pyocyanin than the mutant strain in all commercially available media evaluated. Particularly, pyocyanin production was higher in the complemented than in the parental strain in brain-heart infusion and tryptic soy broths. These results suggested that protein degradation products by AprA were utilized for pyocyanin production. Protein-rich media were used in subsequent validation studies. Similar results were obtained when the basal medium was supplemented with casein or skim milk as the sole organic nitrogen source. However, gelatin failed to induce abundant pyocyanin production in the complemented strain, despite the presence of protein degradation products by AprA as assessed by SDS-PAGE. Thus, gelatin degradation products may not be suitable for pyocyanin synthesis. In conclusion, AprA could contribute to pyocyanin production in the presence of several proteins or peptides. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Evidences of extracellular abiotic degradation of hexadecane through free radical mechanism induced by the secreted phenazine compounds of P. aeruginosa NY3.

    Science.gov (United States)

    Nie, Hongyun; Nie, Maiqian; Wang, Lei; Diwu, Zhenjun; Xiao, Ting; Qiao, Qi; Wang, Yan; Jiang, Xin

    2018-03-02

    The aim of this work was to investigate the effects of secreted extracellular phenazine compounds (PHCs) on the degradation efficiency of alkanes by P. aeruginosa NY3. Under aerobic conditions, the PHCs secreted by P. aeruginosa NY3 initiate the oxidation of alkanes outside cells, in coupling with some reducing agents, such as β-Nicotinamide adenine dinucleotide, reduced disodium salt (NADH) or reduced glutathione (GSH). This reaction might be via free radical reactions similar to Fenton Oxidation Reaction (FOR). P. aeruginosa NY3 secretes pyocyanin (Pyo), 1-hydroxyphenazine (HPE), phenazine-1-carboxylic acid (PCA), and phenazine-1-amide (PCN) simultaneously. The cell-free extracellular fluid containing these four PHCs degrades hexadecane effectively. The observation of Electron Spin Resonance (EPR) signals of superoxide anion radical (O 2 - ), hydroxyl radical (OH) and/or carbon free radicals (R) both in vivo and in vitro suggested the degradation of hexadecane could be via a free radical pathway. Secretion of PHCs has been found to be characteristic of Pseudomonas which is often involved in or related to the degradation of organic pollutants. Our work suggested that certain organic contaminants may be oxidized through ubiquitously extracellular abiotic degradation by the free radicals produced during bio-remediation and bio-treatment. Copyright © 2018. Published by Elsevier Ltd.

  9. Optimization of extracellular catalase production from Aspergillus ...

    African Journals Online (AJOL)

    The studies of the effect of each variable and the establishment of a correlation between the response of enzyme activity and variables revealed that the link is a multiple linear regression form. The optimization was carried out through a simplex algorithm. The amount of extracellular catalase produced by the strain in the ...

  10. Optimization of extracellular catalase production from Aspergillus ...

    African Journals Online (AJOL)

    aghomotsegin

    extracellular catalase produced by the strain in the optimized medium was about four times higher than ... celial and unicellular fungi in synthetic media (Kurakov et .... covering the appropriate range and the broad calibration kit ... This optimization allowed us to define new cultural con- ..... Ann. New York Academy Sci.

  11. Extracellular deoxyribonuclease production by periodontal bacteria.

    Science.gov (United States)

    Palmer, L J; Chapple, I L C; Wright, H J; Roberts, A; Cooper, P R

    2012-08-01

    Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host's immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA-supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Twenty-seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis. © 2011 John Wiley & Sons A/S.

  12. Genome-Wide Survey of Pseudomonas aeruginosa PA14 Reveals a Role for the Glyoxylate Pathway and Extracellular Proteases in the Utilization of Mucin

    Science.gov (United States)

    Flynn, Jeffrey M.; Phan, Chi

    2017-01-01

    ABSTRACT Chronic airway infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. Although this bacterium has been extensively studied for its virulence determinants, biofilm growth, and immune evasion mechanisms, comparatively little is known about the nutrient sources that sustain its growth in vivo. Respiratory mucins represent a potentially abundant bioavailable nutrient source, although we have recently shown that canonical pathogens inefficiently use these host glycoproteins as a growth substrate. However, given that P. aeruginosa, particularly in its biofilm mode of growth, is thought to grow slowly in vivo, the inefficient use of mucin glycoproteins may be relevant to its persistence within the CF airways. To this end, we used whole-genome fitness analysis, combining transposon mutagenesis with high-throughput sequencing, to identify genetic determinants required for P. aeruginosa growth using intact purified mucins as a sole carbon source. Our analysis reveals a biphasic growth phenotype, during which the glyoxylate pathway and amino acid biosynthetic machinery are required for mucin utilization. Secondary analyses confirmed the simultaneous liberation and consumption of acetate during mucin degradation and revealed a central role for the extracellular proteases LasB and AprA. Together, these studies describe a molecular basis for mucin-based nutrient acquisition by P. aeruginosa and reveal a host-pathogen dynamic that may contribute to its persistence within the CF airways. PMID:28507068

  13. Production of extracellular fatty acid using engineered Escherichia coli

    Directory of Open Access Journals (Sweden)

    Liu Hui

    2012-04-01

    Full Text Available Abstract Background As an alternative for economic biodiesel production, the microbial production of extracellular fatty acid from renewable resources is receiving more concerns recently, since the separation of fatty acid from microorganism cells is normally involved in a series of energy-intensive steps. Many attempts have been made to construct fatty acid producing strains by targeting genes in the fatty acid biosynthetic pathway, while few studies focused on the cultivation process and the mass transfer kinetics. Results In this study, both strain improvements and cultivation process strategies were applied to increase extracellular fatty acid production by engineered Escherichia coli. Our results showed overexpressing ‘TesA and the deletion of fadL in E. coli BL21 (DE3 improved extracellular fatty acid production, while deletion of fadD didn’t strengthen the extracellular fatty acid production for an undetermined mechanism. Moreover, the cultivation process controls contributed greatly to extracellular fatty acid production with respect to titer, cell growth and productivity by adjusting the temperature, adding ampicillin and employing on-line extraction. Under optimal conditions, the E. coli strain (pACY-‘tesA-ΔfadL produced 4.8 g L−1 extracellular fatty acid, with the specific productivity of 0.02 g h−1 g−1dry cell mass, and the yield of 4.4% on glucose, while the ratios of cell-associated fatty acid versus extracellular fatty acid were kept below 0.5 after 15 h of cultivation. The fatty acids included C12:1, C12:0, C14:1, C14:0, C16:1, C16:0, C18:1, C18:0. The composition was dominated by C14 and C16 saturated and unsaturated fatty acids. Using the strain pACY-‘tesA, similar results appeared under the same culture conditions and the titer was also much higher than that ever reported previously, which suggested that the supposedly superior strain did not necessarily perform best for the efficient production of desired

  14. Production of bio surfactants (Rhamnolipids) by pseudomonas aeruginosa isolated from colombian sludges

    International Nuclear Information System (INIS)

    Pimienta, A.L; Diaz M, M. P; Carvajal S, F.G; Grosso V, J.L.

    1997-01-01

    The bio surfactant production by strains of Pseudomonas aeruginosa isolated from Colombian hydrocarbon contaminated sludge has been determined. The methodology included the isolation of microorganisms, standardization of batch culture conditions for good surfactant production and characterization of the produced rhamnolipid. Several carbon sources were evaluated with regard to the growth and production curves. The stability of the rhamnolipid was also determined under variable conditions of pH, temperature and salt concentration. The strain Pseudomonas aeruginosa BS 3 showed bio surfactant production capabilities of rhamnolipid resulting in concentrations up to 2 g-dm with surface tensions of 30 - 32 mN-m in batch cultures with commercial nutrients

  15. Evaluation of phytochemicals from medicinal plants of Myrtaceae family on virulence factor production by Pseudomonas aeruginosa.

    Science.gov (United States)

    Musthafa, Khadar Syed; Sianglum, Wipawadee; Saising, Jongkon; Lethongkam, Sakkarin; Voravuthikunchai, Supayang Piyawan

    2017-05-01

    Virulence factors regulated by quorum sensing (QS) play a critical role in the pathogenesis of an opportunistic human pathogen, Pseudomonas aeruginosa in causing infections to the host. Hence, in the present work, the anti-virulence potential of the medicinal plant extracts and their derived phytochemicals from Myrtaceae family was evaluated against P. aeruginosa. In the preliminary screening of the tested medicinal plant extracts, Syzygium jambos and Syzygium antisepticum demonstrated a maximum inhibition in QS-dependent violacein pigment production by Chromobacterium violaceum DMST 21761. These extracts demonstrated an inhibitory activity over a virulence factor, pyoverdin, production by P. aeruginosa ATCC 27853. Gas chromatography-mass spectrometric (GC-MS) analysis revealed the presence of 23 and 12 phytochemicals from the extracts of S. jambos and S. antisepticum respectively. Three top-ranking phytochemicals, including phytol, ethyl linoleate and methyl linolenate, selected on the basis of docking score in molecular docking studies lowered virulence factors such as pyoverdin production, protease and haemolytic activities of P. aeruginosa to a significant level. In addition, the phytochemicals reduced rhamnolipid production by the organism. The work demonstrated an importance of plant-derived compounds as anti-virulence drugs to conquer P. aeruginosa virulence towards the host. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  16. Extracellular polysaccharide production by Thraustochytrid protists

    Digital Repository Service at National Institute of Oceanography (India)

    Jain, R.; Raghukumar, S.; Tharanathan, R.; Bhosle, N.B.

    around groups of cells in stationary cultures. EPS in shake culture filtrates ranged from 0.3 to 1.1 g/L. EPS production, which was studied in greater detail in 2 isolates, SC-1 and CW1, increased with age of cultures, reaching a peak in the stationary...

  17. Electrochemical sensors for identifying pyocyanin production in clinical Pseudomonas aeruginosa isolates.

    Science.gov (United States)

    Sismaet, Hunter J; Pinto, Ameet J; Goluch, Edgar D

    2017-11-15

    In clinical practice, delays in obtaining culture results impact patient care and the ability to tailor antibiotic therapy. Despite the advancement of rapid molecular diagnostics, the use of plate cultures inoculated from swab samples continues to be the standard practice in clinical care. Because the inoculation culture process can take between 24 and 48h before a positive identification test can be run, there is an unmet need to develop rapid throughput methods for bacterial identification. Previous work has shown that pyocyanin can be used as a rapid, redox-active biomarker for identifying Pseudomonas aeruginosa in clinical infections. However, further validation is needed to confirm pyocyanin production occurs in all clinical strains of P. aeruginosa. Here, we validate this electrochemical detection strategy using clinical isolates obtained from patients with hospital-acquired infections or with cystic fibrosis. Square-wave voltammetric scans of 94 different clinical P. aeruginosa isolates were taken to measure the concentration of pyocyanin. The results showed that all isolates produced measureable concentrations of pyocyanin with production rates correlated with patient symptoms and comorbidity. Further bioinformatics analysis confirmed that 1649 genetically sequenced strains (99.9%) of P. aeruginosa possess the two genes (PhzM and PhzS) necessary to produce pyocyanin, supporting the specificity of this biomarker. Confirming the production of pyocyanin by all clinically-relevant strains of P. aeruginosa is a significant step towards validating this strategy for rapid, point-of-care diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection

    DEFF Research Database (Denmark)

    Kolpen, Mette; Kühl, Michael; Bjarnsholt, Thomas

    2014-01-01

    local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O), an intermediate...

  19. Extracellular products of photosynthesis in a tropical environment

    Digital Repository Service at National Institute of Oceanography (India)

    Gomes, H.R.; Pant, A.

    Extracellular products in the Arabian Sea averaged 0.42 g/cm2/day and represented 35% of the total carbon fixed by phytoplankton. No seasonal changes are observed during the two seasons i.e. premonsoon (Jan-Feb) and onset of southwest monsoon (April...

  20. Strain- and Substrate-Dependent Redox Mediator and Electricity Production by Pseudomonas aeruginosa.

    Science.gov (United States)

    Bosire, Erick M; Blank, Lars M; Rosenbaum, Miriam A

    2016-08-15

    Pseudomonas aeruginosa is an important, thriving member of microbial communities of microbial bioelectrochemical systems (BES) through the production of versatile phenazine redox mediators. Pure culture experiments with a model strain revealed synergistic interactions of P. aeruginosa with fermenting microorganisms whereby the synergism was mediated through the shared fermentation product 2,3-butanediol. Our work here shows that the behavior and efficiency of P. aeruginosa in mediated current production is strongly dependent on the strain of P. aeruginosa We compared levels of phenazine production by the previously investigated model strain P. aeruginosa PA14, the alternative model strain P. aeruginosa PAO1, and the BES isolate Pseudomonas sp. strain KRP1 with glucose and the fermentation products 2,3-butanediol and ethanol as carbon substrates. We found significant differences in substrate-dependent phenazine production and resulting anodic current generation for the three strains, with the BES isolate KRP1 being overall the best current producer and showing the highest electrochemical activity with glucose as a substrate (19 μA cm(-2) with ∼150 μg ml(-1) phenazine carboxylic acid as a redox mediator). Surprisingly, P. aeruginosa PAO1 showed very low phenazine production and electrochemical activity under all tested conditions. Microbial fuel cells and other microbial bioelectrochemical systems hold great promise for environmental technologies such as wastewater treatment and bioremediation. While there is much emphasis on the development of materials and devices to realize such systems, the investigation and a deeper understanding of the underlying microbiology and ecology are lagging behind. Physiological investigations focus on microorganisms exhibiting direct electron transfer in pure culture systems. Meanwhile, mediated electron transfer with natural redox compounds produced by, for example, Pseudomonas aeruginosa might enable an entire microbial

  1. Extracellular Palladium Nanoparticle Production using Geobacter sulfurreducens

    KAUST Repository

    Yates, Matthew D.

    2013-09-03

    Sustainable methods are needed to recycle precious metals and synthesize catalytic nanoparticles. Palladium nanoparticles can be produced via microbial reduction of soluble Pd(II) to Pd(0), but in previous tests using dissimilatory metal reducing bacteria (DMRB), the nanoparticles were closely associated with the cells, occupying potential reductive sites and eliminating the potential for cell reuse. The DMRB Geobacter sulfurreducens was shown here to reduce soluble Pd(II) to Pd(0) nanoparticles primarily outside the cell, reducing the toxicity of metal ions, and allowing nanoparticle recovery without cell destruction that has previously been observed using other microorganisms. Cultures reduced 50 ± 3 mg/L Pd(II) with 1% hydrogen gas (v/v headspace) in 6 h incubation tests [100 mg/L Pd(II) initially], compared to 8 ± 3 mg/L (10 mM acetate) without H2. Acetate was ineffective as an electron donor for palladium removal in the presence or absence of fumarate as an electron acceptor. TEM imaging verified that Pd(0) nanoparticles were predominantly in the EPS surrounding cells in H2-fed cultures, with only a small number of particles visible inside the cell. Separation of the cells and EPS by centrifugation allowed reuse of the cell suspensions and effective nanoparticle recovery. These results demonstrate effective palladium recovery and nanoparticle production using G. sulfurreducens cell suspensions and renewable substrates such as H2 gas. © 2013 American Chemical Society.

  2. Production of extracellular proteolytic enzymes by Beauveria bassiana

    Directory of Open Access Journals (Sweden)

    Józefa Chrzanowska

    2014-08-01

    Full Text Available The production of proteolytic enzymes by two strains of Beauveria bassiana 278, B. bassiana 446 and one strain of Ascosphera apis 496 was analysed. It was demonstrated that the strain of B. bassiana 278 proved to be the best producer of basic and acid proteases. The influence of different environmental factors such as nitrogen and carbon sources on the production of extracellular hydrolytic enzymes was assessed. In addition the acid protease from B. bassiana was partially characterized.

  3. Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection.

    Directory of Open Access Journals (Sweden)

    Mette Kolpen

    Full Text Available Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs surround the biofilms and create local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS. We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O, an intermediate in the denitrification pathway. We measured N2O and O2 with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO3(- and NO2(- in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N2O (range 1.4-157.9 µM N2O. The concentration of N2O in the sputum was higher below the oxygenated layers. In 4 samples the N2O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO3(- decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N2O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.

  4. Nitrous oxide production in sputum from cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection.

    Science.gov (United States)

    Kolpen, Mette; Kühl, Michael; Bjarnsholt, Thomas; Moser, Claus; Hansen, Christine Rønne; Liengaard, Lars; Kharazmi, Arsalan; Pressler, Tanja; Høiby, Niels; Jensen, Peter Østrup

    2014-01-01

    Chronic lung infection by Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O), an intermediate in the denitrification pathway. We measured N2O and O2 with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO3(-) and NO2(-) in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N2O (range 1.4-157.9 µM N2O). The concentration of N2O in the sputum was higher below the oxygenated layers. In 4 samples the N2O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO3(-) decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N2O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.

  5. Pseudomonas aeruginosa induces pigment production and enhances virulence in a white phenotypic variant of Staphylococcus aureus.

    Science.gov (United States)

    Antonic, Vlado; Stojadinovic, Alexander; Zhang, Binxue; Izadjoo, Mina J; Alavi, Mohammad

    2013-01-01

    Staphyloxanthin is a virulence factor which protects Staphylococcus aureus in stress conditions. We isolated two pigment variants of S. aureus and one strain of Pseudomonas aeruginosa from a single wound infection. S. aureus variants displayed white and yellow colony phenotypes. The sequence of the operons for staphyloxanthin synthesis indicated that coding and promoter regions were identical between the two pigment variants. Quorum sensing controls pigment synthesis in some bacteria. It is also shown that P. aeruginosa quorum-sensing molecules affect S. aureus transcription. We explored whether the co-infecting P. aeruginosa can affect pigment production in the white S. aureus variant. In co-culture experiments between the white variants and a selected number of Gram-positive and Gram-negative bacteria, only P. aeruginosa induced pigment production in the white variant. Gene expression analysis of the white variant did not indicate upregulation of the crtM and other genes known to be involved in pigment production (sigB, sarA, farnesyl pyrophosphate synthase gene [FPP-synthase], hfq). In contrast, transcription of the catalase gene was significantly upregulated after co-culture. P. aeruginosa-induced pigment synthesis and catalase upregulation correlated with increased resistance to polymyxin B, hydrogen peroxide, and the intracellular environment of macrophages. Our data indicate the presence of silent but functional staphyloxanthin synthesis machinery in a white phenotypic variant of S. aureus which is activated by a co-infecting P. aeruginosa via inter-species communication. Another S. aureus virulence factor, catalase is also induced by this co-infecting bacterium. The resulting phenotypic changes are directly correlated with resistance of the white variant to stressful conditions.

  6. Enterobacter aerogenes metabolites enhance Microcystis aeruginosa biomass recovery for sustainable bioflocculant and biohydrogen production.

    Science.gov (United States)

    Xu, Liang; Zhou, Mo; Ju, Hanyu; Zhang, Zhenxing; Zhang, Jiquan; Sun, Caiyun

    2018-04-07

    We report a recycling bioresource involving harvesting of Microcystis aeruginosa using the bioflocculant (MBF-32) produced by Enterobacter aerogenes followed by the recovery of the harvested M. aeruginosa as the main substrate for the sustainable production of MBF-32 and biohydrogen. The experimental results indicate that the efficiency of bioflocculation exceeded 90% under optimal conditions. The harvested M. aeruginosa was further recycled as the main substrate for the supply of necessary elements. The highest yield (3.6±0.1g/L) of MBF-32 could be obtained from 20g/L of wet biomass of M. aeruginosa with an additional 20g/L of glucose as the extra carbon source. The highest yield of biohydrogen was 35mL of H 2 /g (dw) algal biomass, obtained from 20g/L of wet biomass of M. aeruginosa with an additional 10g/L of glycerol. Transcriptome analyses indicated that MBF-32 was mainly composed of polysaccharide and tyrosine/tryptophan proteins. Furthermore, NADH synthase and polysaccharide export-related genes were found to be up-regulated. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Pseudomonas aeruginosa induces pigment production and enhances virulence in a white phenotypic variant of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Antonic V

    2013-11-01

    Full Text Available Vlado Antonic,1–3 Alexander Stojadinovic,3–5 Binxue Zhang,1–3 Mina J Izadjoo,1–3,5 Mohammad Alavi1–3 1Henry M Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA; 2Diagnostic and Translational Research Center, Gaithersburg, MD, USA; 3Combat Wound Initiative Program, Bethesda, MD, USA; 4Walter Reed National Military Medical Center, Bethesda, MD, USA; 5Uniformed Services University of the Health Sciences, Bethesda, MD, USA Abstract: Staphyloxanthin is a virulence factor which protects Staphylococcus aureus in stress conditions. We isolated two pigment variants of S. aureus and one strain of Pseudomonas aeruginosa from a single wound infection. S. aureus variants displayed white and yellow colony phenotypes. The sequence of the operons for staphyloxanthin synthesis indicated that coding and promoter regions were identical between the two pigment variants. Quorum sensing controls pigment synthesis in some bacteria. It is also shown that P. aeruginosa quorum-sensing molecules affect S. aureus transcription. We explored whether the co-infecting P. aeruginosa can affect pigment production in the white S. aureus variant. In co-culture experiments between the white variants and a selected number of Gram-positive and Gram-negative bacteria, only P. aeruginosa induced pigment production in the white variant. Gene expression analysis of the white variant did not indicate upregulation of the crtM and other genes known to be involved in pigment production (sigB, sarA, farnesyl pyrophosphate synthase gene [FPP-synthase], hfq. In contrast, transcription of the catalase gene was significantly upregulated after co-culture. P. aeruginosa-induced pigment synthesis and catalase upregulation correlated with increased resistance to polymyxin B, hydrogen peroxide, and the intracellular environment of macrophages. Our data indicate the presence of silent but functional staphyloxanthin synthesis machinery in a white phenotypic variant

  8. Mechanisms involved in the evasion of the host defence by Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Kharazmi, A

    1991-01-01

    Pseudomonas aeruginosa, an extracellular opportunistic pathogen, utilizes two major mechanisms to evade the host defence system. One of these mechanisms is the production of a large number of extracellular products, such as proteases, toxins, and lipases. The two proteases, alkaline protease and ...

  9. Production and characterization of di-rhamnolipid produced by Pseudomonas aeruginosa TMN

    International Nuclear Information System (INIS)

    Moussa, T. A. A.; Mohamed, M. S.; Samak, N.

    2014-01-01

    Pseudomonas aeruginosa TMN was used to produce rhamnolipid (RL) from a variety of carbon and nitrogen substrates. The most favorable carbon sources for RL production were glucose and glycerol (both at 40 g/L), giving a RL yield of 0.3 and 0.25 g/L, respectively. Meanwhile, sodium nitrate appeared to be the preferable nitrogen source, resulting in a RL production of 0.34g/L. Rhamnolipid production from P. aeruginosa TMN was affected by temperature, pH and agitation rate, with 37 °C, pH 7 and 200 rpm agitation favorable for rhamnolipid production. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and electro spray ionization-mass spectrometry (ESI-MS) analyses indicated that the purified product contained one type of commonly found rhamnolipid, which is L-rhamnosyl-L-rhamnosyl-β- hydroxydecanoyl-β-hydroxydecanoate. The rhamnolipid product can reduce the surface tension of water to 34 mN/m with a critical micelle concentration of nearly 18.75 mg/L and emulsified kerosene by 46%. P. aeruginosa TMN strain is a potential source of rhamnolipid biosurfactant, which could be used for the development of bioremediation processes in the marine environment. (author)

  10. Production and characterization of di-rhamnolipid produced by Pseudomonas aeruginosa TMN

    Energy Technology Data Exchange (ETDEWEB)

    Moussa, T. A. A.; Mohamed, M. S.; Samak, N., E-mail: mervat_sayed@yahoo.com, E-mail: mervat@sci.cu.edu.eg [Cairo University (Egypt)

    2014-10-15

    Pseudomonas aeruginosa TMN was used to produce rhamnolipid (RL) from a variety of carbon and nitrogen substrates. The most favorable carbon sources for RL production were glucose and glycerol (both at 40 g/L), giving a RL yield of 0.3 and 0.25 g/L, respectively. Meanwhile, sodium nitrate appeared to be the preferable nitrogen source, resulting in a RL production of 0.34g/L. Rhamnolipid production from P. aeruginosa TMN was affected by temperature, pH and agitation rate, with 37 °C, pH 7 and 200 rpm agitation favorable for rhamnolipid production. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and electro spray ionization-mass spectrometry (ESI-MS) analyses indicated that the purified product contained one type of commonly found rhamnolipid, which is L-rhamnosyl-L-rhamnosyl-β- hydroxydecanoyl-β-hydroxydecanoate. The rhamnolipid product can reduce the surface tension of water to 34 mN/m with a critical micelle concentration of nearly 18.75 mg/L and emulsified kerosene by 46%. P. aeruginosa TMN strain is a potential source of rhamnolipid biosurfactant, which could be used for the development of bioremediation processes in the marine environment. (author)

  11. Effects of UV-B radiation on microcystin production of a toxic strain of Microcystis aeruginosa and its competitiveness against a non-toxic strain

    International Nuclear Information System (INIS)

    Yang, Zhen; Kong, Fanxiang; Shi, Xiaoli; Yu, Yang; Zhang, Min

    2015-01-01

    Highlights: • UV-B radiation showed higher inhibition to non-toxin producing than toxin-producing strains on growth and photosynthetic activity. • Both intracellular and extracellular MC contents decreased markedly under UV-B radiation. • Higher resistance to UV-B radiation helped toxin-producing M. aeruginosa to predominate in the competition. - Abstract: Microcystins (MCs) produced by toxic cyanobacteria pose a health hazard to humans and animals. Some environmental factors can alter the MC concentrations by affecting the abundance of toxin-producing strains in a cyanobacteria population and/or their toxin production. In this study, we designed a monoculture and competition experiment to investigate the impacts of UV-B radiation on MC production and the competition between toxin and non-toxin producing strains of Microcystis aeruginosa. UV-B radiation resulted in higher inhibition of the growth and photosynthetic activity of the non-toxin producing strain relative to that observed for the toxin-producing strain. Both intracellular and extracellular MC contents decreased markedly when the toxin-producing strain was exposed to UV-B radiation. In addition, a quantitative real-time PCR assay revealed that the ratio of toxin-producing M. aeruginosa under UV-B exposure was higher than that under PAR alone at an early stage of the experiment. However, its abundance under UV-B exposure was lower compared with the PAR alone treatment after day 12. Our study demonstrated that UV-B radiation has a great impact on the abundance of the toxin-producing strain in the Microcystis population and their toxin production, which suggests that the fluctuation of UV-B radiation affects the MC level of cyanobacteria blooms

  12. Effects of UV-B radiation on microcystin production of a toxic strain of Microcystis aeruginosa and its competitiveness against a non-toxic strain

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Zhen, E-mail: zhyang@niglas.ac.cn; Kong, Fanxiang, E-mail: fxkong@niglas.ac.cn; Shi, Xiaoli; Yu, Yang; Zhang, Min

    2015-02-11

    Highlights: • UV-B radiation showed higher inhibition to non-toxin producing than toxin-producing strains on growth and photosynthetic activity. • Both intracellular and extracellular MC contents decreased markedly under UV-B radiation. • Higher resistance to UV-B radiation helped toxin-producing M. aeruginosa to predominate in the competition. - Abstract: Microcystins (MCs) produced by toxic cyanobacteria pose a health hazard to humans and animals. Some environmental factors can alter the MC concentrations by affecting the abundance of toxin-producing strains in a cyanobacteria population and/or their toxin production. In this study, we designed a monoculture and competition experiment to investigate the impacts of UV-B radiation on MC production and the competition between toxin and non-toxin producing strains of Microcystis aeruginosa. UV-B radiation resulted in higher inhibition of the growth and photosynthetic activity of the non-toxin producing strain relative to that observed for the toxin-producing strain. Both intracellular and extracellular MC contents decreased markedly when the toxin-producing strain was exposed to UV-B radiation. In addition, a quantitative real-time PCR assay revealed that the ratio of toxin-producing M. aeruginosa under UV-B exposure was higher than that under PAR alone at an early stage of the experiment. However, its abundance under UV-B exposure was lower compared with the PAR alone treatment after day 12. Our study demonstrated that UV-B radiation has a great impact on the abundance of the toxin-producing strain in the Microcystis population and their toxin production, which suggests that the fluctuation of UV-B radiation affects the MC level of cyanobacteria blooms.

  13. Algal Production of Extra- and Intra-Cellular Polysaccharides as an Adaptive Response to the Toxin Crude Extract of Microcystis Aeruginosa

    Directory of Open Access Journals (Sweden)

    Mostafa Mohamed El-Sheekh

    2012-11-01

    Full Text Available This is an investigation concerned with studying the possible adaptive response of four different unicellular algae, Anabaena PCC 7120, Oscillatoria angustissima, Scendesmus obliquus and Chlorella vulgaris, to the toxin of Microcystis aeruginosa (Kützing. Theeffects of four different concentrations, 25, 50, 100 and 200 μg mL-1 of microcystins crude extract of M. aeruginosa, on both intra and extra-cellular polysaccharide levels, in log phase,of the four tested algae were studied. The obtained results showed differential increase in the production levels for both intra and extra-cellular polysaccharides by the tested algae,compared with the control. S. obliquus and C. vulgaris showed a resistance to crude toxinhigher than Anabaena PCC 7120 and O. angustissima. The highly production of polysaccharides by green algal species under this toxic stress indicated the involvement of these polysaccharides in protecting the algal cells against toxic species and, reflect thebiological behavior of particular algal species to the environmental stresses.

  14. Enhanced extracellular chitinase production in Pseudomonas fluorescens: biotechnological implications

    Directory of Open Access Journals (Sweden)

    Azhar Alhasawi

    2017-06-01

    Full Text Available Chitin is an important renewable biomass of immense commercial interest. The processing of this biopolymer into value-added products in an environmentally-friendly manner necessitates its conversion into N-acetyl glucosamine (NAG, a reaction mediated by the enzyme chitinase. Here we report on the ability of the soil microbe Pseudomonas fluorescens to secrete copious amounts of chitinase in the spent fluid when cultured in mineral medium with chitin as the sole source of carbon and nitrogen. Although chitinase was detected in various cellular fractions, the enzyme was predominantly localized in the extracellular component that was also rich in NAG and glucosamine. Maximal amounts of chitinase with a specific activity of 80 µmol NAG produced mg–1 protein min–1 was obtained at pH 8 after 6 days of growth in medium with 0.5 g of chitin. In-gel activity assays and Western blot studies revealed three isoenzymes. The enzyme had an optimal activity at pH 10 and a temperature range of 22–38 ℃. It was stable for up to 3 months. Although it showed optimal specificity toward chitin, the enzyme did readily degrade shrimp shells. When these shells (0.1 g were treated with the extracellular chitinase preparation, NAG [3 mmoles (0.003 g-mol] was generated in 6 h. The extracellular nature of the enzyme coupled with its physico-chemical properties make this chitinase an excellent candidate for biotechnological applications.

  15. PRODUCTION OF AN EXTRACELLULAR CELLOBIASE IN SOLID STATE FERMENTATION

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    Ruchi Agrawal

    2013-02-01

    Full Text Available The bioethanol production from lignocellulosic biomass has attracted wide interest globally in last decade. One of the main reasons for the high cost of bioethanol production from lignocellulosic biomass is the expensive enzymes involved in enzymatic hydrolysis of cellulose (cellulase. The utilization of agro-industrial waste as a potential substrate for producing enzymes may serve a dual purpose of reducing the environmental pollution along with producing a high value commercial product. Twelve different agro-industrial wastes were evaluated for extracellular cellobiose or β-glucosidase production by a mutant of Bacillus subtilis on solid state fermentations (SSF. The Citrus sinensis peel waste was found to be the most suitable substrate with highest BGL titre (35 U/gds. Optimum incubation time, inoculum size, moisture content and volume of buffer for enzyme extraction were 72 h, 40 % v/w, 10 mL and 20 mL respectively.

  16. Biosurfactant Production by Pseudomonas aeruginosa and Burkholderia gladioli Isolated from Mangrove Sediments Using Alternative Substrates

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    Karla Maria Catter

    2016-10-01

    Full Text Available Biosurfactants are surface-active agents produced by a variety of microorganisms. To make biosurfactant production economically feasible, several alternative carbon sources have been proposed. This study describes biosurfactant production by strains of Pseudomonas aeruginosa and Burkholderia gladioli isolated from mangrove sediments in Northeastern Brazil and cultured in mineral media enriched with waste cooking oil. The biosurfactants were tested for drop collapse, emulsion formation and stability and surface tension. P. aeruginosa performed better both at lowering the surface tension (from 69 to 28 mN/m and at forming stable emulsions (approximately 80% at 48 hours of culture. The strains tested in this study were found to be efficient biosurfactant producers when cultured on substrates enriched with vegetable oil. DOI: http://dx.doi.org/10.17807/orbital.v8i5.771

  17. Environmental conditions affecting exopolysaccharide production by Pseudomonas aeruginosa, Micrococcus sp., and Ochrobactrum sp.

    Science.gov (United States)

    Kiliç, Nur Koçberber; Dönmez, Gönül

    2008-06-15

    Three different chromium-resistant microorganisms (Pseudomonas aeruginosa, Micrococcus sp., and Ochrobactrum sp.) were tested with regard to their EPS production at different pH levels, temperatures, Cr(VI) concentrations, and incubation periods. The optimum pH level was 7 for P. aeruginosa and Micrococcus sp., while it was 8 for Ochrobactrum sp. according to the highest EPS amount at 100 mg/L Cr(VI) concentration. The highest production of EPSs by the three bacteria was obtained under different environmental conditions. P. aeruginosa produced the highest EPS (863.3 mg/L) after incubation for 96 h on media with 50 mg/L Cr(VI) at 20 degrees C, Micrococcus sp. gave the highest yield (444.6 mg/L) after incubation for 72 h on media with 100 mg/L Cr(VI) at the same temperature, and Ochrobactrum sp. had the highest production (430.5 mg/L) on media with 150 mg/L Cr(VI) at 30 degrees C at the end of 48 h of incubation.

  18. A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non-eDNA networks in Pseudomonas aeruginosa biofilms.

    Science.gov (United States)

    Swearingen, Matthew C; Mehta, Ajeet; Mehta, Amar; Nistico, Laura; Hill, Preston J; Falzarano, Anthony R; Wozniak, Daniel J; Hall-Stoodley, Luanne; Stoodley, Paul

    2016-02-01

    Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Production of Quorum Sensing Inhibitors in Growing Onion Bulbs Infected with Pseudomonas aeruginosa E (HQ324110).

    Science.gov (United States)

    Abd-Alla, Mohamed H; Bashandy, Shymaa R

    2012-01-01

    Eighteen organic compounds were present in growing onion bulbs cultivar Giza 6 infected with P. aeruginosa, but only fourteen of them are present in dry infected onion bulbs; however, four compounds were missing in dry onion. The missing compounds in dry infected onion bulbs are pantolactone, 4,5-dihydro-4,5-dimethylfuran-2(3H)-one, myristic acid, and linoleic acid. All of them were detected in growing onion (living cell) during Pseudomonas aeruginosa infection, and it is hypothesized that it may be produced by plants and act as defence system. Pantolactone and myristic acid were selected to explore their effects on growth and virulence factors of Pseudomonas aeruginosa. Exogenous application of pantolactone and myristic acid significantly inhibited pyocyanin production, protease, and lipase and polygalacturonase activity but did not have any significant effects on bacterial growth. The inhibition of virulence factors without reduction in bacterial growth may be providing strong support that these chemical molecules are general quorum sensing inhibitors than an antibacterial effect. Disruption of quorum sensing of pathogen indicates that this new approach has potential in fighting bacterial infections in human and plants.

  20. Production and characterization of an extracellular lipase from Candida guilliermondii

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    Anne Caroline Defranceschi Oliveira

    2014-12-01

    Full Text Available Extracellular lipases from the endophytic yeast Candida guilliermondii isolated from castor leaves (Ricinus communis L. were produced using low-cost raw materials such as agro-industrial residues and applying them in the esterification of oleic acid for evaluating their potential use in biodiesel production. After partial purification using ammonium sulfate, the enzyme was characterized and presented higher activity (26.8 ± 1.5 U mL-1 in the presence of 5 mmol L-1 NaCl at 30 ºC and pH 6.5. The production through submerged fermentation was formerly performed in 150 mL erlenmeyer flasks and, once the enzyme production was verified, assays in a 14 L bioreactor were conducted, obtaining 18 ± 1.4 U mL-1. The produced enzyme was applied in the oleic acid esterification under different solvents: hexane, cyclohexane or cyclohexanone and different acid:alcohol molar ratios. Higher ester conversion rate (81% was obtained using hexane and the molar ratio of 1:9 was the best conditions using methanol. The results suggest the potential for development of endophytic yeast in the production of biocatalyst through submerged fermentation using agroindustrial residues as culture medium.

  1. Enhancing the Feasibility of Microcystis aeruginosa as a Feedstock for Bioethanol Production under the Influence of Various Factors.

    Science.gov (United States)

    Khan, Muhammad Imran; Lee, Moon Geon; Seo, Hyo Jin; Shin, Jin Hyuk; Shin, Tai Sun; Yoon, Yang Ho; Kim, Min Yong; Choi, Jong Il; Kim, Jong Deog

    2016-01-01

    Microcystis aeruginosa, a freshwater microalga, is capable of producing and accumulating different types of sugars in its biomass which make it a good feedstock for bioethanol production. Present study aims to investigate the effect of different factors increasing growth rate and carbohydrates productivity of M. aeruginosa. MF media (modified BG11 media) and additional ingredients such as aminolevulinic acid (2 mM), lysine (2.28 mM), alanine (1 mM), and Naphthalene acetic acid (1 mM) as cytokine promoted M. aeruginosa growth and sugar contents. Salmonella showed growth-assisting effect on M. aeruginosa. Enhanced growth rate and carbohydrates contents were observed in M. aeruginosa culture grown at 25°C under red LED light of 90 μmolm(-2)s(-1) intensity. More greenish and carbohydrates rich M. aeruginosa biomass was prepared (final OD660 nm = 2.21 and sugar contents 10.39 mM/mL) as compared to control (maximum OD660 nm = 1.4 and sugar contents 3 mM/mL). The final algae biomass was converted to algae juice through a specific pretreatment method. The resulted algae Juice was used as a substrate in fermentation process. Highest yield of bioethanol (50 mM/mL) was detected when Brettanomyces custersainus, Saccharomyces cerevisiae, and Pichia stipitis were used in combinations for fermentation process as compared to their individual fermentation. The results indicated the influence of different factors on the growth rate and carbohydrates productivity of M. aeruginosa and its feasibility as a feedstock for fermentative ethanol production.

  2. Enhancing the Feasibility of Microcystis aeruginosa as a Feedstock for Bioethanol Production under the Influence of Various Factors

    Directory of Open Access Journals (Sweden)

    Muhammad Imran Khan

    2016-01-01

    Full Text Available Microcystis aeruginosa, a freshwater microalga, is capable of producing and accumulating different types of sugars in its biomass which make it a good feedstock for bioethanol production. Present study aims to investigate the effect of different factors increasing growth rate and carbohydrates productivity of M. aeruginosa. MF media (modified BG11 media and additional ingredients such as aminolevulinic acid (2 mM, lysine (2.28 mM, alanine (1 mM, and Naphthalene acetic acid (1 mM as cytokine promoted M. aeruginosa growth and sugar contents. Salmonella showed growth-assisting effect on M. aeruginosa. Enhanced growth rate and carbohydrates contents were observed in M. aeruginosa culture grown at 25°C under red LED light of 90 μmolm−2s−1 intensity. More greenish and carbohydrates rich M. aeruginosa biomass was prepared (final OD660 nm = 2.21 and sugar contents 10.39 mM/mL as compared to control (maximum OD660 nm = 1.4 and sugar contents 3 mM/mL. The final algae biomass was converted to algae juice through a specific pretreatment method. The resulted algae Juice was used as a substrate in fermentation process. Highest yield of bioethanol (50 mM/mL was detected when Brettanomyces custersainus, Saccharomyces cerevisiae, and Pichia stipitis were used in combinations for fermentation process as compared to their individual fermentation. The results indicated the influence of different factors on the growth rate and carbohydrates productivity of M. aeruginosa and its feasibility as a feedstock for fermentative ethanol production.

  3. Production of extracellular laccase from the newly isolated Bacillus ...

    African Journals Online (AJOL)

    This study was carried out with aim of screening for extracellular thermostable laccase producing bacteria. Twenty-two (22) laccase positive strains were isolated from the selected environmental samples while extracellular laccase activity was detected only in six strains namely TM1, TMT1, PK4, PS1, TMS1 and ASP3.

  4. Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene

    International Nuclear Information System (INIS)

    Kokjohn, T.A.; Miller, R.V.

    1987-01-01

    We cloned a 2.3-kilobase-pair fragment of the Pseudomonas aeruginosa PAO chromosome which is capable of complementing recA mutations of Escherichia coli. The recA-complementing activity was further localized to a 1.5-kilobase-pair PvuII-HindIII fragment. Southern blot analysis under conditions of high stringency indicated that DNA sequence homology is shared by the E. coli recA gene and the P. aeruginosa recA analog. The cloned recA analog was shown to restore resistance to methyl methanesulfonate, nitrofurantoin, and UV irradiation to E. coli recA mutants. Upon introduction of the cloned P. aeruginosa gene, these mutants regained recombination proficiency in HfrH-mediated conjugation and the ability to induce lambda prophages and SOS functions (din gene transcription) after exposure to DNA-damaging agents. Lambda prophage carrying a cI ind mutation was not inducible, suggesting that the mechanism of induction of these SOS functions by the P. aeruginosa RecA analog is similar to that by the activated E. coli RecA protein. The product of the recA analog was identified in minicells as a protein of approximately 47,000 daltons. Western blot analysis using anti-E. coli RecA antibody demonstrated that this protein is antigenically cross-reactive with the E. coli recA protein. The recA-containing fragment was cloned into the broad-host-range vector pCP13 and introduced into Rec- strains of P. aeruginosa containing the rec-102 allele. The plasmid was shown to restore recombination proficiency in FP5-mediated conjugations and to restore resistance to UV irradiation and methyl methanesulfonate to these Rec- mutants. It was shown that a wild-type allele of rec-102 is necessary for UV-mediated induction of D3 and F116 prophages. The cloned recA analog restored the UV inducibility of these prophages in rec-102 mutants

  5. Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene

    Energy Technology Data Exchange (ETDEWEB)

    Kokjohn, T.A.; Miller, R.V.

    1987-04-01

    We cloned a 2.3-kilobase-pair fragment of the Pseudomonas aeruginosa PAO chromosome which is capable of complementing recA mutations of Escherichia coli. The recA-complementing activity was further localized to a 1.5-kilobase-pair PvuII-HindIII fragment. Southern blot analysis under conditions of high stringency indicated that DNA sequence homology is shared by the E. coli recA gene and the P. aeruginosa recA analog. The cloned recA analog was shown to restore resistance to methyl methanesulfonate, nitrofurantoin, and UV irradiation to E. coli recA mutants. Upon introduction of the cloned P. aeruginosa gene, these mutants regained recombination proficiency in HfrH-mediated conjugation and the ability to induce lambda prophages and SOS functions (din gene transcription) after exposure to DNA-damaging agents. Lambda prophage carrying a cI ind mutation was not inducible, suggesting that the mechanism of induction of these SOS functions by the P. aeruginosa RecA analog is similar to that by the activated E. coli RecA protein. The product of the recA analog was identified in minicells as a protein of approximately 47,000 daltons. Western blot analysis using anti-E. coli RecA antibody demonstrated that this protein is antigenically cross-reactive with the E. coli recA protein. The recA-containing fragment was cloned into the broad-host-range vector pCP13 and introduced into Rec- strains of P. aeruginosa containing the rec-102 allele. The plasmid was shown to restore recombination proficiency in FP5-mediated conjugations and to restore resistance to UV irradiation and methyl methanesulfonate to these Rec- mutants. It was shown that a wild-type allele of rec-102 is necessary for UV-mediated induction of D3 and F116 prophages. The cloned recA analog restored the UV inducibility of these prophages in rec-102 mutants.

  6. Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

    Directory of Open Access Journals (Sweden)

    Bernardas Morkunas

    2016-07-01

    Full Text Available Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable.

  7. New insights about antibiotic production by Pseudomonas aeruginosa: a gene expression analysis

    Science.gov (United States)

    Gionco, Bárbara; Tavares, Eliandro R.; de Oliveira, Admilton G.; Yamada-Ogatta, Sueli F.; do Carmo, Anderson O.; Pereira, Ulisses de Pádua; Chideroli, Roberta T.; Simionato, Ane S.; Navarro, Miguel O. P.; Chryssafidis, Andreas L.; Andrade, Galdino

    2017-09-01

    The bacterial resistance for antibiotics is one of the most important problems in public health and only a small number of new products are in development. Antagonistic microorganisms from soil are a promising source of new candidate molecules. Products of secondary metabolism confer adaptive advantages for their producer, in the competition for nutrients in the microbial community. The biosynthesis process of compounds with antibiotic activity is the key to optimize their production and the transcriptomic study of microorganisms is of great benefit for the discovery of these metabolic pathways. Pseudomonas aeruginosa LV strain growing in the presence of copper chloride produces a bioactive organometallic compound, which has a potent antimicrobial activity against various microorganisms. The objective of this study was to verify overexpressed genes and evaluate their relation to the organometallic biosynthesis in this microorganism. P. aeruginosa LV strain was cultured in presence and absence of copper chloride. Two methods were used for transcriptomic analysis, genome reference-guided assembly and de novo assembly. The genome referenced analysis identified nine upregulated genes when bacteria were exposed to copper chloride, while the De Novo Assembly identified twelve upregulated genes. Nineteen genes can be related to an increased microbial metabolism for the extrusion process of exceeding intracellular copper. Two important genes are related to the biosynthesis of phenazine and tetrapyrroles compounds, which can be involved in the bioremediation of intracellular copper and biosynthesis of the organometallic compound. Additional studies are being carried out to further prove the function of the described genes and relate them to the biosynthetic pathway of the organometallic compound.

  8. New Insights about Antibiotic Production by Pseudomonas aeruginosa: A Gene Expression Analysis

    Directory of Open Access Journals (Sweden)

    Bárbara Gionco

    2017-09-01

    Full Text Available The bacterial resistance for antibiotics is one of the most important problems in public health and only a small number of new products are in development. Antagonistic microorganisms from soil are a promising source of new candidate molecules. Products of secondary metabolism confer adaptive advantages for their producer, in the competition for nutrients in the microbial community. The biosynthesis process of compounds with antibiotic activity is the key to optimize their production and the transcriptomic study of microorganisms is of great benefit for the discovery of these metabolic pathways. Pseudomonas aeruginosa LV strain growing in the presence of copper chloride produces a bioactive organometallic compound, which has a potent antimicrobial activity against various microorganisms. The objective of this study was to verify overexpressed genes and evaluate their relation to the organometallic biosynthesis in this microorganism. P. aeruginosa LV strain was cultured in presence and absence of copper chloride. Two methods were used for transcriptomic analysis, genome reference-guided assembly and de novo assembly. The genome referenced analysis identified nine upregulated genes when bacteria were exposed to copper chloride, while the De Novo Assembly identified 12 upregulated genes. Nineteen genes can be related to an increased microbial metabolism for the extrusion process of exceeding intracellular copper. Two important genes are related to the biosynthesis of phenazine and tetrapyrroles compounds, which can be involved in the bioremediation of intracellular copper and we suggesting that may involve in the biosynthesis of the organometallic compound. Additional studies are being carried out to further prove the function of the described genes and relate them to the biosynthetic pathway of the organometallic compound.

  9. Growth, extracellular alkaline phosphatase activity, and kinetic characteristic responses of the bloom-forming toxic cyanobacterium, Microcystis aeruginosa, to atmospheric particulate matter (PM2.5, PM2.5-10, and PM>10).

    Science.gov (United States)

    Xu, Ziran; Wang, Shoubing; Wang, Yuanan; Zhang, Jie

    2018-03-01

    Atmospheric particulate matter (APM), commonly seen and widely excited in environment, appears great enough to influence the biochemical processes in aquatic microorganisms and phytoplankton. Understanding the response of cyanobacteria to various factors is fundamental for eutrophication control. To clarify the response of cyanobacteria to APM, the effects of PM 2.5 , PM 2.5-10 , and PM >10 on Microcystis aeruginosa were researched. Variabilities in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular activity, and kinetic parameters of alkaline phosphatase were evaluated by lab-cultured experiments. Results showed that the PM 2.5 had a slight stimulation impact on the growth and enhanced both of the 48- and 72-h extracellular alkaline phosphatase activity (APA), the affinity of alkaline phosphatase for substrate, and the 72-h maximum enzymatic reaction velocity (V max ). Moreover, the stimulations in extracellular APA and V max enhanced with the increasing exposure concentrations. We also found there were no obvious distinctions on the effects of growth and alkaline phosphatase in M. aeruginosa between PM 2.5-10 and PM >10 exposure groups. Obviously, inhibitory effects on growth existed in 4.0 and 8.0 mg/L PM 2.5-10 and 8.0 mg/L PM >10 at 120 h. Furthermore, PM 2.5-10 and PM >10 exerted inhibitory effects on the extracellular APA during the 72-h exposure. Simultaneously, the V max was notably inhibited and the affinity of alkaline phosphatase for substrate was more inseparable compared with control in PM 2.5-10 and PM >10 treatments. Nevertheless, the inhibitors in extracellular APA and kinetic parameters were unrelated to PM 2.5-10 and PM >10 exposure concentrations. Two-way ANOVA results revealed that there were significant interactions between exposure concentration and diameter of APM on the 120-h cell density, soluble protein content, APA, and 72 h APA of M. aeruginosa. These results in our study would be meaningful to further

  10. Utilization of Crude Glycerol as a Substrate for the Production of Rhamnolipid by Pseudomonas aeruginosa.

    Science.gov (United States)

    Eraqi, Walaa A; Yassin, Aymen S; Ali, Amal E; Amin, Magdy A

    2016-01-01

    Biosurfactants are produced by bacteria or yeast utilizing different substrates as sugars, glycerol, or oils. They have important applications in the detergent, oil, and pharmaceutical industries. Glycerol is the product of biodiesel industry and the existing glycerol market cannot accommodate the excess amounts generated; consequently, new markets for refined glycerol need to be developed. The aim of present work is to optimize the production of microbial rhamnolipid using waste glycerol. We have developed a process for the production of rhamnolipid biosurfactants using glycerol as the sole carbon source by a local Pseudomonas aeruginosa isolate that was obtained from an extensive screening program. A factorial design was applied with the goal of optimizing the rhamnolipid production. The highest production yield was obtained after 2 days when cells were grown in minimal salt media at pH 6, containing 1% (v/v) glycerol and 2% (w/v) sodium nitrate as nitrogen source, at 37°C and at 180 rpm, and reached 2.164 g/L after 54 hours (0.04 g/L h). Analysis of the produced rhamnolipids by TLC, HPLC, and FTIR confirmed the nature of the biosurfactant as monorhamnolipid. Glycerol can serve as a source for the production of rhamnolipid from microbial isolates providing a cheap and reliable substrate.

  11. Synergistic effect of 14-alpha-lipoyl andrographolide and various antibiotics on the formation of biofilms and production of exopolysaccharide and pyocyanin by Pseudomonas aeruginosa.

    Science.gov (United States)

    Zeng, Xiangping; Liu, Xiangyang; Bian, Jiang; Pei, Gang; Dai, Huanqin; Polyak, Steven W; Song, Fuhang; Ma, Li; Wang, Yuqiang; Zhang, Lixin

    2011-06-01

    Pseudomonas aeruginosa produces a biofilm that provides the bacteria with an effective barrier against antibiotics. Here, we investigated the synergy of various antibiotics with 14-alpha-lipoyl andrographolide (AL-1), focusing upon synthesis of the biofilm. AL-1 also inhibited the production of the exopolysaccharide and pyocyanin components. We propose that AL-1 may potentially serve as a cotherapy to combat P. aeruginosa.

  12. Inhibition of biofilm formation, quorum sensing and infection in Pseudomonas aeruginosa by natural products-inspired organosulfur compounds.

    Directory of Open Access Journals (Sweden)

    Nathaniel C Cady

    Full Text Available Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO (1 mM. Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control, and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed.

  13. Optimization of rhamnolipid production from Pseudomonas aeruginosa PBS towards application for microbial enhanced oil recovery.

    Science.gov (United States)

    Sharma, Rajni; Singh, Jagdish; Verma, Neelam

    2018-01-01

    The present work reveals the potential of biosurfactant producing P. aeruginosa PBS for microbial enhanced oil recovery (MEOR). The biosurfactant production medium and culture conditions were optimized using response surface methodology. The optimization of media components and process parameters was consecutively executed in two sets of experimental runs designed by central composite rotatable design (CCRD). The maximum biosurfactant yield was attained with 2% fresh inoculum of P. aeruginosa PBS in minimal salt medium (pH 7), possessing 2.17% sodium citrate as C-source and 0.5% yeast extract as N-source, after 48 h upon incubation at 30 °C/150 rpm. Under optimum conditions, biosurfactant yield was increased more than threefold and turned out to be 2.65 g/L as compared to 0.82 g/L under previous conditions. The biosurfactant was characterized as a glycolipid comprising of four rhamnolipid homologs (RhaRhaC 10 C 10 , RhaRhaC 8 C 10 , RhaRhaC 12 C 10 /RhaRhaC 10 C 12 , RhaC 10 C 10 ) by thin layer chromatography, fourier transform infrared spectroscopy, nuclear magnetic resonance and mass spectrometry. The produced biosurfactant was highly efficient for oil recovery application showing extreme reduction in surface tension of medium (71.80 to 23.76 mN/m), immense hydrocarbons emulsification capacity (50-60%) and greater stability at wide range of temperature (4-100 °C) and pH (4-10) along with an excellent (56.18 ± 1.59%) additional oil recovery in sand-pack column lab test.

  14. Enhanced production of extracellular inulinase by the yeast Kluyveromyces marxianus in xylose catabolic state.

    Science.gov (United States)

    Hoshida, Hisashi; Kidera, Kenta; Takishita, Ryuta; Fujioka, Nobuhisa; Fukagawa, Taiki; Akada, Rinji

    2018-06-01

    The production of extracellular proteins by the thermotolerant yeast Kluyveromyces marxianus, which utilizes various sugars, was investigated using media containing sugars such as glucose, galactose, and xylose. SDS-PAGE analysis of culture supernatants revealed abundant production of an extracellular protein when cells were grown in xylose medium. The N-terminal sequence of the extracellular protein was identical to a part of the inulinase encoded by INU1 in the genome. Inulinase is an enzyme hydrolyzing β-2,1-fructosyl bond in inulin and sucrose and is not required for xylose assimilation. Disruption of INU1 in the strain DMKU 3-1042 lost the production of the extracellular protein and resulted in growth defect in sucrose and inulin media, indicating that the extracellular protein was inulinase (sucrase). In addition, six K. marxianus strains among the 16 strains that were analyzed produced more inulinase in xylose medium than in glucose medium. However, expression analysis indicated that the INU1 promoter activity was lower in the xylose medium than in the glucose medium, suggesting that enhanced production of inulinase is controlled in a post-transcriptional manner. The production of inulinase was also higher in cultures with more agitation, suggesting that oxygen supply affects the production of inulinase. Taken together, these results suggest that both xylose and oxygen supply shift cellular metabolism to enhance the production of extracellular inulinase. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  15. Production of biosurfactants from Pseudomonas aeruginosa PA 1 isolated in oil environments

    Directory of Open Access Journals (Sweden)

    L.M. Santa Anna

    2002-04-01

    Full Text Available The potential production of rhamnolipid-type biosurfactants is assessed based on the development of a fermentative process with a strain of Pseudomonas aeruginosa PA1, which was isolated from oil production wastewater in the Northeast of Brazil. These production of molecules using different carbon (n-hexadecane, paraffinic oil, glycerol and babassu oil and nitrogen sources (NaNO3, (NH42SO4 and CH4N2O was studied. The best results were obtained when using glycerol as substrate. A C/N ratio of 60/1 and use of sodium nitrate as nitrogen source resulted in higher production of the rhamnolipid, expressed by rhamnose (3.16 g/L and by the yield in relation to biomass (Yp/x = 0.70 g/g. Additionally, physical-chemical characteristics of the spent broth with and without cells were studied, providing a low critical micelle concentration of 19 mg/L and toxicity values of 13 and 13.8 mg/L using two test organisms, the micro crustacean Daphnia similis and the bacterium Vibrio fisheri (Microtox, respectively.

  16. Heterologous production of Pseudomonas aeruginosa rhamnolipid under anaerobic conditions for microbial enhanced oil recovery.

    Science.gov (United States)

    Zhao, F; Shi, R; Zhao, J; Li, G; Bai, X; Han, S; Zhang, Y

    2015-02-01

    The ex situ application of rhamnolipid to enhance oil recovery is costly and complex in terms of rhamnolipid production and transportation, while in situ production of rhamnolipid is restricted by the oxygen-deficient environments of oil reservoirs. To overcome the oxygen-limiting conditions and to circumvent the complex regulation of rhamnolipid biosynthesis in Pseudomonas aeruginosa, an engineered strain Pseudomonas stutzeri Rhl was constructed for heterologous production of rhamnolipid under anaerobic conditions. The rhlABRI genes for rhamnolipid biosynthesis were cloned into a facultative anaerobic strain Ps. stutzeri DQ1 to construct the engineered strain Rhl. Anaerobic production of rhamnolipid was confirmed by thin layer chromatography and Fourier transform infrared analysis. Rhamnolipid product reduced the air-water surface tension to 30.3 mN m(-1) and the oil-water interfacial tension to 0.169 mN m(-1). Rhl produced rhamnolipid of 1.61 g l(-1) using glycerol as the carbon source. Rhl anaerobic culture emulsified crude oil up to EI24 ≈ 74. An extra 9.8% of original crude oil was displaced by Rhl in the core flooding test. Strain Rhl achieved anaerobic production of rhamnolipid and worked well for enhanced oil recovery in the core flooding model. The rhamnolipid produced by Rhl was similar to that of the donor strain SQ6. This is the first study to achieve anaerobic and heterologous production of rhamnolipid. Results demonstrated the potential feasibility of Rhl as a promising strain to enhance oil recovery through anaerobic production of rhamnolipid. © 2014 The Society for Applied Microbiology.

  17. Growth and microcystin production of a Brazilian Microcystis aeruginosa strain (LTPNA 02 under different nutrient conditions

    Directory of Open Access Journals (Sweden)

    Stella Bortoli

    Full Text Available Cyanobacteria are prokaryotic and photosynthetic organisms, which can produce a wide range of bioactive compounds with different properties; including a variety of toxic compounds, also known as cyanotoxins. In this work, we describe the isolation of seven cyanobacterial strains from two reservoirs in São Paulo State, Brazil. Seven different chemical variants of microcystins (MC-RR, MC-LR, MC-YR, MC-LF, MC-LW, and two demethylated variants, dm-MC-RR and dm-MC-LR were detected in three of the ten isolated strains. One particular Microcystis aeruginosa strain (LTPNA 02 was chosen to evaluate its growth by cell count, and its toxin production under seven different nutritional regimes. We observed different growth behaviors in the logarithmic growth period for only three experiments (p < 0.05. The total growth analysis identified four experiments as different from the control (p < 0.01. Three microcystin variants (MC-RR, MC-LR and MC-YR were quantified by liquid chromatography-tandem mass spectrometry. At the experimental end, the toxin content was unchanged when comparing cell growth in ASM-1 (N:P = 1, MLA and BG-11 (N:P = 10 medium. In all other experiments, the lowest microcystin production was observed from cells grown in Bold 3N medium during the exponential growth phase. The highest microcystin content was observed in cultures using BG-11(N:P = 100 medium.

  18. Optimization and characterization of biosurfactant production from kitchen waste oil using Pseudomonas aeruginosa.

    Science.gov (United States)

    Chen, Chunyan; Sun, Ni; Li, Dongsheng; Long, Sihua; Tang, Xiaoyu; Xiao, Guoqing; Wang, Linyuan

    2018-03-16

    Kitchen waste oil (KWO) from catering industries or households was used as a low-cost carbon source for producing biosurfactants by self-isolated Pseudomonas aeruginosa. Fermentation performance with KWO was superior to those with four other carbon sources, with higher optical density (OD 600 ) of 2.33 and lower interfacial tension of 0.57 mN/m. Culture conditions for biosurfactant production were optimized, with optimal pH of 8.0 and nitrogen source concentration of 2.0 g/L, respectively. The results of infrared spectroscopy and liquid chromatography-mass spectrometry (LC-MS) showed that the biosurfactant was a mixture of six rhamnolipid congeners, among which Rha-Rha-C 10 -C 10 and Rha-C 10 -C 10 were the main components, with mass fraction of approximately 34.20 and 50.86%, respectively. The critical micelle concentration (CMC) obtained was 55.87 mg/L. In addition, the rhamnolipids exhibited excellent tolerance to temperature (20-100 °C), pH (6.0-12.0), and salinity (2-20%; w/v) in a wide range, thereby showing good stability to extreme environmental conditions. The rhamnolipids positively affected oil removal from oil sludge and KWO-contaminated cotton cloth, with removal rate of 34.13 and of 30.92%, respectively. Our results demonstrated that biosurfactant production from KWO was promising, with advantages of good performance, low cost and environmental safety.

  19. Optimization of growth and extracellular glucoamylase production by ...

    African Journals Online (AJOL)

    Candida famata was isolated from traditional Moroccan sourdough. It exhibited high glucoamylase and biomass production. Starch induces high glucoamylase production C. famata with maximum glucoamylase activity at 5 g/L. Glucose stimulates good production in biomass but strongly inhibits glucoamylase production.

  20. Facultative control of matrix production optimizes competitive fitness in Pseudomonas aeruginosa PA14 biofilm models.

    Science.gov (United States)

    Madsen, Jonas S; Lin, Yu-Cheng; Squyres, Georgia R; Price-Whelan, Alexa; de Santiago Torio, Ana; Song, Angela; Cornell, William C; Sørensen, Søren J; Xavier, Joao B; Dietrich, Lars E P

    2015-12-01

    As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O2. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O2-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Production of enantiomerically pure D-Phenylglycine using Pseudomonas aeruginosa 10145 as biocatalyst

    Directory of Open Access Journals (Sweden)

    F. O. M. Alonso

    2008-03-01

    Full Text Available Different bacterial strains were screened to detect nitrilase and/or nitrile hidratase/amidase activities towards benzonitrile, to be used as biocatalyst to produce enantiomerically pure non-proteinogenic amino acids using amino nitriles as starting material. The best biocatalyst found was Pseudomonas aeruginosa 10145, which showed high enzyme activities. Whole cells were used as catalyst for the transformation of 2-phenyl-2-amino-acetonitrile for the corresponding D-phenylglycine. The percentage conversion was followed by chiral HPLC. After 1 hour reaction 18% of 2-phenyl-2-amino-acetonitrile was converted into D-phenylglycine with an enantiomeric excess of over 95%. When an inducer was added to the media, an increase in nitrile hydrolyzing activities was detected, hence leading to total conversion of (R-2-phenyl-2-amino-acetonitrile to the corresponding amino acid in 30 min reaction. The isolated yield of the target product was 50% and its characterization was performed by polarimetry, chiral HPLC, IR-FT spectroscopy and GC-MS.

  2. Genes Required for Free Phage Production are Essential for Pseudomonas aeruginosa Chronic Lung Infections.

    Science.gov (United States)

    Lemieux, Andrée-Ann; Jeukens, Julie; Kukavica-Ibrulj, Irena; Fothergill, Joanne L; Boyle, Brian; Laroche, Jérôme; Tucker, Nicholas P; Winstanley, Craig; Levesque, Roger C

    2016-02-01

    The opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infection in patients with cystic fibrosis. The Liverpool Epidemic Strain LESB58 is highly resistant to antibiotics, transmissible, and associated with increased morbidity and mortality. Its genome contains 6 prophages and 5 genomic islands. We constructed a polymerase chain reaction (PCR)-based signature-tagged mutagenesis library of 9216 LESB58 mutants and screened the mutants in a rat model of chronic lung infection. A total of 162 mutants were identified as defective for in vivo maintenance, with 11 signature-tagged mutagenesis mutants having insertions in prophage and genomic island genes. Many of these mutants showed both diminished virulence and reduced phage production. Transcription profiling by quantitative PCR and RNA-Seq suggested that disruption of these prophages had a widespread trans-acting effect on the transcriptome. This study demonstrates that temperate phages play a pivotal role in the establishment of infection through modulation of bacterial host gene expression. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  3. Production of extracellular lipase by a new strain Staphylococcus ...

    African Journals Online (AJOL)

    SAM

    2014-07-09

    Jul 9, 2014 ... mesophilic and solvent tolerant lipase with industrial potential. Key words: ..... amount of enzyme production indicating the inducible nature of the .... biodiesel production, oleochemical industry, polymer syn- thesis and ...

  4. Production of extracellular lipase by a new strain Staphylococcus ...

    African Journals Online (AJOL)

    Based on morphological, biochemical and 16S rRNA sequence analysis, the potent isolate was identified as Staphylococcus aureus. The lipase production of the isolate was increased by improving the conditions of production medium. Maximum lipase production (8.11 U/ml) was achieved when 2% punnakka oil was ...

  5. Production and partial purification of extracellular tannase by ...

    African Journals Online (AJOL)

    End-product repression was also studied with inclusion of gallic acid to the growth medium. Enrichment with various additives of metal ions and detergents resulted in inhibition of tannase production. The enzyme was partially purified using ammonium sulfate precipitation followed by the use of DEAE-cellulose. SDS-PAGE ...

  6. Production of extra-cellular polymer in Azotobacter and biosorption ...

    African Journals Online (AJOL)

    Two Azotobacter strains were isolated from alkaline and acid soils. The production of alginate and exopolymer from these two strains showed that, strain AC2 produced high polymer in 2% beet molasses or 1% sucrose broth and addition of nitrogen sources (yeast extract) reduced production of this polymer. The optimum ...

  7. Biofilm and metallo beta-lactamase production among the strains of Pseudomonas aeruginosa and Acinetobacter spp. at a Tertiary Care Hospital in Kathmandu, Nepal

    Directory of Open Access Journals (Sweden)

    Bandana Baniya

    2017-11-01

    Full Text Available Abstract Introduction Pseudomonas aeruginosa and Acinetobacter spp. are found to be associated with biofilm and metallo-β-lactamase production and are the common causes of serious infections mainly in hospitalized patients. So, the main aims of this study were to determine the rates of biofilm production and metallo beta-lactamase production (MBL among the strains of Pseudomonas aeruginosa and Acinetobacter spp. isolated from hospitalized patients. Methods A total of 85 P. aeruginosa isolates and 50 Acinetobacter spp. isolates isolated from different clinical specimens from patients admitted to Shree Birendra Hospital, Kathmandu, Nepal from July 2013 to May 2014 were included in this study. The bacterial isolates were identified with the help of biochemical tests. Modified Kirby-Bauer disc diffusion technique was used for antimicrobial susceptibility testing. Combined disc diffusion technique was used for the detection of MBL production, while Congo red agar method and tube adherence method were used for detection of biofilm production. Results Around 16.4% of P. aeruginosa isolates and 22% of the strains of Acinetobacter spp. were metallo β-lactamase producers. Out of 85 P. aeruginosa isolates, 23 (27.05% were biofilm producers according to tube adherence test while, only 13 (15.29% were biofilm producers as per Congo red agar method. Similarly, out of 50 Acinetobacter spp. 7 (14% isolates were biofilm producers on the basis of tube adherence test, while only 5 (10% were positive for biofilm production by Congo red agar method. Highest rates of susceptibility of P. aeruginosa as well as Acinetobacter spp. were seen toward colistin. Conclusion In our study, biofilm production and metallo beta-lactamase production were observed among Pseudomonas aeruginosa and Acinetobacter spp. However, no statistically significant association could be established between biofilm production and metallo beta-lactamase production.

  8. In Situ Magnetic Separation for Extracellular Protein Production

    DEFF Research Database (Denmark)

    Kappler, T.; Cerff, Martin; Ottow, Kim Ekelund

    2009-01-01

    A new approach for in situ product removal from bioreactors is presented in which high-gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non-porous bacitracin linked magnetic adsorbents were...... was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we Could show that protease yield...... in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in No production processes which are often limited due to weak downstream operations, Potential limitations encountered during a bioprocess can be overcome...

  9. Production of Extracellular Anti-leukaemic Enzyme L- asparaginase ...

    African Journals Online (AJOL)

    Erah

    asparaginase, Solid-state media, TGY broth, TFY broth, SDS-PAGE, ... to contamination, ease of product extraction ... producing property of isolates; organic nitrate ... water, to 100 ml, and contained in a 250 ml .... carbon utilization and nitrogen utilization.

  10. Selection and optimization of extracellular lipase production using ...

    African Journals Online (AJOL)

    Pedro

    2014-01-22

    Jan 22, 2014 ... Erlenmeyer flasks containing 50 ml medium cultive solution of (g L-1 of distilled water): yeast ..... screening of alkaline lipase-production fungi from Brazil savanna soil. World J. Microb. Biot. ... rhamnosus. Int. J. Food Microbiol.

  11. Optimization of extracellular lípase production by Y

    African Journals Online (AJOL)

    lipase production by Yarrowia lipolytica using response surface ..... The distance of the axial points was 1.414, calculated ..... factors and amino acids, which are required for cell ... increase in the oxygen transfer rate, usually as a result of.

  12. Optimization of growth and extracellular glucoamylase production by ...

    African Journals Online (AJOL)

    SERVER

    2007-11-19

    Nov 19, 2007 ... Starch induces high glucoamylase production C. famata with maximum glucoamylase ... at different pH and temperatures of incubation (Spenser-. Martins and ..... of the raw starch-digesting α-amylase of Bacillus sp. IMD 435.

  13. Effective Biosurfactants Production by Pseudomonas aeruginosa and its Efficacy on Different Oils

    Directory of Open Access Journals (Sweden)

    Sarita Kumari

    2010-07-01

    Full Text Available A rhamnolipid producing bacterium, Pseudomonas aeruginosa was isolated from contaminated soil with oily wastes. The Pseudomonas aeruginosa grown with glucose and corn oil as a carbon source produced bio-surfactant. This biosurfactant was purified by procedures that included chloroform-ethanol extraction and 0.05M bicarbonate treatments. The active compound was identified as rhamnolipid by using thin layer chromatography. The emulsification activity of bio-surfactant, the coconut oil responded better than the olive oil, groundnut oil and sunflower oil and gave a maximum level of 1 cm.

  14. Tasco®: A Product of Ascophyllum nodosum Enhances Immune Response of Caenorhabditis elegans Against Pseudomonas aeruginosa Infection

    Directory of Open Access Journals (Sweden)

    Franklin Evans

    2012-01-01

    Full Text Available The effects of Tasco®, a product made from the brown seaweed (Ascophyllum nodosum were tested for the ability to protect Caenorhabditis elegans against Pseudomonas aeruginosa infection. A water extract of Tasco® (TWE reduced P. aeruginosa inflicted mortality in the nematode. The TWE, at a concentration of 300 µg/mL, offered the maximum protection and induced the expression of innate immune response genes viz.; zk6.7 (Lypases, lys-1 (Lysozyme, spp-1 (Saponin like protein, f28d1.3 (Thaumatin like protein, t20g5.7 (Matridin SK domain protein, abf-1 (Antibacterial protein and f38a1.5 (Lectin family protein. Further, TWE treatment also affected a number of virulence components of the P. aeuroginosa and reduced its secreted virulence factors such as lipase, proteases and toxic metabolites; hydrogen cyanide and pyocyanin. Decreased virulence factors were associated with a significant reduction in expression of regulatory genes involved in quorum sensing, lasI, lasR, rhlI and rhlR. In conclusion, the TWE-treatment protected the C. elegans against P. aeruginosa infection by a combination of effects on the innate immunity of the worms and direct effects on the bacterial quorum sensing and virulence factors.

  15. Production of Extracellular Anti-leukaemic Enzyme Lasparaginase ...

    African Journals Online (AJOL)

    Production of L-asparaginase was carried out in three different media, namely, solid-state media, Tryptone Glucose Yeast extract (TGY) broth and Tryptone Fructose Yeast extract (TFY) broth.. The enzyme was purified to near homogeneity by ammonium sulphate precipitation, dialysis, gel filtration on Sephadex G-100 ...

  16. Production of extracellular laccase from the newly isolated Bacillus ...

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-08-24

    Aug 24, 2016 ... enzyme production were observed at the temperature of 37°C, pH 7.5, 10% inoculum size ... samples, namely, the treated and untreated effluents from textile .... 47°C), carbon at 1% level (glucose, galactose, fructose, lactose,.

  17. Selection and optimization of extracellular lipase production using ...

    African Journals Online (AJOL)

    The aim of this study was to isolate and select lipase-producing microorganisms originated from different substrates, as well as to optimize the production of microbial lipase by submerged fermentation under different nutrient conditions. Of the 40 microorganisms isolated, 39 showed a halo around the colonies and 4 were ...

  18. Production of extracellular chitinase Beauveria bassiana under submerged fermentation conditions

    Science.gov (United States)

    Elawati, N. E.; Pujiyanto, S.; Kusdiyantini, E.

    2018-05-01

    Chitinase-producing microbes have attracted attention as one of the potential agents for control of phytopathogenic fungi and insect pests. The fungus that potentially produces chitinase is Beauveria bassiana. This study aims to determine the growth curve and chitinase activities of B. bassiana isolated from Helopeltis antonii insects after application. Method of measuring growth curve was done by dry cell period method, while for measurement of enzyme activity done by measuring absorbance at spectrophotometer. The results showed optimum growth time of B. bassiana with the highest cell count of 0.031 g on day 4 which was log phase, while the highest enzyme activity was 0,585 U / mL on the 4th day for 7 days incubation. Based on these results when correlated growth with enzyme production, chitinase enzyme products are produced in log phase and categorized as primary metabolism.

  19. Production of microbial rhamnolipid by Pseudomonas aeruginosa MM1011 for ex situ enhanced oil recovery.

    Science.gov (United States)

    Amani, Hossein; Müller, Markus Michael; Syldatk, Christoph; Hausmann, Rudolf

    2013-07-01

    Recently, several investigations have been carried out on the in situ bacteria flooding, but the ex situ biosurfactant production and addition to the sand pack as agents for microbial enhanced oil recovery (MEOR) has little been studied. In order to develop suitable technology for ex situ MEOR processes, it is essential to carry out tests about it. Therefore, this work tries to fill the gap. The intention of this study was to investigate whether the rhamnolipid mix could be produced in high enough quantities for enhanced oil recovery in the laboratory scale and prove its potential use as an effective material for field application. In this work, the ability of Pseudomonas aeruginosa MM1011 to grow and produce rhamnolipid on sunflower as sole carbon source under nitrogen limitation was shown. The production of Rha-C10-C10 and Rha2-C10-C10 was confirmed by thin-layer chromatography and high-performance liquid chromatography analysis. The rhamnolipid mixture obtained was able to reduce the surface and interfacial tension of water to 26 and 2 mN/m, respectively. The critical micelle concentration was 120 mg/L. Maximum rhamnolipid production reached to about 0.7 g/L in a shake flask. The yield of rhamnolipid per biomass (Y RL/x ), rhamnolipid per sunflower oil (Y RL/s ), and the biomass per sunflower oil (Y x/s ) for shake flask were obtained about 0.01, 0.0035, and 0.035 g g(-1), respectively. The stability of the rhamnolipid at different salinities, pH and temperature, and also, its emulsifying activity has been investigated. It is an effective surfactant at very low concentrations over a wide range of temperatures, pHs, and salt concentrations, and it also has the ability to emulsify oil, which is essential for enhanced oil recovery. With 120 mg/L rhamnolipid, 27 % of original oil in place was recovered after water flooding from a sand pack. This result not only suggests rhamnolipids as appropriate model biosurfactants for MEOR, but it even shows the potential as a

  20. Variation in hydrogen cyanide production between different strains of Pseudomonas aeruginosa

    Czech Academy of Sciences Publication Activity Database

    Gilchrist, F. J.; Alcock, A.; Belcher, J.; Brady, M.; Jones, A.; Smith, D.; Španěl, Patrik; Webb, K.; Lenney, W.

    2011-01-01

    Roč. 38, č. 2 (2011), s. 409-414 ISSN 0903-1936 Institutional research plan: CEZ:AV0Z40400503 Keywords : microbiology * pseudomonas aeruginosa Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 5.895, year: 2011

  1. Extracellular membrane vesicles in blood products-biology and clinical relevance

    Directory of Open Access Journals (Sweden)

    Emilija Krstova Krajnc

    2016-01-01

    Full Text Available Extracellular membrane vesicles are fragments shed from plasma membranes off all cell types that are undergoing apoptosis or are being subjected to various types of stimulation or stress.  Even in the process of programmed cell death (apoptosis, cell fall apart of varying size vesicles. They expose phosphatidylserine (PS on the outer leaflet of their membrane, and bear surface membrane antigens reflecting their cellular origin. Extracellular membrane vesicles have been isolated from many types of biological fluids, including serum, cerebrospinal fluid, urine, saliva, tears and conditioned culture medium. Flow cytometry is one of the many different methodological approaches that have been used to analyze EMVs. The method attempts to characterize the EMVs cellular origin, size, population, number, and structure. EMVs are present and accumulate in blood products (erythrocytes, platelets as well as in fresh frozen plasma during storage. The aim of this review is to highlight the importance of extracellular vesicles as a cell-to-cell communication system and the role in the pathogenesis of different diseases. Special emphasis will be given to the implication of extracellular membrane vesicles in blood products and their clinical relevance. Although our understanding of the role of  EMVs in disease is far from comprehensive, they display promise as biomarkers for different diseases in the future and also as a marker of quality and safety in the quality control of blood products.

  2. Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9 Purificação e caracterização de uma lipase extracelular produzida por uma nova cepa: Pseudomonas aeruginosa SRT9

    Directory of Open Access Journals (Sweden)

    Prita S. Borkar

    2009-06-01

    Full Text Available An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16 fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/minrespectively.Uma lipase extracelular foi isolada e purificada a partir de um caldo de cultura de Pseudomonas aeruginosa SRT9 até homogeneidade visível empregando-se precipitação com sulfato de amônia, seguida de técnicas cromatográficas em colunas de fenil sefarose CL-4B e Mono Q HR 5/5, obtendo-se um fator de purificação de 98 vezes, e atividade especifica de 12307,8 U/mg. Por SDS_PAGE, estimou-se que o peso molecular da lipase purificada é 29kDa, com um ponto isoelétrico de 4,5. A lipase apresentou atividade máxima em uma ampla faixa de temperatura e pH, com ótimos a 55ºC e pH 6,9. A lípase foi mais ativa sobre triacilglicerois de cadeia longa (C14-C16. A lipase foi fortemente inibida por EDTA, o que sugere que a

  3. Biosurfactant production by Pseudomonas aeruginosa MSIC02 in cashew apple juice using a 24 full factorial experimental design

    Directory of Open Access Journals (Sweden)

    Rocha Maria Valderez Ponte

    2014-01-01

    Full Text Available In this work, the production of biosurfactants from cashew apple juice by P. aeruginosa MSIC02 was investigate by carrying out a 24 full factorial experimental design, using temperature, glucose concentration from cashew apple juice, phosphorous concentration and cultivation time as variables. The response variable was the percentage of reduction in surface tension in the cell-free culture medium, since it indicates the surface-active agent production. Maximum biosurfactant production, equivalent to a 58% reduction in surface tension, was obtained at 37°C, with glucose concentration of 5.0 g/L and no phosphorous supplementation. Surface tension reduction was significant, since low values were observed in the cell-free medium (27.50 dyne/cm, indicating that biosurfactant was produced. The biosurfactant emulsified different hydrophobic sources and showed stability in the face of salinity, exposure to high temperatures and extreme pH conditions. These physiochemical properties demonstrate the potential for using biosurfactants produced by P. aeruginosa MSIC02 in various applications.

  4. Disinfection by-product formation during chlor(am)ination of algal organic matters (AOM) extracted from Microcystis aeruginosa: effect of growth phases, AOM and bromide concentration.

    Science.gov (United States)

    Chen, Juxiang; Gao, Naiyun; Li, Lei; Zhu, Mingqiu; Yang, Jing; Lu, Xian; Zhang, Yansen

    2017-03-01

    Algae organic matter (AOM), including extracellular organic matter (EOM) and intracellular organic matter (IOM), has caused a series of problems to the water quality, among which formation of disinfection by-products (DBPs) during subsequent chlor(am)ination process was especially serious and concerned. This study characterized physicochemical properties of the EOM and IOM solution extracted from different growth phases of Microcystis aeruginosa and investigated the corresponding formation potential of DBPs during chlor(am)ination process. Besides, the effects of initial concentration of xEOM, IOM, and Br - on the yields of disinfection by-product formation potential were studied. The results indicated that the specific UV absorbance (SUVA 254 ) values of IOM and EOM (1.09 and 2.66 L/mg m) were considerably lower than that of natural organic matter (NOM) (4.79 L/mg m). Fluorescence dates showed the soluble microbial by-product was dominant in both EOM and IOM, and the tryptophan was the main component of AOM. From the excitation-emission matrix figure of EOM and IOM, we found that the content of the high molecular weight protein substance in IOM was higher than EOM. During chlorination of EOM and IOM, the yields of four kinds of DBPs followed the order trichloroethene (TCM) > 1,1-DCP > dichloride acetonitrile (DCAN) > trichloronitromethane (TCNM), while the order was TCM > DCAN > TCNM > 1,1-DCP during chloramination process. The bromine substitution factor (BSF) value increased with the increasing of the concentration of Br - . When the concentration of Br - was 500 μg/L, the BSF values of chlorination EOM and IOM were 51.1 and 68.4%, respectively. As the concentration of Br - increased, the formation of Cl-DBPs was inhibited and the formation of Br-DBPs was promoted. Graphical abstract ᅟ.

  5. Extracellular production and degradation of superoxide in the coral Stylophora pistillata and cultured Symbiodinium.

    Directory of Open Access Journals (Sweden)

    Eldad Saragosti

    2010-09-01

    Full Text Available Reactive oxygen species (ROS are thought to play a major role in cell death pathways and bleaching in scleractinian corals. Direct measurements of ROS in corals are conspicuously in short supply, partly due to inherent problems with ROS quantification in cellular systems.In this study we characterized the dynamics of the reactive oxygen species superoxide anion radical (O(2(- in the external milieu of the coral Stylophora pistillata. Using a sensitive, rapid and selective chemiluminescence-based technique, we measured extracellular superoxide production and detoxification activity of symbiont (non-bleached and aposymbiont (bleached corals, and of cultured Symbiodinium (from clades A and C. Bleached and non-bleached Stylophora fragments were found to produce superoxide at comparable rates of 10(-11-10(-9 mol O(2(- mg protein(-1 min(-1 in the dark. In the light, a two-fold enhancement in O(2(- production rates was observed in non-bleached corals, but not in bleached corals. Cultured Symbiodinium produced superoxide in the dark at a rate of . Light was found to markedly enhance O(2(- production. The NADPH Oxidase inhibitor Diphenyleneiodonium chloride (DPI strongly inhibited O(2(- production by corals (and more moderately by algae, possibly suggesting an involvement of NADPH Oxidase in the process. An extracellular O(2(- detoxifying activity was found for bleached and non-bleached Stylophora but not for Symbiodinium. The O(2(- detoxifying activity was partially characterized and found to resemble that of the enzyme superoxide dismutase (SOD.The findings of substantial extracellular O(2(- production as well as extracellular O(2(- detoxifying activity may shed light on the chemical interactions between the symbiont and its host and between the coral and its environment. Superoxide production by Symbiodinium possibly implies that algal bearing corals are more susceptible to an internal build-up of O(2(-, which may in turn be linked to oxidative stress

  6. Bioaugmentation of oil reservoir indigenous Pseudomonas aeruginosa to enhance oil recovery through in-situ biosurfactant production without air injection.

    Science.gov (United States)

    Zhao, Feng; Li, Ping; Guo, Chao; Shi, Rong-Jiu; Zhang, Ying

    2018-03-01

    Considering the anoxic conditions within oil reservoirs, a new microbial enhanced oil recovery (MEOR) technology through in-situ biosurfactant production without air injection was proposed. High-throughput sequencing data revealed that Pseudomonas was one of dominant genera in Daqing oil reservoirs. Pseudomonas aeruginosa DQ3 which can anaerobically produce biosurfactant at 42 °C was isolated. Strain DQ3 was bioaugmented in an anaerobic bioreactor to approximately simulate MEOR process. During bioaugmentation process, although a new bacterial community was gradually formed, Pseudomonas was still one of dominant genera. Culture-based data showed that hydrocarbon-degrading bacteria and biosurfactant-producing bacteria were activated, while sulfate reducing bacteria were controlled. Biosurfactant was produced at simulated reservoir conditions, decreasing surface tension to 33.8 mN/m and emulsifying crude oil with EI 24  = 58%. Core flooding tests revealed that extra 5.22% of oil was displaced by in-situ biosurfactant production. Bioaugmenting indigenous biosurfactant producer P. aeruginosa without air injection is promising for in-situ MEOR applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Extracellular peptidase hunting for improvement of protein production in plant cells and roots

    Directory of Open Access Journals (Sweden)

    Jérôme eLallemand

    2015-02-01

    Full Text Available Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion, in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA and human serum immunoglobulins G (hIgGs. Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

  8. Production of extracellular lipases by Rhizopus oligosporus in a stirred fermentor

    Directory of Open Access Journals (Sweden)

    Tehreema Iftikhar

    2010-12-01

    Full Text Available The present investigation deals with the kinetics of submerged extracellular lipases fermentation by both wild and mutant strains of Rhizopus oligosporus var. microsporus in a laboratory scale stirred fermentor. Other parameters studied were inoculum size, pH, agitation and rate of aeration. It was found that the growth and lipases production was increased gradually and reached its maximum 9.07± 0.42ª U mL-1 (W and 42.49 ± 3.91ª U mL-1 (M after 30h of fermentation for both wild and mutant strain. There is overall increase of 109% (W and 124% (M in the production of extracellular lipases as compared to shake flask. Another significant finding of the present study is that the fermentation period is reduced to 30 h in case of wild and 23 h in case of mutant from 48 h in shake flask studies. The specific productivity of mutant strain (qp = 377.3 U/g cells/h was several folds higher than wild strain. The specific production rate and growth coefficient revealed the hyperproducibility of extracellular lipases using mutant IIB-63NTG-7.

  9. Dark production of extracellular superoxide by the coral Porites astreoides and representative symbionts

    Directory of Open Access Journals (Sweden)

    Tong Zhang

    2016-11-01

    Full Text Available The reactive oxygen species (ROS superoxide has been implicated in both beneficial and detrimental processes in coral biology, ranging from pathogenic disease resistance to coral bleaching. Despite the critical role of ROS in coral health, there is a distinct lack of ROS measurements and thus an incomplete understanding of underpinning ROS sources and production mechanisms within coral systems. Here, we quantified in situ extracellular superoxide concentrations at the surfaces of aquaria-hosted Porites astreoides during a diel cycle. High concentrations of superoxide (~10’s of nM were present at coral surfaces, and these levels did not change significantly as a function of time of day. These results indicate that the coral holobiont produces extracellular superoxide in the dark, independent of photosynthesis. As a short-lived anion at physiological pH, superoxide has a limited ability to cross intact biological membranes. Further, removing surface mucus layers from the P. astreoides colonies did not impact external superoxide concentrations. We therefore attribute external superoxide derived from the coral holobiont under these conditions to the activity of the coral host epithelium, rather than mucus-derived epibionts or internal sources such as endosymbionts (e.g., Symbiodinium. However, endosymbionts likely contribute to internal ROS levels via extracellular superoxide production. Indeed, common coral symbionts, including multiple strains of Symbiodinium (clades A to D and the bacterium Endozoicomonas montiporae LMG 24815, produced extracellular superoxide in the dark and at low light levels. Further, representative P. astreoides symbionts, Symbiodinium CCMP2456 (clade A and E. montiporae, produced similar concentrations of superoxide alone and in combination with each other, in the dark and low light, and regardless of time of day. Overall, these results indicate that healthy, non-stressed P. astreoides and representative symbionts produce

  10. Glycine metabolism by Pseudomonas aeruginosa: hydrogen cyanide biosynthesis

    International Nuclear Information System (INIS)

    Castric, P.A.

    1977-01-01

    Hydrogen cyanide (HCN) production by Pseudomonas aeruginosa in a synthetic medium is stimulated by the presence of glycine. Methionine enhances this stimulation but will not substitute for glycine as a stimulator of cyanogenesis. Threonine and phenylalanine are effective substitutes for glycine in the stimulation of HCN production. Glycine, threonine, and serine are good radioisotope precursors of HCN, but methionine and phenylalanine are not. Cell extracts of P. aeruginosa convert [ 14 C]threonine to [ 14 C]glycine. H14CN is produced with low dilution of label from either [1- 14 C]glycine or [2- 14 C]glycine, indicating a randomization of label either in the primary or secondary metabolism of glycine. When whole cells were fed [1,2- 14 C]glycine, cyanide and bicarbonate were the only radioactive extracellular products observed

  11. High-Throughput Genetic Screen Reveals that Early Attachment and Biofilm Formation Are Necessary for Full Pyoverdine Production by Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Donghoon Kang

    2017-09-01

    Full Text Available Pseudomonas aeruginosa is a re-emerging, multidrug-resistant, opportunistic pathogen that threatens the lives of immunocompromised patients, patients with cystic fibrosis, and those in critical care units. One of the most important virulence factors in this pathogen is the siderophore pyoverdine. Pyoverdine serves several critical roles during infection. Due to its extremely high affinity for ferric iron, pyoverdine gives the pathogen a significant advantage over the host in their competition for iron. In addition, pyoverdine can regulate the production of multiple bacterial virulence factors and perturb host mitochondrial homeostasis. Inhibition of pyoverdine biosynthesis decreases P. aeruginosa pathogenicity in multiple host models. To better understand the regulation of pyoverdine production, we developed a high-throughput genetic screen that uses the innate fluorescence of pyoverdine to identify genes necessary for its biosynthesis. A substantial number of hits showing severe impairment of pyoverdine production were in genes responsible for early attachment and biofilm formation. In addition to genetic disruption of biofilm, both physical and chemical perturbations also attenuated pyoverdine production. This regulatory relationship between pyoverdine and biofilm is particularly significant in the context of P. aeruginosa multidrug resistance, where the formation of biofilm is a key mechanism preventing access to antimicrobials and the immune system. Furthermore, we demonstrate that the biofilm inhibitor 2-amino-5,6-dimethylbenzimidazole effectively attenuates pyoverdine production and rescues Caenorhabditis elegans from P. aeruginosa-mediated pathogenesis. Our findings suggest that targeting biofilm formation in P. aeruginosa infections may have multiple therapeutic benefits and that employing an unbiased, systems biology-based approach may be useful for understanding the regulation of specific virulence factors and identifying novel anti

  12. Production and characterization of rhamnolipid biosurfactant from waste frying coconut oil using a novel Pseudomonas aeruginosa D.

    Science.gov (United States)

    George, S; Jayachandran, K

    2013-02-01

    To improve biosurfactant production economics by the utilization of potential low-cost materials. In an attempt to utilize cost-effective carbon sources in the fermentative production of biosurfactants, various pure and waste frying oils were screened by a standard biosurfactant producing strain. Considering the regional significance, easy availability and the economical advantages, waste frying coconut oil was selected as the substrate for further studies. On isolation of more competent strains that could use waste frying coconut oil efficiently as a carbon source, six bacterial strains were isolated on cetyltrimethyl ammonium bromide-methylene blue agar plate, from a soil sample collected from the premises of a coconut oil mill. Among these, Pseudomonas aeruginosa D was selected as the potential producer of rhamnolipid. Spectrophotometric method, TLC, methylene blue active substance assay, drop collapse technique, surface tension measurement by Du Nouy ring method and emulsifying test confirmed the rhamnolipid producing ability of the selected strain and various process parameters were optimized for the production of maximum amount of biosurfactant. Rhamnolipid components purified and separated by ethyl acetate extraction, preparative silica gel column chromatography, HPLC and TLC were characterized by fast atom bombardment mass spectrometry as a mixture of dirhamnolipids and monorhamnolipids. The rhamnolipid homologues detected were Rha-Rha-C(10) -C(10) , Rha-C(12) -C(10) and Rha-C(10) -C(8) /Rha-C(8) -C(10) . These results indicated the possibility of waste frying coconut oil to be used as a very effective alternate substrate for the economic production of rhamnolipid by a newly isolated Ps. aeruginosa D. Results of this study throws light on the alternate use of already used cooking oil as high-energy source for producing a high value product like rhamnolipid. This would provide options for the food industry other than the recycling and reuse of waste frying

  13. Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa.

    Science.gov (United States)

    Cowles, Kimberly N; Gitai, Zemer

    2010-06-01

    Spatial organization of bacterial proteins influences many cellular processes, including division, chromosome segregation and motility. Virulence-associated proteins also localize to specific destinations within bacterial cells. However, the functions and mechanisms of virulence factor localization remain largely unknown. In this work, we demonstrate that polar assembly of the Pseudomonas aeruginosa PAO1 type IV pilus is regulated by surface association in a manner that affects gene transcription, protein levels and protein localization. We also uncover one mechanism for this regulation that acts through the actin homologue MreB. Inactivation of MreB leads to mislocalization of the pilus retraction ATPase PilT, mislocalization of the pili themselves and a reduction in motility. Furthermore, the role of MreB in polar localization of PilT is modulated by surface association, corroborating our results that environmental factors influence the regulation of pilus production. Specifically, MreB mediates both the initiation and maintenance of PilT localization when cells are grown in suspension but only affects the initiation of localization when cells are grown on a surface. Together, these results suggest that the bacterial cytoskeleton provides a mechanism for the polar localization of P. aeruginosa pili and demonstrate that protein localization may represent an important aspect of virulence factor regulation in bacterial pathogens.

  14. Production of extracellular proteases by Mucor circinelloides using D-glucose as carbon source / substrate

    Directory of Open Access Journals (Sweden)

    Andrade Vânia Sousa

    2002-01-01

    Full Text Available Recently, some Mucorales species have been reported as protease producers. The production of extracellular proteases by Mucor circinelloides using glucose as substrate was studied. Experiments were carried out with different D-glucose concentrations (40, 60 and 80 g/L. Biomass, pH and protease activity were determined. Although biomass production had reached best yields for the medium containing D-glucose in a concentration of 80 g/L, the enzymatic production was higher when the substrate concentration was reduced to 40 g/L. The yield factor for product on cell growth and the yield factor for product on carbon substrate were higher when the microorganism grew in medium containing 40 g/L glucose. The kinetics parameters suggest that this strain seems to be promising as an alternative microorganism for protease production.

  15. Production of Monascus pigments as extracellular crystals by cell suspension culture.

    Science.gov (United States)

    Lu, Fengling; Liu, Lujie; Huang, Yaolin; Zhang, Xuehong; Wang, Zhilong

    2018-01-01

    It is generally accepted that Monascus pigments are predominantly cell-bound, including both intracellular and surface-bound pigments. This long-term misconception was corrected in the present work. Production of extracellular crystal pigments by submerged culture of Monascus sp. was confirmed by microscopic observation and collection of Monascus pigments from extracellular broth by direct membrane filtration. Following up the new fact, the bioactivity of mycelia as whole-cell biocatalyst for biosynthesis and biodegradation of Monascus pigments had been detailedly examined in both an aqueous solution and a nonionic surfactant micelle aqueous solution. Based on those experimental results, cell suspension culture in an aqueous medium was developed as a novel strategy for accumulation of high concentration of Monascus pigments. Thus, glucose feeding during submerged culture in the aqueous medium was carried out successfully and high orange Monascus pigments concentration of near 4 g/L was achieved.

  16. Intra and extracellular nuclease production by Aspergillus niger and Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Ferreira Adlane V. B.

    1998-01-01

    Full Text Available Intra and extracellular nuclease production by strains of Aspergillus niger and Aspergillus nidulans was estimated using a modified DNAse test agar and cell-free extract assays. Differences in the production of nucleases by A. niger and A. nidulans were observed. These observations suggest that the DNAse test agar can be helpful for a quick screening for some types of nucleases in filamentous fungi. The assays using cell-free extracts can also be useful for initial characterization of other types of nucleases.

  17. Production of extracellular lipase by the phytopathogenic fungus Fusarium solani FS1

    OpenAIRE

    Maia, Maria de Mascena Diniz; Morais, Marcia Maria Camargo de; Morais Jr., Marcos Antonio de; Melo, Eduardo Henrique Magalhães; Lima Filho, José Luiz de

    1999-01-01

    A Brazilian strain of Fusarium solani was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.l-1 of lipase after 72 hours of cultivation at 25oC in shake-flask at 120 rpm in a medium containing 3% (w/v) peptone plus 0.5% (v/v) olive oil. Glucose (1% w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3% (w/v) resulted in a reduced lipase production while increased olive oil concentration (above 0.5%) did n...

  18. Membrane-anchored MucR mediates nitrate-dependent regulation of alginate production in Pseudomonas aeruginosa

    KAUST Repository

    Wang, Yajie

    2015-04-29

    Alginates exhibit unique material properties suitable for medical and industrial applications. However, if produced by Pseudomonas aeruginosa, it is an important virulence factor in infection of cystic fibrosis patients. The alginate biosynthesis machinery is activated by c-di-GMP imparted by the inner membrane protein, MucR. Here, it was shown that MucR impairs alginate production in response to nitrate in P. aeruginosa. Subsequent site-specific mutagenesis of MucR revealed that the second MHYT sensor motif (MHYT II, amino acids 121–124) of MucR sensor domain was involved in nitrate sensing. We also showed that both c-di-GMP synthesizing and degrading active sites of MucR were important for alginate production. Although nitrate and deletion of MucR impaired alginate promoter activity and global c-di-GMP levels, alginate yields were not directly correlated with alginate promoter activity or c-di-GMP levels, suggesting that nitrate and MucR modulate alginate production at a post-translational level through a localized pool of c-di-GMP. Nitrate increased pel promoter activity in the mucR mutant while in the same mutant the psl promoter activity was independent of nitrate. Nitrate and deletion of mucR did not impact on swarming motility but impaired attachment to solid surfaces. Nitrate and deletion of mucR promoted the formation of biofilms with increased thickness, cell density, and survival. Overall, this study provided insight into the functional role of MucR with respect to nitrate-mediated regulation of alginate biosynthesis. © 2015 Springer-Verlag Berlin Heidelberg

  19. HIF-1α is essential for effective PMN bacterial killing, antimicrobial peptide production and apoptosis in Pseudomonas aeruginosa keratitis.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Berger

    Full Text Available Hypoxia-inducible factor (HIF-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals mice after ocular infection with Pseudomonas (P. aeruginosa. Furthermore, the current studies focused on the neutrophil (PMN, the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG, the role of HIF-1α was assessed in P. aeruginosa-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production.

  20. Effects of light wavelengths on extracellular and capsular polysaccharide production by Nostoc flagelliforme.

    Science.gov (United States)

    Han, Pei-pei; Sun, Ying; Jia, Shi-ru; Zhong, Cheng; Tan, Zhi-lei

    2014-05-25

    The influences of different wavelengths of light (red 660nm, yellow 590nm, green 520nm, blue 460nm, purple 400nm) and white light on extracellular polysaccharide (EPS) and capsular polysaccharide (CPS) production by Nostoc flagelliforme in liquid culture were demonstrated in this study. The results showed that, compared with white light, red and blue lights significantly increased both EPS and CPS production while yellow light reduced their production; purple and green lights stimulated EPS production but inhibited CPS formation. Nine constituent monosaccharides and one uronic acid were detected in both EPS and CPS, and their ratios showed significant differences among treatment with different light wavelengths. However, the advanced structure of EPS and CPS from various light conditions did not present obvious difference through Fourier transform infrared spectroscopy and X-ray diffraction characterization. These findings establish a basis for development of high-yielding polysaccharide production process and understanding their regulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Reprint of “Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus”

    Energy Technology Data Exchange (ETDEWEB)

    Borghese, Roberto, E-mail: roberto.borghese@unibo.it [Dept. of Pharmacy and Biotechnology, University of Bologna (Italy); Brucale, Marco [Institute for the Study of Nanostructured Materials (CNR-ISMN), Rome (Italy); Fortunato, Gianuario [Dept. of Pharmacy and Biotechnology, University of Bologna (Italy); Lanzi, Massimiliano [Dept. of Industrial Chemistry “Toso Montanari”, University of Bologna (Italy); Mezzi, Alessio [Institute for the Study of Nanostructured Materials (CNR-ISMN), Rome (Italy); Valle, Francesco; Cavallini, Massimiliano [Institute for the Study of Nanostructured Materials (CNR-ISMN), Bologna (Italy); Zannoni, Davide [Dept. of Pharmacy and Biotechnology, University of Bologna (Italy)

    2017-02-15

    Highlights: • Tellurite is reduced by R. capsulatus as cytosolic tellurium nanoprecipitates TeNPs. • Lawsone allows R. capsulatus to produce extracellular TeNPs. • Extracellular TeNPs production depends on the carbon source used for cells growth. • Both lawsone concentration and the incubation time determine the TeNPs size. • Extracellular TeNPs are coated with extracellular polymeric substances (EPS). - Abstract: The toxic oxyanion tellurite (TeO{sub 3}{sup 2−}) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te{sup 0} in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te{sup 0} nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1 mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600–700 nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te{sup 0} to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents.

  2. Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

    Science.gov (United States)

    Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

    2017-08-01

    Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105 + , CD44 + , CD14 - , CD34 - , CD45 - , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. Ecological screening of lipolytic cultures and process optimization for extracellular lipase production from fungal hyperproducer

    International Nuclear Information System (INIS)

    Iftikhar, T.; Niaz, M.; Anwer, M.; Abbas, S.Q.; Saleem, M.; Jabeen, R.

    2011-01-01

    Present investigation describes the biosynthesis of extracellular lipases by various local fungal strains isolated from various lipid rich habitats of Faisalabad. The isolated cultures of Aspergillus niger, Penicillium chrysogenum, Rhizopus microsporus, Mucor mucedo, Alternaria alternata, Trichophyton sp., Fusarium semitectum, E (un-identified), Curvularia sp., Aspergillus flavus, G (un-identified), F (Mucor sp.) and H (Synnematous) were identified and screened for the extracelluler lipases production. Different environmental parameters such as pH, temperature, inoculum size, amount of substrate and incubation time were optimized for the selected hyper producer. It was found that maximum production of lipases by Trichophyton sp., was obtained after 48 h of batch fermentation. Similarly, the diluent pH of 7.0 and incubation temperature of 30 deg. C were found optimum for enzyme production by the microorganism. The maximum production of lipases during the course of present studies was 65.20 +- 1.13a U/g. (author)

  4. Isolation and identification of biosurfactant-producing strains from the genus Pseudomonas aeruginosa and antibacterial effects of biosurfactant production in vitro

    Directory of Open Access Journals (Sweden)

    Salman Ahmady-Asbchin

    2013-01-01

    Full Text Available Introduction: Biosurfactants are amphiphilic biological compounds produced extracellularly or as part of the cell membranes by a variety of microorganisms. Because of their use in various industries, they are of a particular importance. The aim of this study was to identify a strain of bacteria of the genus Pseudomonas aeruginosa biosurfactant producers. Materials and methods: In this study, different samples of oil, water and soil contaminated with oil were prepared. Hemolytic activity, emulsification activity and measurement of surface tension were used and selected strains were identified by biochemical tests. The nature and effect of antibacterial biosurfactant was evaluated for strain selection.Results: In this study, eighty eight bacterial strains were isolated. Twenty four strains were isolated from the isolated strains with hemolytic activity. Among which, 14 strains have emulsification activity more than 70% and at last four strains reached surface tension to be less than 40 mN/m. Selected strain based on biochemical tests was recognized as a Pseudomonas aeruginosa. The nature of biosurfactant was determined by TLC, and proved to be of glycolipid kind. Therefore, the produced biosurfactant of the selected strain had antibacterial activity against six bacterial infectious. Sensitive bacteria to the effects of biosurfactant extract of Pseudomonas aeruginosa83, was Staphylococcus aureus and the most resistant bacteria to these extract, was the Proteus mirabilis. The results of MIC, MBC showed that MIC of the extract in concentration of 63 and 125 mg/ml on Escherichia coli, Staphylococcus epidermidis and Staphylococcus aureus respectively. Also, the MBC were extract in concentration of 63 and 125mg/ml on Staphylococcus epidermidis and Staphylococcus aureus respectively.Discussion and conclusion: Pseudomonas aeruginosa had high potential in reducing the surface tension and biosurfactant extracted had high antibacterial effects. Therefore, it

  5. Medium factors on anaerobic production of rhamnolipids by Pseudomonas aeruginosa SG and a simplifying medium for in situ microbial enhanced oil recovery applications.

    Science.gov (United States)

    Zhao, Feng; Zhou, Jidong; Han, Siqin; Ma, Fang; Zhang, Ying; Zhang, Jie

    2016-04-01

    Aerobic production of rhamnolipid by Pseudomonas aeruginosa was extensively studied. But effect of medium composition on anaerobic production of rhamnolipid by P. aeruginosa was unknown. A simplifying medium facilitating anaerobic production of rhamnolipid is urgently needed for in situ microbial enhanced oil recovery (MEOR). Medium factors affecting anaerobic production of rhamnolipid were investigated using P. aeruginosa SG (Genbank accession number KJ995745). Medium composition for anaerobic production of rhamnolipid by P. aeruginosa is different from that for aerobic production of rhamnolipid. Both hydrophobic substrate and organic nitrogen inhibited rhamnolipid production under anaerobic conditions. Glycerol and nitrate were the best carbon and nitrogen source. The commonly used N limitation under aerobic conditions was not conducive to rhamnolipid production under anaerobic conditions because the initial cell growth demanded enough nitrate for anaerobic respiration. But rhamnolipid was also fast accumulated under nitrogen starvation conditions. Sufficient phosphate was needed for anaerobic production of rhamnolipid. SO4(2-) and Mg(2+) are required for anaerobic production of rhamnolipid. Results will contribute to isolation bacteria strains which can anaerobically produce rhamnolipid and medium optimization for anaerobic production of rhamnolipid. Based on medium optimization by response surface methodology and ions composition of reservoir formation water, a simplifying medium containing 70.3 g/l glycerol, 5.25 g/l NaNO3, 5.49 g/l KH2PO4, 6.9 g/l K2HPO4·3H2O and 0.40 g/l MgSO4 was designed. Using the simplifying medium, 630 mg/l of rhamnolipid was produced by SG, and the anaerobic culture emulsified crude oil to EI24 = 82.5 %. The simplifying medium was promising for in situ MEOR applications.

  6. Production of extracellular lipases by Rhizopus oligosporus in a stirred fermentor

    OpenAIRE

    Iftikhar, Tehreema; Niaz, Mubashir; Zia, Muhammad Anjum; Haq, Ikram ul

    2010-01-01

    The present investigation deals with the kinetics of submerged extracellular lipases fermentation by both wild and mutant strains of Rhizopus oligosporus var. microsporus in a laboratory scale stirred fermentor. Other parameters studied were inoculum size, pH, agitation and rate of aeration. It was found that the growth and lipases production was increased gradually and reached its maximum 9.07± 0.42ª U mL-1 (W) and 42.49 ± 3.91ª U mL-1 (M) after 30h of fermentation for both wild and mutant s...

  7. Improvement in extracellular protease production by the marine antarctic yeast Rhodotorula mucilaginosa L7.

    Science.gov (United States)

    Chaud, Luciana C S; Lario, Luciana D; Bonugli-Santos, Rafaella C; Sette, Lara D; Pessoa Junior, Adalberto; Felipe, Maria das Graças de A

    2016-12-25

    Microorganisms from extreme and restrictive eco systems, such as the Antarctic continent, are of great interest due to their ability to synthesize products of commercial value. Among these, enzymes from psychrotolerant and psychrophilic microorganisms offer potential economical benefits due to their high activity at low and moderate temperatures. The cold adapted yeast Rhodotorula mucilaginosa L7 was selected out of 97 yeasts isolated from Antarctica as having the highest extracellular proteolytic activity in preliminary tests. The present study was aimed at evaluating the effects of nutrient composition (peptone, rice bran extract, ammonium sulfate, sodium chloride) and physicochemical parameters (temperature and pH) on its proteolytic activity. A 2 6-2 fractional factorial design experiment followed by a central composite design (CCD 2 3 ) was performed to optimize the culture conditions and improve the extracellular proteolytic activity. The results indicated that the presence of peptone in the medium was the most influential factor in protease production. Enzymatic activity was enhanced by the interaction between low glucose and peptone concentrations. The optimization of culture conditions with the aid of mathematical modeling enabled a c. 45% increase in proteolytic activity and at the same time reduced the amount of glucose and peptone required for the culture. Thus culture conditions established in this work may be employed in the biotechnological production of this protease. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix.

    Science.gov (United States)

    Friedman, Lisa; Kolter, Roberto

    2004-07-01

    Pseudomonas aeruginosa forms biofilms, which are cellular aggregates encased in an extracellular matrix. Molecular genetics studies of three common autoaggregative phenotypes, namely wrinkled colonies, pellicles, and solid-surface-associated biofilms, led to the identification of two loci, pel and psl, that are involved in the production of carbohydrate-rich components of the biofilm matrix. The pel gene cluster is involved in the production of a glucose-rich matrix material in P. aeruginosa strain PA14 (L. Friedman and R. Kolter, Mol. Microbiol. 51:675-690, 2004). Here we investigate the role of the pel gene cluster in P. aeruginosa strain ZK2870 and identify a second genetic locus, termed psl, involved in the production of a mannose-rich matrix material. The 11 predicted protein products of the psl genes are homologous to proteins involved in carbohydrate processing. P. aeruginosa is thus able to produce two distinct carbohydrate-rich matrix materials. Either carbohydrate-rich matrix component appears to be sufficient for mature biofilm formation, and at least one of them is required for mature biofilm formation in P. aeruginosa strains PA14 and ZK2870. Copyright 2004 American Society for Microbiology

  9. Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

    Directory of Open Access Journals (Sweden)

    Luyan Ma

    2009-03-01

    Full Text Available Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

  10. Design and synthesis of a biotinylated chemical probe for detecting the molecular targets of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin.

    Science.gov (United States)

    Baker, Ysobel R; Galloway, Warren R J D; Hodgkinson, James T; Spring, David R

    2013-09-25

    Pseudomonas aeruginosa is a human pathogen associated with a variety of life-threatening nosocomial infections. This organism produces a range of virulence factors which actively cause damage to host tissues. One such virulence factor is pyocyanin, known to play a crucial role in the pathogenesis of P. aeruginosa infections. Previous studies had identified a novel compound capable of strongly inhibiting the production of pyocyanin. It was postulated that this inhibition results from modulation of an intercellular communication system termed quorum sensing, via direct binding of the compound with the LasR protein receptor. This raised the possibility that the compound could be an antagonist of quorum sensing in P. aeruginosa, which could have important implications as this intercellular signaling mechanism is known to regulate many additional facets of P. aeruginosa pathogenicity. However, there was no direct evidence for the binding of the active compound to LasR (or any other targets). Herein we describe the design and synthesis of a biotin-tagged version of the active compound. This could potentially be used as an affinity-based chemical probe to ascertain, in a direct fashion, the active compound's macromolecular biological targets, and thus better delineate the mechanism by which it reduces the level of pyocyanin production.

  11. Extracellular methionine amino peptidase (MAP production by Streptomyces gedanensis in solid-state fermentation

    Directory of Open Access Journals (Sweden)

    Raji Rahulan

    2014-04-01

    Full Text Available A bioprocess was developed for extracellular MAP production from Streptomyces gedanensis by solid-state fermentation. Response surface methodology of Box Behken Design was performed to evaluate the interaction effects of most significant variables {inoculum size, (NH42SO4 concentration, MgSO4.7H2O and tryptone on MAP production after the single parameter optimization and it resulted a maximum MAP production of 55.26 IU/g PUF after 120 h of fermentation. The concentrated crude MAP displayed a pH and temperature optimum of 8.5 and 50°C. By analyzing the thermal stability, the MAP was found to be stable in a temperature range of 50 to 55°C but lost about 50% of its activity at 65°C after 30 min. This is a first report of this kind of study for MAP.

  12. Single Cell Responses to Spatially Controlled Photosensitized Production of Extracellular Singlet Oxygen

    DEFF Research Database (Denmark)

    Pedersen, Brian Wett; Sinks, Louise E.; Breitenbach, Thomas

    2011-01-01

    The response of individual HeLa cells to extracellularly produced singlet oxygen was examined. The spatial domain of singlet oxygen production was controlled using the combination of a membrane-impermeable Pd porphyrin-dendrimer, which served as a photosensitizer, and a focused laser, which served...... to localize the sensitized production of singlet oxygen. Cells in close proximity to the domain of singlet oxygen production showed morphological changes commonly associated with necrotic cell death. The elapsed post-irradiation “waiting period” before necrosis became apparent depended on (a) the distance...... between the cell membrane and the domain irradiated, (b) the incident laser fluence and, as such, the initial concentration of singlet oxygen produced, and (c) the lifetime of singlet oxygen. The data imply that singlet oxygen plays a key role in this process of light-induced cell death. The approach...

  13. Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques

    DEFF Research Database (Denmark)

    Woods, Alan A; Linton, Stuart M; Davies, Michael Jonathan

    2003-01-01

    Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has...... been interpreted in terms of the occurrence of two oxidative mechanisms, one involving oxygen-derived radicals catalysed by trace transition metal ions, and a second involving chlorinating species (HOCl or Cl2), generated by the haem enzyme myeloperoxidase (MPO). As MPO is released extracellularly...... for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions...

  14. Production of chelating agents by Pseudomonas aeruginosa grown in the presence of thorium and uranium

    International Nuclear Information System (INIS)

    Premuzic, E.T.; Lin, M.; Francis, A.J.; Schubert, J.

    1986-01-01

    Chelating agents produced by microorganisms enhance the dissolution of iron increasing the mobility and bioavailability of the metal. Since some similarities exist in the biological behavior of ferric, thorium and uranyl ions, microorganisms resistant to these metals and which grow in their presence may produce sequestering agents of Th and U, and other metals in a manner similar to the complexation of iron by siderophores. The ability of P. aeruginosa to elaborate sequestering agents in medium containing thorium or uranium salts was tested. Uranium has a stronger inhibitory effect on growth of the organism than thorium at similar concentrations. Analyses of the culture media have shown, that relative to the control, and under the experimental conditions used, the microorganisms have produced several new chelating agents for thorium and uranium. Extracts containing these chelating agents have been tested for their decorporation potential. In vitro mouse liver bioassay and in vivo mouse toxicity tests indicate that their efficiency is comparable to DTPA and DFOA and that they are virtually non-toxic to mice. The bacterially produced compounds resemble, but are not identical to the known iron chelating siderophores isolated from microorganisms. Some of their chemical properties are also discussed. (author)

  15. Biofilm production by clinical isolates of Pseudomonas aeruginosa and structural changes in LasR protein of isolates non biofilm-producing

    Directory of Open Access Journals (Sweden)

    Jailton Lobo da Costa Lima

    2018-03-01

    Full Text Available Introduction: Biofilm production is an important mechanism for the survival of Pseudomonas aeruginosa and its relationship with antimicrobial resistance represents a challenge for patient therapeutics. P. aeruginosa is an opportunistic pathogen frequently associated to nosocomial infections, especially in imunocompromised hosts. Objectives: Analyze the phenotypic biofilm production in P. aeruginosa isolates, describe clonal profiles, and analyze quorum sensing (QS genes and the occurrence of mutations in the LasR protein of non-biofilm producing isolates. Methods: Isolates were tested for biofilm production by measuring cells adherence to the microtiter plates. Clonal profile analysis was carried out through ERIC-PCR, QS genes were by specific PCR. Results: The results showed that 77.5% of the isolates were considered biofilm producers. The results of genotyping showed 38 distinct genetic profiles. As for the occurrence of the genes, 100% of the isolates presented the lasR, rhlI and rhlR genes, and 97.5%, presented the lasI gene. In this study nine isolates were not biofilm producers. However, all presented the QS genes. Amplicons related to genes were sequenced in three of the nine non-biofilm-producing isolates (all presenting different genetic similarity profile and aligned to the sequences of those genes in P. aeruginosa strain PAO1 (standard biofilm-producing strain. Alignment analysis showed an insertion of three nucleotides (T, C and G causing the addition of an amino acid valine in the sequence of the LasR protein, in position 53. Conclusion: The modeling of the resulting LasR protein showed a conformational change in its structure, suggesting that this might be the reason why these isolates are unable to produce biofilm. Keywords: Pseudomonas aeruginosa, Biofilm, Multiresistance, Quorum sensing (QS

  16. Extracellular laccase production and phenolic degradation by an olive mill wastewater isolate

    Directory of Open Access Journals (Sweden)

    R. Kumar

    2018-03-01

    Full Text Available Olive mill wastewater (OMWW presents a challenge to the control of effluents due to the presence of a high organic load, antimicrobial agents (monomeric-polymeric phenols, volatile acids, polyalcohols, and tannins, salinity and acidity. In this study, the production of extracellular laccase, monomeric or polymeric phenol, from an OMWW isolate based on its ability to biodegrade phenols and gallic acid as a model of phenolic compounds in OMWW was investigated. Phylogenetic analysis of the 16S RNA gene sequences identified the bacterial isolate (Acinetobacter REY as being closest to Acinetobacter pittii. This isolate exhibited a constitutive production of extracellular laccase with an activity of 1.5 and 1.3 U ml/L when supplemented with the inducers CuSO4 and CuSO4+phenols, respectively. Batch experiments containing minimal media supplemented with phenols or gallic acid as the sole carbon and energy source were performed in order to characterize their phenolic biodegradability. Acinetobacter REY was capable of biodegrading up to 200 mg/L of phenols and gallic acid both after 10 h and 72 h, respectively.

  17. Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Youngwoo Choi

    2017-01-01

    Full Text Available The hypothesis of autoimmune involvement in asthma has received much recent interest. Autoantibodies, such as anti-cytokeratin (CK 18, anti-CK19, and anti-α-enolase antibodies, react with self-antigens and are found at high levels in the sera of patients with severe asthma (SA. However, the mechanisms underlying autoantibody production in SA have not been fully determined. The present study was conducted to demonstrate that neutrophil extracellular DNA traps (NETs, cytotoxic molecules released from neutrophils, are a key player in the stimulation of airway epithelial cells (AECs to produce autoantigens. This study showed that NETs significantly increased the intracellular expression of tissue transglutaminase (tTG but did not affect that of CK18 in AECs. NETs induced the extracellular release of both tTG and CK18 in a concentration-dependent manner. Moreover, NETs directly degraded intracellular α-enolase into small fragments. However, antibodies against neutrophil elastase (NE or myeloperoxidase (MPO attenuated the effects of NETs on AECs. Furthermore, each NET isolated from healthy controls (HC, nonsevere asthma (NSA, and SA had different characteristics. Taken together, these findings suggest that AECs exposed to NETs may exhibit higher autoantigen production, especially in SA. Therefore, targeting of NETs may represent a new therapy for neutrophilic asthma with a high level of autoantigens.

  18. Optimization of environmental factors for improved production of rhamnolipid biosurfactant by Pseudomonas aeruginosa RS29 on glycerol.

    Science.gov (United States)

    Saikia, Rashmi Rekha; Deka, Suresh; Deka, Manab; Sarma, Hemen

    2012-08-01

    A biosurfactant producing Pseudomonas aeruginosa RS29 (identified on the basis of 16S rDNA analysis) with good foaming and emulsification properties has been isolated from crude oil contaminated sites. Optimization of different environmental factors was carried out with an objective to achieve maximum production of biosurfactant. Production of biosurfactant was estimated in terms of surface tension reduction and emulsification (E24) index. It was recorded that the isolated strain produced highest biosurfactant after 48 h of incubation at 37.5 °C, with a pH range of 7-8 and at salinity biosurfactant (Surface tension, 26.3 and 26.4 mN/m and E24 index, 80 and 79% respectively). The CMC of the biosurfactant was 90 mg/l. Maximum biomass (6.30 g/l) and biosurfactant production (0.80 g/l) were recorded at an optimal C/N ratio of 12.5. Biochemical analysis and FTIR spectra confirmed that the biosurfactant was rhamnolipid in nature. GC-MS analysis revealed the presence of C(8) and C(10) fatty acid components in the purified biosurfactant. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

    DEFF Research Database (Denmark)

    Jensen, T; Aliouat, E M; Lundgren, B

    1998-01-01

    The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide...... generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

  20. Characterization of an extracellular β-glucosidase from Dekkera bruxellensis for resveratrol production

    Directory of Open Access Journals (Sweden)

    Hsiao-Ping Kuo

    2018-01-01

    Full Text Available Polygonum cuspidatum is a widely grown crop with a rich source of polydatin (also called piceid for resveratrol production. Resveratrol is produced from piceid via enzymatic cleavage of the sugar moiety of piceid. In this study, Dekkera bruxellensis mutants were selected based on their high p-nitrophenyl-β-d-glucopyranoside and piceid conversion activities. The enzyme responsible for piceid conversion was a heterodimeric protein complex that was predominantly secreted to the extracellular medium and consisted of two subunits at an equal ratio with molecular masses of 30.5 kDa and 48.3 kDa. The two subunits were identified as SCW4p and glucan-β-glucosidase precursor in D. bruxellensis. Both proteins were individually expressed in Saccharomyces cerevisiae exg1Δ mutants, which lack extracellular β-glucosidase activity, to confirm each protein's enzymatic activities. Only the glucan-β-glucosidase precursor was shown to be a secretory protein with piceid deglycosylation activity. Our pilot experiments of piceid bioconversion demonstrate the possible industrial applications for this glucan-β-glucosidase precursor in the future.

  1. Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization

    International Nuclear Information System (INIS)

    Smith, J.B.; Dwyer, S.D.; Smith, L.

    1989-01-01

    Removing extracellular Na+ (Na+o) evoked a large increase in cytosolic free Ca2+ concentration ([Ca2+]i in human skin fibroblasts. Decreasing [Na+]o from 120 to 14 mM caused the half-maximal peak increase in [Ca2+]i. Removing Na+o strongly stimulated 45 Ca2+ efflux and decreased total cell Ca2+ by about 40%. Bradykinin caused changes in [Ca2+]i, total Ca2+, and 45 Ca2+ fluxes similar to those evoked by removing Na+o. Prior stimulation of the cells with bradykinin prevented Na+o removal from increasing [Ca2+]i and vice versa. Na+o removal rapidly increased [ 3 H]inositol polyphosphate production. Loading the cells with Na+ had no effect on the increase in 45 Ca2+ efflux produced by Na+o removal. Therefore, decreasing [Na+]o probably stimulates a receptor(s) which is sensitive to extracellular, not intracellular, Na+. Removing Na+o also mobilized intracellular Ca2+ in smooth muscle and endothelial cells cultured from human umbilical and dog coronary arteries, respectively

  2. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering.

    Science.gov (United States)

    El-Amin, S F; Lu, H H; Khan, Y; Burems, J; Mitchell, J; Tuan, R S; Laurencin, C T

    2003-03-01

    The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition. We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS. In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal

  3. The effect of sub-inhibitory concentrations of rifaximin on urease production and on other virulence factors expressed by Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus.

    Science.gov (United States)

    Ricci, Annalisa; Coppo, Erika; Barbieri, Ramona; Debbia, Eugenio A; Marchese, Anna

    2017-04-01

    Rifaximin, a topical derivative of rifampin, inhibited urease production and other virulence factors at sub-MIC concentrations in strains involved in hepatic encephalopathy and the expression of methicillin resistance in Staphylococcus aureus. In particular, urease production was affected in all Proteus mirabilis and Klebsiella pneumoniae strains as well as in all tested Pseudomonas aeruginosa isolates. Other exotoxins, synthesized by P. aeruginosa, such as protease, gelatinase, lipase, lecithinase and DNAse were also not metabolized in the presence of rifaximin. This antibiotic inhibited pigment production in both P. aeruginosa and Chromobacterium violaceum, a biosensor control strain. Lastly, rifaximin affected haemolysin production in S. aureus and was able to restore cefoxitin susceptibility when the strain was cultured in the presence of sub-MICs of the drug. The present findings confirm and extend previous observations about the beneficial effects of rifaximin for the treatment of gastrointestinal diseases, since in this anatomic site, it reaches a large array of concentrations which prevents enterobacteria from thriving and/or producing their major virulence factors.

  4. Extra-cellular isoamylase production by Rhizopus oryzae in solid-state fermentation of agro wastes

    Directory of Open Access Journals (Sweden)

    Barnita Ghosh

    2011-10-01

    Full Text Available Extra-cellular isoamylase was produced by Rhizopus oryzae PR7 in solid-state fermentations of various agro wastes, among which millet, oat, tapioca, and arum (Colocasia esculenta showed promising results. The highest amount of enzyme production was obtained after 72 h of growth at 28°C. The optimum pH for enzyme production was - 8.0. Among the various additives tested, enzyme production increased with ions such as Ca2+, Mg2+ and also with cysteine, GSH, and DTT. The enzyme synthesis was reduced in the presence of thiol inhibitors like Cu2+ and pCMB. The surfactants like Tween-40, Tween-80 and Triton X-100 helped in enhancing the enzyme activity. The production could be further increased by using the combinations of substrates. The ability to produce high amount of isoamylase within a relatively very short period and the capability of degrading wastes could make the strain suitable for commercial production of the enzyme.

  5. Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

    DEFF Research Database (Denmark)

    Christiansen, Torben; Nielsen, Jens

    2002-01-01

    The production of the extracellular alkaline protease Savinase(R) (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum...

  6. A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

    Science.gov (United States)

    Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

    2000-01-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

  7. NG2 proteoglycan increases mesangial cell proliferation and extracellular matrix production

    International Nuclear Information System (INIS)

    Xiong Jing; Wang Yang; Zhu, Zhonghua; Liu Jianshe; Wang Yumei; Zhang Chun; Hammes, Hans-Peter; Lang, Florian; Feng Yuxi

    2007-01-01

    As a membrane-spanning protein, NG2 chondroitin sulfate proteoglycan interacts with molecules on both sides of plasma membrane. The present study explored the role of NG2 in the pathogenesis of diabetic nephropathy. In the normal kidneys, NG2 was observed predominantly in glomerular mesangium, Bowman's capsule and interstitial vessels. Both mRNA and protein expression in kidneys was significantly higher in strepozotocin-induced diabetic rats than that in normal rats. In the cultured rat mesangial cell line HBZY-1, overexpression of NG2 promoted mesangial cell proliferation and extracellular matrix (ECM) production, such as type VI collagen and laminin. Furthermore, target knockdown of NG2 resulted in decreased cell proliferation and ECM formation. The observations suggest that NG2 is up-regulated in diabetic nephropathy. It actively participates in the development and progression of glomerulosclerosis by stimulating proliferation of mesangial cells and deposition of ECM

  8. Effect of eosinophils activated with Alternaria on the production of extracellular matrix from nasal fibroblasts.

    Science.gov (United States)

    Shin, Seung-Heon; Ye, Mi-Kyung; Choi, Sung-Yong; Kim, Yee-Hyuk

    2016-06-01

    Eosinophils and fibroblasts are known to play major roles in the pathogenesis of nasal polyps. Fungi are commonly found in nasal secretion and are associated with airway inflammation. To investigate whether activated eosinophils by airborne fungi can influence the production of extracellular matrix (ECM) from nasal fibroblasts. Inferior turbinate and nasal polyp fibroblasts were stimulated with Alternaria or Aspergillus, respectively, for 24 hours and ECM messenger RNA (mRNA) and protein expressions were measured. Eosinophils isolated from healthy volunteers were stimulated with Alternaria or Aspergillus for 4 hours then superoxide, eosinophil peroxidase, and transforming growth factor β1 were measured. Then activated eosinophils were cocultured with nasal fibroblasts for 24 hours, and ECM mRNA expressions were measured. Alternaria strongly enhanced ECM mRNA expression and protein production from nasal fibroblasts. Alternaria also induced the production of superoxide, eosinophil peroxidase, and transforming growth factor β1 from eosinophils, and activated eosinophils enhanced ECM mRNA expression when they were cocultured without the Transwell insert system. Eosinophils activated with Alternaria enhanced ECM mRNA expression from nasal polyp fibroblasts. Alternaria plays an important role in tissue fibrosis in the pathogenesis of nasal polyps by directly or indirectly influencing the production of ECM from nasal fibroblasts. Copyright © 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  9. Pseudomonas aeruginosa biofilm infections

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim

    2014-01-01

    Bacteria in natural, industrial and clinical settings predominantly live in biofilms, i.e., sessile structured microbial communities encased in self-produced extracellular matrix material. One of the most important characteristics of microbial biofilms is that the resident bacteria display...... a remarkable increased tolerance toward antimicrobial attack. Biofilms formed by opportunistic pathogenic bacteria are involved in devastating persistent medical device-associated infections, and chronic infections in individuals who are immune-compromised or otherwise impaired in the host defense. Because...... the use of conventional antimicrobial compounds in many cases cannot eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. The present review is focussed on the important opportunistic pathogen and biofilm model organism Pseudomonas aeruginosa. Initially...

  10. Production of polyol oils from soybean oil by Pseudomonas aeruginosa E03-12.

    Science.gov (United States)

    Soy-polyols are important starting materials for the manufacture of polymers such as polyurethane. We have been trying to develop a bioprocess for the production of polyol oils directly from soybean oil. We reported earlier the polyol products produced from soybean oil by Acinetobacter haemolyticus ...

  11. Enhancement of sludge reduction and methane production by removing extracellular polymeric substances from waste activated sludge.

    Science.gov (United States)

    Nguyen, Minh Tuan; Mohd Yasin, Nazlina Haiza; Miyazaki, Toshiki; Maeda, Toshinari

    2014-12-01

    The management of waste activated sludge (WAS) recycling is a concern that affects the development of the future low-carbon society, particularly sludge reduction and biomass utilization. In this study, we investigated the effect of removing extracellular polymeric substances (EPS), which play important roles in the adhesion and flocculation of WAS, on increased sludge disintegration, thereby enhancing sludge reduction and methane production by anaerobic digestion. EPS removal from WAS by ethylenediaminetetraacetic acid (EDTA) significantly enhanced sludge reduction, i.e., 49 ± 5% compared with 27 ± 1% of the control at the end the digestion process. Methane production was also improved in WAS without EPS by 8881 ± 109 CH4 μmol g(-1) dry-weight of sludge. Microbial activity was determined by denaturing gradient gel electrophoresis and real-time polymerase chain reaction, which showed that the hydrolysis and acetogenesis stages were enhanced by pretreatment with 2% EDTA, with a larger methanogenic community and better methane production. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Iron Oxide Nanoparticles Stimulates Extra-Cellular Matrix Production in Cellular Spheroids

    Directory of Open Access Journals (Sweden)

    Megan Casco

    2017-01-01

    Full Text Available Nanotechnologies have been integrated into drug delivery, and non-invasive imaging applications, into nanostructured scaffolds for the manipulation of cells. The objective of this work was to determine how the physico-chemical properties of magnetic nanoparticles (MNPs and their spatial distribution into cellular spheroids stimulated cells to produce an extracellular matrix (ECM. The MNP concentration (0.03 mg/mL, 0.1 mg/mL and 0.3 mg/mL, type (magnetoferritin, shape (nanorod—85 nm × 425 nm and incorporation method were studied to determine each of their effects on the specific stimulation of four ECM proteins (collagen I, collagen IV, elastin and fibronectin in primary rat aortic smooth muscle cell. Results demonstrated that as MNP concentration increased there was up to a 6.32-fold increase in collagen production over no MNP samples. Semi-quantitative Immunohistochemistry (IHC results demonstrated that MNP type had the greatest influence on elastin production with a 56.28% positive area stain compared to controls and MNP shape favored elastin stimulation with a 50.19% positive area stain. Finally, there are no adverse effects of MNPs on cellular contractile ability. This study provides insight on the stimulation of ECM production in cells and tissues, which is important because it plays a critical role in regulating cellular functions.

  13. Production of an extracellular thermohalophilic lipase from a moderately halophilic bacterium, Salinivibrio sp. strain SA-2.

    Science.gov (United States)

    Amoozegar, Mohammad Ali; Salehghamari, Ensieh; Khajeh, Khosro; Kabiri, Mahbube; Naddaf, Saied

    2008-06-01

    Fifty strains of moderately halophilic bacteria were isolated from various salty environments in Iran. A strain designated as SA-2 was shown to be the best producer of extracellular lipase and was selected for further studies. Biochemical and physiological characterization along with 16S rDNA sequence analysis placed SA-2 in the genus Salinivibrio. The optimum salt, pH, temperature and aeration for enzyme production were 0.1 M KCl, pH 8, 35 degrees C and 150 rpm, respectively. The enzyme production was synchronized bacterial growth and reached a maximum level during the early-stationary phase in the basal medium containing 1 M NaCl. Triacylglycerols enhanced lipase production, while carbohydrates had inhibitory effects on it. The maximum lipase activity was obtained at pH 7.5, 50 degrees C and CaCl(2) concentration of 0.01 M. The enzyme was stable at pH range of 7.5-8 and retained 90% of its activity at 80 degrees C for 30 min. Different concentrations of NaNO(3), Na(2)SO(4), KCl and NaCl had no affect on lipase stability for 3 h. These results suggest that the lipase secreted by Salinivibrio sp. strain SA-2 is industrially important from the perspective of its tolerance to a broad temperature range, its moderate thermoactivity and its high tolerance to a wide range of salt concentrations (0-3 M NaCl).

  14. Enhanced active extracellular polysaccharide production from Ganoderma formosanum using computational modeling.

    Science.gov (United States)

    Hsu, Kai-Di; Wu, Shu-Pei; Lin, Shin-Ping; Lum, Chi-Chin; Cheng, Kuan-Chen

    2017-10-01

    Extracellular polysaccharide (EPS) is one of the major bioactive ingredients contributing to the health benefits of Ganoderma spp. In this study, response surface methodology was applied to determine the optimal culture conditions for EPS production of Ganoderma formosanum. The optimum medium composition was found to be at initial pH 5.3, 49.2 g/L of glucose, and 4.9 g/L of yeast extract by implementing a three-factor-three-level Box-Behnken design. Under this condition, the predicted yield of EPS was up to 830.2 mg/L, which was 1.4-fold higher than the one from basic medium (604.5 mg/L). Furthermore, validating the experimental value of EPS production depicted a high correlation (100.4%) with the computational prediction response model. In addition, the percentage of β-glucan, a well-recognized bioactive polysaccharide, in EPS was 53±5.5%, which was higher than that from Ganoderma lucidum in a previous study. Moreover, results of monosaccharide composition analysis indicated that glucose was the major component of G. formosanum EPS, supporting a high β-glucan percentage in EPS. Taken together, this is the first study to investigate the influence of medium composition for G. formosanum EPS production as well as its β-glucan composition. Copyright © 2017. Published by Elsevier B.V.

  15. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

    Science.gov (United States)

    Laverty, Garry; Gorman, Sean P.; Gilmore, Brendan F.

    2014-01-01

    Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation. PMID:25438014

  16. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

    Directory of Open Access Journals (Sweden)

    Garry Laverty

    2014-07-01

    Full Text Available Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl, pellicle Formation (Pel and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

  17. Changes in muscle fiber contractility and extracellular matrix production during skeletal muscle hypertrophy.

    Science.gov (United States)

    Mendias, Christopher L; Schwartz, Andrew J; Grekin, Jeremy A; Gumucio, Jonathan P; Sugg, Kristoffer B

    2017-03-01

    Skeletal muscle can adapt to increased mechanical loads by undergoing hypertrophy. Transient reductions in whole muscle force production have been reported during the onset of hypertrophy, but contractile changes in individual muscle fibers have not been previously studied. Additionally, the extracellular matrix (ECM) stores and transmits forces from muscle fibers to tendons and bones, and determining how the ECM changes during hypertrophy is important in understanding the adaptation of muscle tissue to mechanical loading. Using the synergist ablation model, we sought to measure changes in muscle fiber contractility, collagen content, and cross-linking, and in the expression of several genes and activation of signaling proteins that regulate critical components of myogenesis and ECM synthesis and remodeling during muscle hypertrophy. Tissues were harvested 3, 7, and 28 days after induction of hypertrophy, and nonoverloaded rats served as controls. Muscle fiber specific force (sF o ), which is the maximum isometric force normalized to cross-sectional area, was reduced 3 and 7 days after the onset of mechanical overload, but returned to control levels by 28 days. Collagen abundance displayed a similar pattern of change. Nearly a quarter of the transcriptome changed over the course of overload, as well as the activation of signaling pathways related to hypertrophy and atrophy. Overall, this study provides insight into fundamental mechanisms of muscle and ECM growth, and indicates that although muscle fibers appear to have completed remodeling and regeneration 1 mo after synergist ablation, the ECM continues to be actively remodeling at this time point. NEW & NOTEWORTHY This study utilized a rat synergist ablation model to integrate changes in single muscle fiber contractility, extracellular matrix composition, activation of important signaling pathways in muscle adaption, and corresponding changes in the muscle transcriptome to provide novel insight into the basic

  18. High-level extracellular production and characterization of Candida antarctica lipase B in Pichia pastoris.

    Science.gov (United States)

    Eom, Gyeong Tae; Lee, Seung Hwan; Song, Bong Keun; Chung, Keun-Wo; Kim, Young-Wun; Song, Jae Kwang

    2013-08-01

    The gene encoding lipase B from Candida antarctica (CalB) was expressed in Pichia pastoris after it was synthesized by the recursive PCR and cloned into the Pichia expression plasmid, pPICZαA. The CalB was successfully secreted in the recombinant P. pastoris strain X-33 with an apparent molecular weight of 34 kDa. For 140 h flask culture, the dry cell weight and the extracellular lipase activity reached at 5.4 g/l and 57.9 U/l toward p-nitrophenyl palmitate, respectively. When we performed the fed-batch fermentation using a methanol feeding strategy for 110 h, the dry cell weight and the extracellular lipase activity were increased to 135.7 g/l and 11,900 U/l; the CalB protein concentration was 1.18 g/l of culture supernatant. The characteristics of CalB recovered from the P. pastoris culture were compared with the commercial form of CalB produced in Aspergillus oryzae. The kinetic constants and specific activity, the effects of activity and stability on temperature and pH, the glycosylation extent, the degree of immobilization on macroporous resin and the yield of esterification reaction between oleic acid and n-butanol were almost identical to each other. Therefore, we successfully proved that the Pichia-based expression system for CalB in this study was industrially promising compared with one of the most efficient production systems. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. The Crc protein participates in down-regulation of the Lon gene to promote rhamnolipid production and rhl quorum sensing in Pseudomonas aeruginosa.

    Science.gov (United States)

    Yang, Nana; Ding, Shuting; Chen, Feifei; Zhang, Xue; Xia, Yongjie; Di, Hongxia; Cao, Qiao; Deng, Xin; Wu, Min; Wong, Catherine C L; Tian, Xiao-Xu; Yang, Cai-Guang; Zhao, Jing; Lan, Lefu

    2015-05-01

    Rhamnolipid acts as a virulence factor during Pseudomonas aeruginosa infection. Here, we show that deletion of the catabolite repression control (crc) gene in P. aeruginosa leads to a rhamnolipid-negative phenotype. This effect is mediated by the down-regulation of rhl quorum sensing (QS). We discover that a disruption of the gene encoding the Lon protease entirely offsets the effect of crc deletion on the production of both rhamnolipid and rhl QS signal C4-HSL. Crc is unable to bind lon mRNA in vitro in the absence of the RNA chaperon Hfq, while Crc contributes to Hfq-mediated repression of the lon gene expression at a posttranscriptional level. Deletion of crc, which results in up-regulation of lon, significantly reduces the in vivo stability and abundance of the RhlI protein that synthesizes C4-HSL, causing the attenuation of rhl QS. Lon is also capable of degrading the RhlI protein in vitro. In addition, constitutive expression of rhlI suppresses the defects of the crc deletion mutant in rhamnolipid, C4-HSL and virulence on lettuce leaves. This study therefore uncovers a novel posttranscriptional regulatory cascade, Crc-Hfq/Lon/RhlI, for the regulation of rhamnolipid production and rhl QS in P. aeruginosa. © 2015 John Wiley & Sons Ltd.

  20. Utilization of Crude Glycerol as a Substrate for the Production of Rhamnolipid by Pseudomonas aeruginosa

    OpenAIRE

    Eraqi, Walaa A.; Yassin, Aymen S.; Ali, Amal E.; Amin, Magdy A.

    2016-01-01

    Biosurfactants are produced by bacteria or yeast utilizing different substrates as sugars, glycerol, or oils. They have important applications in the detergent, oil, and pharmaceutical industries. Glycerol is the product of biodiesel industry and the existing glycerol market cannot accommodate the excess amounts generated; consequently, new markets for refined glycerol need to be developed. The aim of present work is to optimize the production of microbial rhamnolipid using waste glycerol. We...

  1. Physiological responses and toxin production of Microcystis aeruginosa in short-term exposure to solar UV radiation.

    Science.gov (United States)

    Hernando, Marcelo; Minaglia, Melina Celeste Crettaz; Malanga, Gabriela; Houghton, Christian; Andrinolo, Darío; Sedan, Daniela; Rosso, Lorena; Giannuzzi, Leda

    2018-01-17

    The aim of this study was to evaluate the effects of short-term (hours) exposure to solar UV radiation (UVR, 280-400 nm) on the physiology of Microcystis aeruginosa. Three solar radiation treatments were implemented: (i) PAR (PAR, 400-700 nm), (ii) TUVA (PAR + UVAR, 315-700 nm) and (iii) TUVR (PAR + UVAR + UVBR, 280-700 nm). Differential responses of antioxidant enzymes and the reactive oxygen species (ROS) production to UVR were observed. Antioxidant enzymes were more active at high UVR doses. However, different responses were observed depending on the exposure to UVAR or UVBR and the dose level. No effects were observed on the biomass, ROS production or increased activity of superoxide dismutase (SOD) and catalase (CAT) compared to the control when UVR + PAR doses were lower than 9875 kJ m -2 . For intermediate doses, UVR + PAR doses between 9875 and 10 275 kJ m -2 , oxidative stress increased while resistance was imparted through SOD and CAT in the cells exposed to UVAR. Despite the increased antioxidant activity, biomass decrease and photosynthesis inhibition were observed, but no effects were observed with added exposure to UVBR. At the highest doses (UVR + PAR higher than 10 275 kJ m -2 ), the solar UVR caused decreased photosynthesis and biomass with only activation of CAT by UVBR and SOD and CAT by UVAR. In addition, for such doses, a significant decrease of microcystins (MCs, measured as MC-LR equivalents) was observed as a consequence of UVAR. This study facilitates our understanding of the SOD and CAT protection according to UVAR and UVBR doses and cellular damage and reinforces the importance of UVR as an environmental stressor. In addition, our results support the hypothesized antioxidant function of MCs.

  2. Extracellular Xylanolytic and Pectinolytic Hydrolase Production by Aspergillus flavus Isolates Contributes to Crop Invasion

    Directory of Open Access Journals (Sweden)

    Jay E. Mellon

    2015-08-01

    Full Text Available Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates.

  3. Eosinophils Regulate Interferon Alpha Production in Plasmacytoid Dendritic Cells Stimulated with Components of Neutrophil Extracellular Traps.

    Science.gov (United States)

    Skrzeczynska-Moncznik, Joanna; Zabieglo, Katarzyna; Bossowski, Jozef P; Osiecka, Oktawia; Wlodarczyk, Agnieszka; Kapinska-Mrowiecka, Monika; Kwitniewski, Mateusz; Majewski, Pawel; Dubin, Adam; Cichy, Joanna

    2017-03-01

    Eosinophils constitute an important component of helminth immunity and are not only associated with various allergies but are also linked to autoinflammatory disorders, including the skin disease psoriasis. Here we demonstrate the functional relationship between eosinophils and plasmacytoid dendritic cells (pDCs) as related to skin diseases. We previously showed that pDCs colocalize with neutrophil extracellular traps (NETs) in psoriatic skin. Here we demonstrate that eosinophils are found in psoriatic skin near neutrophils and NETs, suggesting that pDC responses can be regulated by eosinophils. Eosinophils inhibited pDC function in vitro through a mechanism that did not involve cell contact but depended on soluble factors. In pDCs stimulated by specific NET components, eosinophil-conditioned media attenuated the production of interferon α (IFNα) but did not affect the maturation of pDCs as evidenced by the unaltered expression of the costimulatory molecules CD80 and CD86. As pDCs and IFNα play a key role in autoimmune skin inflammation, these data suggest that eosinophils may influence autoinflammatory responses through their impact on the production of IFNα by pDCs.

  4. Solid state fermentation for extracellular polysaccharide production by Lactobacillus confusus with coconut water and sugar cane juice as renewable wastes.

    Science.gov (United States)

    Seesuriyachan, Phisit; Techapun, Charin; Shinkawa, Hidenori; Sasaki, Ken

    2010-01-01

    Extracellular polysaccharide (EPS) production by Lactobacillus confusus in liquid and solid state fermentation was carried out using coconut water and sugarcane juice as renewable wastes. High concentrations of EPS of 62 (sugarcane juice) and 18 g/l of coconut water were produced in solid state fermentation when nitrogen sources were reduced 5-fold from the original medium.

  5. Production of rhamnolipids by Pseudomonas aeruginosa is inhibited by H2S but resumes in a co-culture with P. stutzeri: applications for microbial enhanced oil recovery.

    Science.gov (United States)

    Zhao, Feng; Ma, Fang; Shi, Rongjiu; Zhang, Jie; Han, Siqin; Zhang, Ying

    2015-09-01

    Sulfate-reducing bacteria and H2S exist widely in oil production systems, and in situ production of rhamnolipids is promising for microbial enhanced oil recovery (MEOR). However, information of the effect of S(2-) on rhamnolipids production is scarce. Two facultative anaerobic rhamnolipids-producing bacterial strains, Pseudomonas aeruginosa SG and WJ-1, were used. Above 10 mg S(2-)/l, both cell growth and rhamnolipids production were inhibited. A large inoculum (9%, v/v) failed to completely relieve the inhibitory effect of 10 mg S(2-)/l. Below 30 mg S(2-)/l, both strains resumed rhamnolipid production through co-culturing with the denitrifying and sulphide-removing strain Pseudomonas stutzeri DQ1. H2S has a direct but reversible inhibitory effect on rhamnolipids production. Control of H2S in oilfields is indispensable to MEOR, and the co-culture method is effective in restoring rhamnolipid production in presence of S(2-).

  6. Mesenchymal stromal cells improve human islet function through released products and extracellular matrix.

    Science.gov (United States)

    Arzouni, Ahmed A; Vargas-Seymour, Andreia; Rackham, Chloe L; Dhadda, Paramjeet; Huang, Guo-Cai; Choudhary, Pratik; Nardi, Nance; King, Aileen J F; Jones, Peter M

    2017-12-01

    The aims of the present study were (i) to determine whether the reported beneficial effects of mesenchymal stromal cells (MSCs) on mouse islet function extend to clinically relevant human tissues (islets and MSCs), enabling translation into improved protocols for clinical human islet transplantation; and (ii) to identify possible mechanisms through which human MSCs influence human islet function. Human islets were co-cultured with human adipose tissue-derived MSCs (hASCs) or pre-treated with its products - extracellular matrix (ECM) and annexin A1 (ANXA1). Mouse islets were pre-treated with mouse MSC-derived ECM. Islet insulin secretory function was assessed in vitro by radioimmunoassay. Quantitative RT-PCR was used to screen human adipMSCs for potential ligands of human islet G-protein-coupled receptors. We show that co-culture with hASCs improves human islet secretory function in vitro , as measured by glucose-stimulated insulin secretion, confirming previous reports using rodent tissues. Furthermore, we demonstrate that these beneficial effects on islet function can be partly attributed to the MSC-derived products ECM and ANXA1. Our results suggest that hASCs have the potential to improve the quality of human islets isolated for transplantation therapy of Type 1 diabetes. Furthermore, it may be possible to achieve improvements in human islet quality in a cell-free culture system by using the MSC-derived products ANXA1 and ECM. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  7. Facultative control of matrix production optimizes competitive fitness in Pseudomonas aeruginosa PA14 biofilm models

    DEFF Research Database (Denmark)

    Madsen, Jonas Stenløkke; Lin, Yu Cheng; Squyres, Georgia R.

    2015-01-01

    response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage...... in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation...... to the colony edge produced mutants with clear competitive advantages against the wild type in this O2-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure...

  8. Characterization of Pseudomonas aeruginosa Chitinase, a Gradually Secreted Protein

    NARCIS (Netherlands)

    Folders, J. (Jindra); Algra, J. (Jon); Roelofs, M.S. (Marc); Loon, L.C. van; Tommassen, J.P.M.; Bitter, Wilbert

    2001-01-01

    The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino

  9. Inactivation of the rhlA gene in Pseudomonas aeruginosa prevents rhamnolipid production, disabling the protection against polymorphonuclear leukocytes

    DEFF Research Database (Denmark)

    van Gennip, Maria; Christensen, Louise Dahl; Alhede, Morten

    2009-01-01

    Many of the virulence factors produced by the opportunistic human pathogen Pseudomonas aeruginosa are quorum-sensing (QS) regulated. Among these are rhamnolipids, which have been shown to cause lysis of several cellular components of the human immune system, e.g. monocyte-derived macrophages and ...

  10. Production of N-acyl-L-homoserine lactones by P. aeruginosa isolates from chronic lung infections associated with cystic fibrosis

    DEFF Research Database (Denmark)

    Geisenberger, O; Givskov, M; Riedel, K

    2000-01-01

    The N-acyl-L-homoserine lactones (AHLs) produced by sequential Pseudomonas aeruginosa isolates from chronically infected patients with cystic fibrosis were analyzed by thin-layer chromatography. It is demonstrated that both the amounts and the types of molecules synthesized by isolates from...

  11. Lipolytic Potential of Aspergillus japonicus LAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

    Science.gov (United States)

    Souza, Lívia Tereza Andrade; Oliveira, Jamil S.; dos Santos, Vera L.; Regis, Wiliam C. B.; Santoro, Marcelo M.; Resende, Rodrigo R.

    2014-01-01

    Lipolytic potential of Aspergillus japonicus LAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu2+, Ni2+, and Al3+ showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. The KM and V max values of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications. PMID:25530954

  12. Evaluation of extracellular products and mutagenicity in cyanobacteria cultures separated from a eutrophic reservoir

    International Nuclear Information System (INIS)

    Huang, W.-J.; Lai, C.-H.; Cheng, Y.-L.

    2007-01-01

    The algal extracellular products (ECPs) in three cultures of cyanobacteria species (Anabaena, Microcystis, and Oscillatoria) dominating the eutrophic reservoir populations and their toxins have been investigated in the present work. Using gas chromatography coupled with high-resolution electron-impact mass spectrometry (GC/EI-MS) and high performance anion-exchange chromatography (HPAEC) techniques, more than 20 compounds were found in the algal culture (including cells and filtrates) extracts. The main identified ECPs were classified to polysaccharides, hydrocarbons, and aldehydes. Odor causing substances such as trans-1,10-dimethyl-trans-9-decalol (geosmin) and 2-methylisoborneol (2-MIB)were also found in the algal cultures. The potential mutagenicity of the algal suspensions was also studied with the Ames test. The organic extracts of the algal suspension from the axenic cultures were mutagenicity in TA98 without S9 mix and in TA100 with and without S9 mix. The results indicate that the ECPs of three algae species dominating the eutrophic reservoir were mutagenic clearly in the bacterial test

  13. Extracellular microvesicle production by human eosinophils activated by “inflammatory” stimuli

    Directory of Open Access Journals (Sweden)

    Praveen Akuthota

    2016-10-01

    Full Text Available A key function of human eosinophils is to secrete cytokines, chemokines and cationic proteins, trafficking and releasing these mediators for roles in inflammation and other immune responses. Eosinophil activation leads to secretion of pre-synthesized granule-stored mediators through different mechanisms, but the ability of eosinophils to secrete extracellular vesicles (EVs, very small vesicles with preserved membrane topology, is still poorly understood. In the present work, we sought to identify and characterize EVs released from human eosinophils during different conditions: after a culturing period or after isolation and stimulation with inflammatory stimuli, which are known to induce eosinophil activation and secretion: CCL11 (eotaxin-1 and tumor necrosis factor alpha (TNF-α. EV production was investigated by nanoscale flow cytometry, conventional transmission electron microscopy (TEM and pre-embedding immunonanogold EM. The tetraspanins CD63 and CD9 were used as EV biomarkers for both flow cytometry and ultrastructural immunolabeling. Nanoscale flow cytometry showed that human eosinophils produce EVs in culture and that a population of EVs expressed detectable CD9, while CD63 was not consistently detected. When eosinophils were stimulated immediately after isolation and analyzed by TEM, EVs were clearly identified as microvesicles (MVsoutwardly budding off the plasma membrane. Both CCL11 and TNF-α induced significant increases of MVs compared to unstimulated cells.TNF-α induced amplified release of MVs more than CCL11. Eosinophil MV diameters varied from 20-1000 nm. Immunonanogold EM revealed clear immunolabeling for CD63 and CD9 on eosinophil MVs, although not all MVs were labeled. Altogether, we identified, for the first time, that human eosinophils secrete MVs and that this production increases in response to inflammatory stimuli. This is important to understand the complex secretory activities of eosinophils underlying immune

  14. Optimization of the production of extracellular α-amylase by Kluyveromyces marxianus IF0 0288 by response surface methodology

    Directory of Open Access Journals (Sweden)

    Panagiota-Yiolanda Stergiou

    2014-06-01

    Full Text Available The aim of this work was to study the production of extracellular α-amylase by Kluyveromyces marxianus IF0 0288 using optimized nutritional and cultural conditions in a complex yeast medium under aerobic batch fermentation. By applying the conventional "one-variable-at-a-time" approach and the response surface methodology, the effect of four fermentation parameters (type of carbon source, initial culture pH, temperature, and incubation time on the growth and α-amylase production was evaluated. The production of α-amylase during 60 h of fermentation increased 13-fold under optimized conditions (1% starch, pH 6.0, 30ºC in comparison to the conventional optimization method. The initial pH value of 6.13 and temperature of 30.3ºC were optimal conditions by the response surface methodology, leading to further improvement (up to 13-fold in the production of extracellular α-amylase. These results constituted first evidence that K. marxianus could be potentially used as an effective source of extracellular α-amylase.

  15. The exit strategy: Pharmacological modulation of extracellular matrix production and deposition for better aqueous humor drainage.

    Science.gov (United States)

    Pattabiraman, Padmanabhan P; Toris, Carol B

    2016-09-15

    Primary open angle glaucoma (POAG) is an optic neuropathy and an irreversible blinding disease. The etiology of glaucoma is not known but numerous risk factors are associated with this disease including aging, elevated intraocular pressure (IOP), race, myopia, family history and use of steroids. In POAG, the resistance to the aqueous humor drainage is increased leading to elevated IOP. Lowering the resistance and ultimately the IOP has been the only way to slow disease progression and prevent vision loss. The primary drainage pathway comprising of the trabecular meshwork (TM) is made up of relatively large porous beams surrounded by extracellular matrix (ECM). Its juxtacanalicular tissue (JCT) or the cribriform meshwork is made up of cells embedded in dense ECM. The JCT is considered to offer the major resistance to the aqueous humor outflow. This layer is adjacent to the endothelial cells forming Schlemm's canal, which provides approximately 10% of the outflow resistance. The ECM in the TM and the JCT undergoes continual remodeling to maintain normal resistance to aqueous humor outflow. It is believed that the TM is a major contributor of ECM proteins and evidence points towards increased ECM deposition in the outflow pathway in POAG. It is not clear how and from where the ECM components emerge to hinder the normal aqueous humor drainage. This review focuses on the involvement of the ECM in ocular hypertension and glaucoma and the mechanisms by which various ocular hypotensive drugs, both current and emerging, target ECM production, remodeling, and deposition. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo.

    Science.gov (United States)

    Westman, Johannes; Papareddy, Praveen; Dahlgren, Madelene W; Chakrakodi, Bhavya; Norrby-Teglund, Anna; Smeds, Emanuel; Linder, Adam; Mörgelin, Matthias; Johansson-Lindbom, Bengt; Egesten, Arne; Herwald, Heiko

    2015-12-01

    The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.

  17. Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

    International Nuclear Information System (INIS)

    Matsuzaki, Shinichi; Ishizuka, Tamotsu; Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo; Komachi, Mayumi; Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko; Dobashi, Kunio; Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki; Mori, Masatomo; Okajima, Fumikazu

    2011-01-01

    Highlights: → The involvement of extracellular acidification in airway remodeling was investigated. → Extracellular acidification alone induced CTGF production in human ASMCs. → Extracellular acidification enhanced TGF-β-induced CTGF production in human ASMCs. → Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. → OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-β-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G q/11 protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP 3 ) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G q/11 protein and inositol-1,4,5-trisphosphate-induced Ca 2+ mobilization in human ASMCs.

  18. Two schemes for production of biosurfactant from Pseudomonas aeruginosa MR01: Applying residues from soybean oil industry and silica sol-gel immobilized cells.

    Science.gov (United States)

    Bagheri Lotfabad, Tayebe; Ebadipour, Negisa; Roostaazad, Reza; Partovi, Maryam; Bahmaei, Manochehr

    2017-04-01

    Rhamnolipids are the most common biosurfactants and P. aeruginosa strains are the most frequently studied microorganisms for the production of rhamnolipids. Eco-friendly advantages and promising applications of rhamnolipids in various industries are the major reasons for pursuing the economic production of these biosurfactants. This study shows that cultivation of P. aeruginosa MR01 in medium contained inexpensive soybean oil refinery wastes which exhibited similar levels and homologues of rhamnolipids. Mass spectrometry indicated that the Rha-C10-C10 and Rha-Rha-C10-C10 constitute the main rhamnolipids in different cultures of MR01 including one of oil carbon source analogues. Moreover, rhamnolipid mixtures extracted from different cultures showed critical micelle concentrations (CMC) in the range of ≃24 to ≃36mg/l with capability to reduce the surface tension of aqueous solution from 72 to ≃27-32mN/m. However, the sol-gel technique using tetraethyl orthosilicate (TEOS) was used as a gentler method in order to entrap the P. aeruginosa MR01 cells in mold silica gels. Immobilized cells can be utilized several times in consecutive fermentation batches as well as in flow fermentation processes. In this way, reusability of the cells may lead to a more economical fermentation process. Approximately 90% of cell viability was retained during the silica sol-gel immobilization and ≃84% of viability of immobilized cells was preserved for 365days of immobilization and storage of the cells in phosphate buffer at 4°C and 25°C. Moreover, mold gels showed good mechanical stability during the seven successive fermentation batches and the entrapped cells were able to efficiently preserve their biosurfactant-producing potential. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. HIF-1? Is Essential for Effective PMN Bacterial Killing, Antimicrobial Peptide Production and Apoptosis in Pseudomonas aeruginosa Keratitis

    OpenAIRE

    Berger, Elizabeth A.; McClellan, Sharon A.; Vistisen, Kerry S.; Hazlett, Linda D.

    2013-01-01

    Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with Pseudomonas (P.) aeruginosa. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antag...

  20. Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

    Science.gov (United States)

    Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I

    2015-03-04

    Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.

  1. Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeast Rhodotorula mucilaginosa L7.

    Science.gov (United States)

    Lario, Luciana Daniela; Chaud, Luciana; Almeida, María das Graças; Converti, Attilio; Durães Sette, Lara; Pessoa, Adalberto

    2015-11-01

    The production, purification, and characterization of an extracellular protease released by Rhodotorula mucilaginosa L7 were evaluated in this study. This strain was isolated from an Antarctic marine alga and previously selected among others based on the capacity to produce the highest extracellular proteolytic activity in preliminary tests. R. mucilaginosa L7 was grown in Saboraud-dextrose medium at 25 °C, and the cell growth, pH of the medium, extracellular protease production and the glucose and protein consumption were determined as a function of time. The protease was then purified, and the effects of pH, temperature, and salt concentration on the catalytic activity and enzyme stability were determined. Enzyme production started at the beginning of the exponential phase of growth and reached a maximum after 48 h, which was accompanied by a decrease in the pH as well as reductions of the protein and glucose concentrations in the medium. The purified protease presented optimal catalytic activity at pH 5.0 and 50 °C. Finally, the enzyme was stable in the presence of high concentrations of NaCl. These characteristics are of interest for future studies and may lead to potential biotechnological applications that require enzyme activity and stability under acidic conditions and/or high salt concentrations. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  2. Does Extracellular DNA Production Vary in Staphylococcal Biofilms Isolated From Infected Implants versus Controls?

    Science.gov (United States)

    Zatorska, Beata; Groger, Marion; Moser, Doris; Diab-Elschahawi, Magda; Lusignani, Luigi Segagni; Presterl, Elisabeth

    2017-08-01

    Prosthetic implant infections caused by Staphylococcus aureus and epidermidis are major challenges for early diagnosis and treatment owing to biofilm formation on the implant surface. Extracellular DNA (eDNA) is actively excreted from bacterial cells in biofilms, contributing to biofilm stability, and may offer promise in the detection or treatment of such infections. (1) Does DNA structure change during biofilm formation? (2) Are there time-dependent differences in eDNA production during biofilm formation? (3) Is there differential eDNA production between clinical and control Staphylococcal isolates? (4) Is eDNA production correlated to biofilm thickness? We investigated eDNA presence during biofilm formation in 60 clinical and 30 control isolates of S aureus and S epidermidis. The clinical isolates were isolated from patients with infections of orthopaedic prostheses and implants: 30 from infected hip prostheses and 30 from infected knee prostheses. The control isolates were taken from healthy volunteers who had not been exposed to antibiotics and a hospital environment during the previous 3 and 12 months, respectively. Control S epidermidis was isolated from the skin of the antecubital fossa, and control S aureus was isolated from the nares. For the biofilm experiments the following methods were used to detect eDNA: (1) fluorescent staining with 4',6-diamidino-2-phenylindole (DAPI), (2) eDNA extraction using a commercial kit, and (3) confocal laser scanning microscopy for 24-hour biofilm observation using propidium iodide and concanavalin-A staining; TOTO ® -1 and SYTO ® 60 staining were used for observation and quantification of eDNA after 6 and 24 hours of biofilm formation. Additionally antibiotic resistance was described. eDNA production as observed by confocal laser scanning microscopy was greater in clinical isolates than controls (clinical isolates mean ± SD: 1.84% ± 1.31%; control mean ± SD: 1.17% ± 1.37%; p biofilm formation. After 24 hours, the

  3. Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

    Science.gov (United States)

    Spengler, Julia; Lugonja, Božo; Jimmy Ytterberg, A.; Zubarev, Roman A.; Creese, Andrew J.; Pearson, Mark J.; Grant, Melissa M.; Milward, Michael; Lundberg, Karin; Buckley, Christopher D.; Filer, Andrew; Raza, Karim; Cooper, Paul R.; Chapple, Iain L.

    2015-01-01

    Objective In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA–protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Results Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA. PMID:26245941

  4. Heterologous co-expression of accA, fabD, and thioesterase genes for improving long-chain fatty acid production in Pseudomonas aeruginosa and Escherichia coli.

    Science.gov (United States)

    Lee, Sunhee; Jeon, Eunyoung; Jung, Yeontae; Lee, Jinwon

    2012-05-01

    The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl-acyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3-1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli.

  5. A functional genomics study of extracellular protease production by Aspergillus niger

    NARCIS (Netherlands)

    Braaksma, Machtelt

    2010-01-01

    The objective of the project described in this thesis was to study the complex induction of extracellular proteases in the filamentous fungus Aspergillus niger using information gathered with functional genomics technologies. A special emphasis is given to the requirements for performing a

  6. Optimization of Cultural Conditions for Production of Extracellular Polymeric Substances (EPS) by Serpentine Rhizobacterium Cupriavidus pauculus KPS 201

    OpenAIRE

    Arundhati Pal; A. K. Paul

    2013-01-01

    Extracellular polymeric substances (EPS) are complex biopolymers produced by a wide array of microorganisms for protection against dessication, aggregation, adhesion, and expression of virulence. Growth associated production of EPS by Ni-resistant Cupriavidus pauculus KPS 201 was determined in batch culture using sodium gluconate as the sole carbon source. The optimum pH and temperature for EPS production were 6.5 and 25°C, respectively. Optimal EPS yield (118 μg/mL) was attained at 0.35% Na-...

  7. Extracellular Polysaccharide Production in a Scytonemin-Deficient Mutant of Nostoc punctiforme Under UVA and Oxidative Stress.

    Science.gov (United States)

    Soule, Tanya; Shipe, Dexter; Lothamer, Justin

    2016-10-01

    Some cyanobacteria can protect themselves from ultraviolet radiation by producing sunscreen pigments. In particular, the sheath pigment scytonemin protects cells against long-wavelength UVA radiation and is only found in cyanobacteria which are capable of extracellular polysaccharide (EPS) production. The presence of a putative glycosyltransferase encoded within the scytonemin gene cluster, along with the localization of scytonemin and EPS to the extracellular sheath, prompted us to investigate the relationship between scytonemin and EPS production under UVA stress. In this study, it was hypothesized that there would be a relationship between the biosynthesis of scytonemin and EPS under both UVA and oxidative stress, since the latter is a by-product of UVA radiation. EPS production was measured following exposure of wild-type Nostoc punctiforme and the non-scytonemin-producing strain SCY59 to UVA and oxidative stress. Under UVA, SCY59 produced significantly more EPS than the unstressed controls and the wild type, while both strains produced more EPS under oxidative stress compared to the controls. The results suggest that EPS secretion occurs in response to the oxidative stress by-product of UVA rather than as a direct response to UVA radiation.

  8. A functional genomics study of extracellular protease production by Aspergillus niger

    OpenAIRE

    Braaksma, Machtelt

    2010-01-01

    The objective of the project described in this thesis was to study the complex induction of extracellular proteases in the filamentous fungus Aspergillus niger using information gathered with functional genomics technologies. A special emphasis is given to the requirements for performing a successful systems biology study and addressing the challenges met in analyzing the large, information-rich data sets generated with functional genomics technologies. The role that protease activity plays i...

  9. Phosphate starvation triggers production and secretion of an extracellular lipoprotein in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Sophie Le Blastier

    Full Text Available Life in oligotrophic environments necessitates quick adaptive responses to a sudden lack of nutrients. Secretion of specific degradative enzymes into the extracellular medium is a means to mobilize the required nutrient from nearby sources. The aquatic bacterium Caulobacter crescentus must often face changes in its environment such as phosphate limitation. Evidence reported in this paper indicates that under phosphate starvation, C. crescentus produces a membrane surface-anchored lipoprotein named ElpS subsequently released into the extracellular medium. A complete set of 12 genes encoding a type II secretion system (T2SS is located adjacent to the elpS locus in the C. crescentus genome. Deletion of this T2SS impairs release of ElpS in the environment, which surprisingly remains present at the cell surface, indicating that the T2SS is not involved in the translocation of ElpS to the outer membrane but rather in its release. Accordingly, treatment with protease inhibitors prevents release of ElpS in the extracellular medium suggesting that ElpS secretion relies on a T2SS-secreted protease. Finally, secretion of ElpS is associated with an increase in alkaline phosphatase activity in culture supernatants, suggesting a role of the secreted protein in inorganic phosphate mobilization. In conclusion, we have shown that upon phosphate starvation, C. crescentus produces an outer membrane bound lipoprotein, ElpS, which is further cleaved and released in the extracellular medium in a T2SS-dependent manner. Our data suggest that ElpS is associated with an alkaline phosphatase activity, thereby allowing the bacterium to gather inorganic phosphates from a poor environment.

  10. High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.

    Science.gov (United States)

    Zhang, Kang; Su, Lingqia; Duan, Xuguo; Liu, Lina; Wu, Jing

    2017-02-20

    We recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, P amyQ from Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536. Using the enzyme β-cyclodextrin glycosyltransferase (β-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the P amyQ' promoter produced the greatest extracellular β-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter P HpaII -P amyQ' produced the highest extracellular β-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter P HpaII -P amyQ' also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of β-CGTase using the dual-promoter P HpaII -P amyQ' system was investigated in a 3-L fermenter. Extracellular expression of β-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications. The dual-promoter P HpaII -P amyQ' system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.

  11. Inhibition of Quorum Sensing-Controlled Virulence Factor Production in Pseudomonas aeruginosa PAO1 by Ayurveda Spice Clove (Syzygium Aromaticum Bud Extract

    Directory of Open Access Journals (Sweden)

    Kok-Gan Chan

    2012-03-01

    Full Text Available Quorum sensing controls the virulence determinants in most proteobacteria. In this work, the hexane, chloroform and methanol extracts of an Ayurveda spice, namely clove (Syzygium aromaticum, shown anti-quorum sensing activity. Hexane and methanol extracts of clove inhibited the response of C. violaceum CV026 to exogenously supplied N‑hexanoylhomoserine lactone, in turn preventing violacein production. Chloroform and methanol extracts of clove significantly reduced bioluminescence production by E. coli [pSB1075] grown in the presence of N-(3-oxododecanoyl-L-homoserine lactone. We demonstrated that clove extract inhibited quorum sensing-regulated phenotypes in Pseudomonas aeruginosa PA01, including expression of lecA::lux (by hexane extract, swarming (maximum inhibition by methanol extract, pyocyanin (maximum inhibition by hexane extract. This study shows that the presence of natural compounds that exhibit anti-quorum sensing activity in the clove extracts may be useful as the lead of anti-infective drugs.

  12. Effects of Iron on DNA Release and Biofilm Development by Pseudomonas Aeruginosa

    DEFF Research Database (Denmark)

    Yang, Liang; Barken, Kim Bundvig; Skindersø, Mette Elena

    2007-01-01

    Extracellular DNA is one of the major matrix components in Pseudomonas aeruginosa biofilms. It functions as an intercellular connector and plays a role in stabilization of the biofilms. Evidence that DNA release in P. aeruginosa PAO1 biofilms is controlled by the las-rhl and pqs quorum-sensing sy......Extracellular DNA is one of the major matrix components in Pseudomonas aeruginosa biofilms. It functions as an intercellular connector and plays a role in stabilization of the biofilms. Evidence that DNA release in P. aeruginosa PAO1 biofilms is controlled by the las-rhl and pqs quorum......-sensing systems has been previously presented. This paper provides evidence that DNA release in P. aeruginosa PAO1 biofilms is also under iron regulation. Experiments involving cultivation of P. aeruginosa in microtitre trays suggested that pqs expression, DNA release and biofilm formation were favoured in media...

  13. Biosynthesis of Gold Nanoparticles Using Pseudomonas Aeruginosa

    International Nuclear Information System (INIS)

    Abd El-Aziz, M.; Badr, Y.; Mahmoud, M. A.

    2007-01-01

    Pseudomonas aeruginosa were used for extracellular biosynthesis of gold nanoparticles (Au NPs). Consequently, Au NPs were formed due to reduction of gold ion by bacterial cell supernatant of P. aeruginos ATCC 90271, P. aeruginos (2) and P. aeruginos (1). The UV-Vis. and fluorescence spectra of the bacterial as well as chemical prepared Au NPs were recorded. Transmission electron microscopy (TEM) micrograph showed the formation of well-dispersed gold nanoparticles in the range of 15-30 nm. The process of reduction being extracellular and may lead to the development of an easy bioprocess for synthesis of Au NPs

  14. Improving Fructose Utilization and Butanol Production by Clostridium acetobutylicum via Extracellular Redox Potential Regulation and Intracellular Metabolite Analysis.

    Science.gov (United States)

    Chen, Li-Jie; Wu, You-Duo; Xue, Chuang; Bai, Feng-Wu

    2017-10-01

    Jerusalem artichoke (JA) can grow well in marginal lands with high biomass yield, and thus is a potential energy crop for biorefinery. The major biomass of JA is from tubers, which contain inulin that can be easily hydrolyzed into a mixture of fructose and glucose, but fructose utilization for producing butanol as an advanced biofuel is poor compared to glucose-based ABE fermentation by Clostridium acetobutylicum. In this article, the impact of extracellular redox potential (ORP) on the process is studied using a mixture of fructose and glucose to simulate the hydrolysate of JA tubers. When the extracellular ORP is controlled above -460 mV, 13.2 g L -1 butanol is produced from 51.0 g L -1 total sugars (40.1 g L -1 fructose and 10.9 g L -1 glucose), leading to dramatically increased butanol yield and butanol/ABE ratio of 0.26 g g -1 and 0.67, respectively. Intracellular metabolite and q-PCR analysis further indicate that intracellular ATP and NADH availabilities are significantly improved together with the fructose-specific PTS expression at the lag phase, which consequently facilitate fructose transport, metabolic shift toward solventogenesis and carbon flux redistribution for butanol biosynthesis. Therefore, the extracellular ORP control can be an effective strategy to improve butanol production from fructose-based feedstock. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Intercellular communication in Helicobacter pylori: luxS is essential for the production of an extracellular signaling molecule.

    Science.gov (United States)

    Forsyth, M H; Cover, T L

    2000-06-01

    Individual bacteria of numerous species can communicate and coordinate their actions via the production, release, and detection of extracellular signaling molecules. In this study, we used the Vibrio harveyi luminescence bioassay to determine whether Helicobacter pylori produces such a factor. Cell-free conditioned media from H. pylori strains 60190 and 26695 each induced >100-fold-greater luminescence in V. harveyi than did sterile culture medium. The H. pylori signaling molecule had a molecular mass of 100-fold-greater luminescence in the V. harveyi bioassay than did conditioned medium from either mutant strain. Production of the signaling molecule was restored in an H. pylori luxS null mutant strain by complementation with a single intact copy of luxS placed in a heterologous site on the chromosome. In addition, Escherichia coli DH5alpha produced autoinducer activity following the introduction of an intact copy of luxS from H. pylori. Production of the signaling molecule by H. pylori was growth phase dependent, with maximal production occurring in the mid-exponential phase of growth. Transcription of H. pylori vacA also was growth phase dependent, but this phenomenon was not dependent on luxS activity. These data indicate that H. pylori produces an extracellular signaling molecule related to AI-2 from V. harveyi. We speculate that this signaling molecule may play a role in regulating H. pylori gene expression.

  16. Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

    Science.gov (United States)

    Tanaka, T; Kawata, M

    1988-08-01

    We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU.

  17. Blue light treatment of Pseudomonas aeruginosa: Strong bactericidal activity, synergism with antibiotics and inactivation of virulence factors.

    Science.gov (United States)

    Fila, Grzegorz; Kawiak, Anna; Grinholc, Mariusz Stanislaw

    2017-08-18

    Pseudomonas aeruginosa is among the most common pathogens responsible for both acute and chronic infections of high incidence and severity. Additionally, P. aeruginosa resistance to conventional antimicrobials has increased rapidly over the past decade. Therefore, it is crucial to explore new therapeutic options, particularly options that specifically target the pathogenic mechanisms of this microbe. The ability of a pathogenic bacterium to cause disease is dependent upon the production of agents termed 'virulence factors', and approaches to mitigate these agents have gained increasing attention as new antibacterial strategies. Although blue light irradiation is a promising alternative approach, only limited and preliminary studies have described its effect on virulence factors. The current study aimed to investigate the effects of lethal and sub-lethal doses of blue light treatment (BLT) on P. aeruginosa virulence factors. We analyzed the inhibitory effects of blue light irradiation on the production/activity of several virulence factors. Lethal BLT inhibited the activity of pyocyanin, staphylolysin, pseudolysin and other proteases, but sub-lethal BLT did not affect the production/expression of proteases, phospholipases, and flagella- or type IV pili-associated motility. Moreover, a eukaryotic cytotoxicity test confirmed the decreased toxicity of blue light-treated extracellular P. aeruginosa fractions. Finally, the increased antimicrobial susceptibility of P. aeruginosa treated with sequential doses of sub-lethal BLT was demonstrated with a checkerboard test. Thus, this work provides evidence-based proof of the susceptibility of drug-resistant P. aeruginosa to BLT-mediated killing, accompanied by virulence factor reduction, and describes the synergy between antibiotics and sub-lethal BLT.

  18. Resistência antimicrobiana de Pseudomonas aeruginosa isolados de pescado e de cortes e de miúdos de frango Antimicrobial resistance in Pseudomonas aeruginosa isolated from fish and poultry products

    Directory of Open Access Journals (Sweden)

    Adriana de Araújo Maia

    2009-03-01

    Full Text Available Pseudomonas aeruginosa isolados de peixes de água doce e de frangos foram submetidos ao teste de suscetibilidade aos antimicrobianos utilizando quatorze drogas, com o objetivo de determinar e confrontar os padrões de suscetibilidade deste microrganismo. As cepas oriundas de peixes pertenciam à coleção do Laboratório de Bacterioses/IV/UFRRJ. Para o isolamento das cepas, foram selecionados miúdos (fígado e cortes (coxa e sobrecoxa de frangos adquiridos em estabelecimentos comerciais no município do Rio de Janeiro. A metodologia de isolamento incluiu o enriquecimento em água peptonada, seguido de semeadura em Agar EMB e Agar GSP. Para as cepas oriundas de peixes, procedeu-se à reativação em água peptonada, seguida de reisolamento em Agar EMB. Colônias sugestivas foram transferidas para Agar TSI e LIA para avaliação das características metabólicas. A capacidade de produção de pigmento verde-azulado foi avaliada em Agar Mueller-Hinton e a da enzima citocromo-oxidase, em Agar Nutriente. O teste de suscetibilidade a antimicrobianos realizado nas 63 cepas revelou maiores percentuais de resistência para NAL e NIT (96,8%, TCY (93,6%, AMC (92,1%, CHL (90,5% e SXT (85,7%, destacando-se a multirresistência dos isolados. A totalidade das cepas oriundas de frangos apresentou sensibilidade a CAZ e IPM e nos isolados de peixes a ATM, CAZ, IPM e AMK.Pseudomonas aeruginosa strains isolated from fish and chicken products were analyzed using the Antimicrobial Susceptibility Test with 14 drugs to evaluate the susceptibility patterns of this microorganism. Strains isolated from two different sources were evaluated. The fish strains belonged to the collection of Bacterioses Laboratory/IV/UFRRJ, and those from chicken specimens were isolated from the liver and chicken carcasses traded in commercial establishments in the city of Rio de Janeiro. The isolation methodology included enrichment in Peptone Water and then spread on EMB Agar and GSP Agar

  19. The influence of heavy metals on the production of extracellular polymer substances in the processes of heavy metal ions elimination.

    Science.gov (United States)

    Mikes, J; Siglova, M; Cejkova, A; Masak, J; Jirku, V

    2005-01-01

    Wastewaters from a chemical industry polluted by heavy metal ions represent a hazard for all living organisms. It can mean danger for ecosystems and human health. New methods are sought alternative to traditional chemical and physical processes. Active elimination process of heavy metals ions provided by living cells, their components and extracellular products represents a potential way of separating toxic heavy metals from industrial wastewaters. While the abilities of bacteria to remove metal ions in solution are extensively used, fungi have been recognized as a promising kind of low-cost adsorbents for removal of heavy-metal ions from aqueous waste sources. Yeasts and fungi differ from each other in their constitution and in their abilities to produce variety of extracellular polymeric substances (EPS) with different mechanisms of metal interactions. The accumulation of Cd(2+), Cr(6+), Pb(2+), Ni(2+) and Zn(2+) by yeasts and their EPS was screened at twelve different yeast species in microcultivation system Bioscreen C and in the shaking Erlenmayer's flasks. This results were compared with the production of yeast EPS and the composition of yeast cell walls. The EPS production was measured during the yeast growth and cell wall composition was studied during the cultivations in the shaking flasks. At the end of the process extracellular polymers and their chemical composition were isolated and amount of bound heavy metals was characterized. The variable composition and the amount of the EPS were found at various yeast strains. It was influenced by various compositions of growth medium and also by various concentrations of heavy metals. It is evident, that the amount of bound heavy metals was different. The work reviews the possibilities of usage of various yeast EPS and components of cell walls in the elimination processes of heavy metal ions. Further the structure and properties of yeasts cell wall and EPS were discussed. The finding of mechanisms mentioned

  20. Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate.

    Science.gov (United States)

    Mahanta, Nilkamal; Gupta, Anshu; Khare, S K

    2008-04-01

    Deoiled Jatropha seed cake was assessed for its suitability as substrate for enzyme production by solid-state fermentation (SSF). Solvent tolerant Pseudomonas aeruginosa PseA strain previously reported by us was used for fermentation. The seed cake supported good bacterial growth and enzyme production (protease, 1818 U/g of substrate and lipase, 625 U/g of substrate) as evident by its chemical composition. Maximum protease and lipase production was observed at 50% substrate moisture, a growth period of 72 and 120 h, and a substrate pH of 6.0 and 7.0, respectively. Enrichment with maltose as carbon source increased protease and lipase production by 6.3- and 1.6-fold, respectively. Nitrogen supplementation with peptone for protease and NaNO(3) for lipase production also enhanced the enzyme yield reaching 11,376 U protease activity and 1084 U lipase activity per gram of Jatropha seed cake. These results demonstrated viable approach for utilization of this huge biomass by solid-state fermentation for the production of industrial enzymes. This offers significant benefit due to low cost and abundant availability of cake during biodiesel production.

  1. Extracellular Lipase and Protease Production from a Model Drinking Water Bacterial Community Is Functionally Robust to Absence of Individual Members.

    Directory of Open Access Journals (Sweden)

    Graham G Willsey

    Full Text Available Bacteria secrete enzymes into the extracellular space to hydrolyze macromolecules into constituents that can be imported for microbial nutrition. In bacterial communities, these enzymes and their resultant products can be modeled as community property. Our goal was to investigate the impact of individual community member absence on the resulting community production of exoenzymes (extracellular enzymes involved in lipid and protein hydrolysis. Our model community contained nine bacteria isolated from the potable water system of the International Space Station. Bacteria were grown in static conditions individually, all together, or in all combinations of eight species and exoproduct production was measured by colorimetric or fluorometric reagents to assess short chain and long chain lipases, choline-specific phospholipases C, and proteases. The exoenzyme production of each species grown alone varied widely, however, the enzyme activity levels of the mixed communities were functionally robust to absence of any single species, with the exception of phospholipase C production in one community. For phospholipase C, absence of Chryseobacterium gleum led to increased choline-specific phospholipase C production, correlated with increased growth of Burkholderia cepacia and Sphingomonas sanguinis. Because each individual species produced different enzyme activity levels in isolation, we calculated an expected activity value for each bacterial mixture using input levels or known final composition. This analysis suggested that robustness of each exoenzyme activity is not solely mediated by community composition, but possibly influenced by bacterial communication, which is known to regulate such pathways in many bacteria. We conclude that in this simplified model of a drinking water bacterial community, community structure imposes constraints on production and/or secretion of exoenzymes to generate a level appropriate to exploit a given nutrient environment.

  2. Polysaccharides serve as scaffold of biofilms formed by mucoid Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Yang, Liang; Hengzhuang, Wang; Wu, Hong

    2012-01-01

    Chronic lung infection by mucoid Pseudomonas aeruginosa is one of the major pathologic features in patients with cystic fibrosis. Mucoid P. aeruginosa is notorious for its biofilm forming capability and resistance to immune attacks. In this study, the roles of extracellular polymeric substances f...

  3. Myogenic Progenitor Cells Control Extracellular Matrix Production by Fibroblasts during Skeletal Muscle Hypertrophy.

    Science.gov (United States)

    Fry, Christopher S; Kirby, Tyler J; Kosmac, Kate; McCarthy, John J; Peterson, Charlotte A

    2017-01-05

    Satellite cells, the predominant stem cell population in adult skeletal muscle, are activated in response to hypertrophic stimuli and give rise to myogenic progenitor cells (MPCs) within the extracellular matrix (ECM) that surrounds myofibers. This ECM is composed largely of collagens secreted by interstitial fibrogenic cells, which influence satellite cell activity and muscle repair during hypertrophy and aging. Here we show that MPCs interact with interstitial fibrogenic cells to ensure proper ECM deposition and optimal muscle remodeling in response to hypertrophic stimuli. MPC-dependent ECM remodeling during the first week of a growth stimulus is sufficient to ensure long-term myofiber hypertrophy. MPCs secrete exosomes containing miR-206, which represses Rrbp1, a master regulator of collagen biosynthesis, in fibrogenic cells to prevent excessive ECM deposition. These findings provide insights into how skeletal stem and progenitor cells interact with other cell types to actively regulate their extracellular environments for tissue maintenance and adaptation. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Inhibition of Streptococcus mutans biofilm formation, extracellular polysaccharide production, and virulence by an oxazole derivative.

    Science.gov (United States)

    Chen, Lulu; Ren, Zhi; Zhou, Xuedong; Zeng, Jumei; Zou, Jing; Li, Yuqing

    2016-01-01

    Dental caries, a biofilm-related oral disease, is a result of disruption of the microbial ecological balance in the oral environment. Streptococcus mutans, which is one of the primary cariogenic bacteria, produces glucosyltransferases (Gtfs) that synthesize extracellular polysaccharides (EPSs). The EPSs, especially water-insoluble glucans, contribute to the formation of dental plaque, biofilm stability, and structural integrity, by allowing bacteria to adhere to tooth surfaces and supplying the bacteria with protection against noxious stimuli and other environmental attacks. The identification of novel alternatives that selectively inhibit cariogenic organisms without suppressing oral microbial residents is required. The goal of the current study is to investigate the influence of an oxazole derivative on S. mutans biofilm formation and the development of dental caries in rats, given that oxazole and its derivatives often exhibit extensive and pharmacologically important biological activities. Our data shows that one particular oxazole derivative, named 5H6, inhibited the formation of S. mutans biofilms and prevented synthesis of extracellular polysaccharides by antagonizing Gtfs in vitro, without affecting the growth of the bacteria. In addition, topical applications with the inhibitor resulted in diminished incidence and severity of both smooth and sulcal surface caries in vivo with a lower percentage of S. mutans in the animals' dental plaque compared to the control group (P mutans.

  5. Extraction of extracellular lipids from chemoautotrophic bacteria Serratia sp. ISTD04 for production of biodiesel.

    Science.gov (United States)

    Bharti, Randhir K; Srivastava, Shaili; Thakur, Indu Shekhar

    2014-08-01

    A CO2 sequestering bacterial strain, Serratia sp. ISTD04, that produces a significant amount of extracellular lipids was isolated from marble mine rocks. (14)C labeling analysis revealed that the rate of assimilation of CO2 by the strain is 0.756×10(-9)μmolCO2fixedcell(-1)h(-1). It was found to produce 466mg/l of extracellular lipid which was characterized using (1)H NMR. After transesterification of lipids, the total saturated and unsaturated FAME was found to be 51% and 49% respectively. The major FAME contained in the biodiesel were palmitic acid methyl ester (C16:0), oleic acid methyl ester (C18:1) and 10-nonadecenoic acid methyl ester (C19:1). Biodiesel produced by Serratia sp. ISTD04 is balanced in terms of FAME composition of good quality. It also contained higher proportion of oleic acid (35%) which makes it suitable for utilization in existing engines. Thus, the strain can be harnessed commercially to sequester CO2 into biodiesel. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Molecular Determinants of the Thickened Matrix in a Dual-Species Pseudomonas aeruginosa and Enterococcus faecalis Biofilm.

    Science.gov (United States)

    Lee, Keehoon; Lee, Kang-Mu; Kim, Donggeun; Yoon, Sang Sun

    2017-11-01

    Biofilms are microbial communities that inhabit various surfaces and are surrounded by extracellular matrices (ECMs). Clinical microbiologists have shown that the majority of chronic infections are caused by biofilms, following the introduction of the first biofilm infection model by J. W. Costerton and colleagues (J. Lam, R. Chan, K. Lam, and J. W. Costerton, Infect Immun 28:546-556, 1980). However, treatments for chronic biofilm infections are still limited to surgical removal of the infected sites. Pseudomonas aeruginosa and Enterococcus faecalis are two frequently identified bacterial species in biofilm infections; nevertheless, the interactions between these two species, especially during biofilm growth, are not clearly understood. In this study, we observed phenotypic changes in a dual-species biofilm of P. aeruginosa and E. faecalis , including a dramatic increase in biofilm matrix thickness. For clear elucidation of the spatial distribution of the dual-species biofilm, P. aeruginosa and E. faecalis were labeled with red and green fluorescence, respectively. E. faecalis was located at the lower part of the dual-species biofilm, while P. aeruginosa developed a structured biofilm on the upper part. Mutants with altered exopolysaccharide (EPS) productions were constructed in order to determine the molecular basis for the synergistic effect of the dual-species biofilm. Increased biofilm matrix thickness was associated with EPSs, not extracellular DNA. In particular, Pel and Psl contributed to interspecies and intraspecies interactions, respectively, in the dual-species P. aeruginosa and E. faecalis biofilm. Accordingly, targeting Pel and Psl might be an effective part of eradicating P. aeruginosa polymicrobial biofilms. IMPORTANCE Chronic infection is a serious problem in the medical field. Scientists have observed that chronic infections are closely associated with biofilms, and the vast majority of infection-causing biofilms are polymicrobial. Many studies

  7. Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuzaki, Shinichi [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Ishizuka, Tamotsu, E-mail: tamotsui@showa.gunma-u.ac.jp [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Komachi, Mayumi [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Dobashi, Kunio [Gunma University Graduate School of Health Sciences, Maebashi 371-8511 (Japan); Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Mori, Masatomo [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan)

    2011-10-07

    Highlights: {yields} The involvement of extracellular acidification in airway remodeling was investigated. {yields} Extracellular acidification alone induced CTGF production in human ASMCs. {yields} Extracellular acidification enhanced TGF-{beta}-induced CTGF production in human ASMCs. {yields} Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. {yields} OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-{beta}-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G{sub q/11} protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP{sub 3}) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G{sub q/11} protein and inositol-1,4,5-trisphosphate-induced Ca{sup 2+} mobilization in human ASMCs.

  8. Production and Characterization of an Extracellular Acid Protease from Thermophilic Brevibacillus sp. OA30 Isolated from an Algerian Hot Spring

    Directory of Open Access Journals (Sweden)

    Mohamed Amine Gomri

    2018-04-01

    Full Text Available Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium. Strain OA30 showed multiple extracellular proteolytic enzymes and protease 32-F38 was purified by chromatographic methods and its biochemical characteristics were studied. Strain OA30 was affiliated with Brevibacillus thermoruber species. Protease 32-F38 had an estimated molecular weight of 64.6 kDa and was optimally active at 50 °C. It showed a great thermostability after 240 min and its optimum pH was 6.0. Protease 32-F38 was highly stable in the presence of different detergents and solvents and was inhibited by metalloprotease inhibitors. The results of this work suggest that protease 32-F38 might have interesting biotechnological applications.

  9. Characterization of Co-Cultivation of Cyanobacteria on Growth, Productions of Polysaccharides and Extracellular Proteins, Nitrogenase Activity, and Photosynthetic Activity.

    Science.gov (United States)

    Xue, Chuizhao; Wang, Libo; Wu, Tong; Zhang, Shiping; Tang, Tao; Wang, Liang; Zhao, Quanyu; Sun, Yuhan

    2017-01-01

    Cyanobacteria as biofertilizers are benefit to reduce the use of chemical fertilizers and reestablish the ecological system in soil. In general, several strains of cyanobacteria were involved in the biofertilizers. The co-cultivation of cyanobacteria were characterized on growth profile, production of polysaccharides and extracellular proteins, nitrogenase activity, and photosynthetic activity for three selected N 2 -fixing cyanobacteria, Anabaena cylindrica (B1611 and F243) and Nostoc sp. (F280). After eight-day culture, the highest dry weights were obtained in F280 pure culture and co-cultivation of B1611 and F280. Higher production of extracellular proteins and cell-bonding polysaccharides (CPS) were observed in co-cultivations compared with pure culture. The highest released polysaccharides (RPS) contents were obtained in pure culture of F280 and co-cultivation of F280 and F243. Galactose and glucose were major components of CPS and RPS in all samples. Trehalose was a specific component of RPS in F280 pure culture. Based on the monosaccharide contents of CPS and RPS, F280 was the dominant species in the related treatments of co-cultivation. The nitrogenase activities in all treatments exhibited a sharp rise at the late stage while a significant decrease existed when three cyanobacteria strains were mixed. Photosynthetic activities for all treatments were determined with rapid light curve, and the related parameters were estimated.

  10. Production and Characterization of an Extracellular Acid Protease from Thermophilic Brevibacillus sp. OA30 Isolated from an Algerian Hot Spring.

    Science.gov (United States)

    Gomri, Mohamed Amine; Rico-Díaz, Agustín; Escuder-Rodríguez, Juan-José; El Moulouk Khaldi, Tedj; González-Siso, María-Isabel; Kharroub, Karima

    2018-04-12

    Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium. Strain OA30 showed multiple extracellular proteolytic enzymes and protease 32-F38 was purified by chromatographic methods and its biochemical characteristics were studied. Strain OA30 was affiliated with Brevibacillus thermoruber species. Protease 32-F38 had an estimated molecular weight of 64.6 kDa and was optimally active at 50 °C. It showed a great thermostability after 240 min and its optimum pH was 6.0. Protease 32-F38 was highly stable in the presence of different detergents and solvents and was inhibited by metalloprotease inhibitors. The results of this work suggest that protease 32-F38 might have interesting biotechnological applications.

  11. Withania somnifera attenuates acid production, acid tolerance and extra-cellular polysaccharide formation of Streptococcus mutans biofilms.

    Science.gov (United States)

    Pandit, Santosh; Song, Kwang-Yeob; Jeon, Jae-Gyu

    2014-01-01

    Withania somnifera (Ashwagandha) is a plant of the Solanaceae family. It has been widely used as a remedy for a variety of ailments in India and Nepal. The plant has also been used as a controlling agent for dental diseases. The aim of the present study was to evaluate the activity of the methanol extract of W. somnifera against the physiological ability of cariogenic biofilms and to identify the components of the extract. To determine the activity of the extract, assays for sucrose-dependent bacterial adherence, glycolytic acid production, acid tolerance, and extracellular polysaccharide formation were performed using Streptococcus mutans biofilms. The viability change of S. mutans biofilms cells was also determined. A phytochemical analysis of the extract was performed using TLC and LC/MS/MS. The extract showed inhibitory effects on sucrose-dependent bacterial adherence (≥ 100 μg/ml), glycolytic acid production (≥ 300 μg/ml), acid tolerance (≥ 300 μg/ml), and extracellular polysaccharide formation (≥ 300 μg/ml) of S. mutans biofilms. However, the extract did not alter the viability of S. mutans biofilms cells in all concentrations tested. Based on the phytochemical analysis, the activity of the extract may be related to the presence of alkaloids, anthrones, coumarines, anthraquinones, terpenoids, flavonoids, and steroid lactones (withanolide A, withaferin A, withanolide B, withanoside IV, and 12-deoxy withastramonolide). These data indicate that W. somnifera may be a potential agent for restraining the physiological ability of cariogenic biofilms.

  12. Gentamicin in Pseudomonas aeruginosa

    African Journals Online (AJOL)

    infections by Ps. aeruginosa is contra-indicated. In our study only 2,3 % of the Ps. aeruginosa strains were resistant to gentamicin (MIC 25 Ilg/ml). In view of the synergy reported for combined gentamicin and carbeni- cillin therapy," a combination of these two drugs may be recommended in the treatment of all Pseudomonas.

  13. Stimulation of rhamnolipid biosurfactants production in Pseudomonas aeruginosa AK6U by organosulfur compounds provided as sulfur sources.

    Science.gov (United States)

    Ismail, Wael; Shammary, Sultanah Al; El-Sayed, Wael S; Obuekwe, Christian; El Nayal, Ashraf M; Abdul Raheem, Abdul Salam; Al-Humam, Abdulmohsen

    2015-09-01

    A Pseudomonas aeruginosa AK6U strain produced rhamnolipid biosurfactants to variable extents when grown on MgSO 4 or organosulfur compounds as sulfur sources and glucose as a carbon source. Organosulfur cultures produced much higher biosurfactants amounts compared to the MgSO 4 cultures. The surface tension of the growth medium was reduced from 72 mN/m to 54 and 30 mN/m in cultures containing MgSO 4 and 4,6-dimethyldibenzothiophene (4,6-DM-DBT), respectively. AK6U cultures produced different rhamnolipid congener profiles depending on the provided sulfur source. The dibenzothiophene (DBT) culture produced more diverse and a higher number of rhamnolipid congeners as compared to the DBT-sulfone and MgSO 4 cultures. The number of mono-rhamnolipid congeners in the DBT culture was also higher than that detected in the DBT-sulfone and MgSO 4 cultures. Di-rhamnolipids dominated the congener profiles in all the analyzed cultures. The sulfur source can have a profound impact on the quality and quantity of the produced biosurfactants.

  14. Stimulation of rhamnolipid biosurfactants production in Pseudomonas aeruginosa AK6U by organosulfur compounds provided as sulfur sources

    Directory of Open Access Journals (Sweden)

    Wael Ismail

    2015-09-01

    Full Text Available A Pseudomonas aeruginosa AK6U strain produced rhamnolipid biosurfactants to variable extents when grown on MgSO4 or organosulfur compounds as sulfur sources and glucose as a carbon source. Organosulfur cultures produced much higher biosurfactants amounts compared to the MgSO4 cultures. The surface tension of the growth medium was reduced from 72 mN/m to 54 and 30 mN/m in cultures containing MgSO4 and 4,6-dimethyldibenzothiophene (4,6-DM-DBT, respectively. AK6U cultures produced different rhamnolipid congener profiles depending on the provided sulfur source. The dibenzothiophene (DBT culture produced more diverse and a higher number of rhamnolipid congeners as compared to the DBT-sulfone and MgSO4 cultures. The number of mono-rhamnolipid congeners in the DBT culture was also higher than that detected in the DBT-sulfone and MgSO4 cultures. Di-rhamnolipids dominated the congener profiles in all the analyzed cultures. The sulfur source can have a profound impact on the quality and quantity of the produced biosurfactants.

  15. Extracellular ATP drives breast cancer cell migration and metastasis via S100A4 production by cancer cells and fibroblasts.

    Science.gov (United States)

    Liu, Ying; Geng, Yue-Hang; Yang, Hui; Yang, Han; Zhou, Yan-Ting; Zhang, Hong-Quan; Tian, Xin-Xia; Fang, Wei-Gang

    2018-05-04

    Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis. Copyright © 2018. Published by Elsevier B.V.

  16. Fluorescent Labeling of Collagen Production by Cells for Noninvasive Imaging of Extracellular Matrix Deposition.

    Science.gov (United States)

    Bardsley, Katie; Yang, Ying; El Haj, Alicia J

    2017-04-01

    Extracellular matrix (ECM) is an essential component of tissues and provides both integrity and biological cues for cells. Collagen is one of the major proteins found within the ECM and therefore is an essential component of all engineered tissues. Therefore, in this article, we present a method for the online real-time monitoring of collagen deposition in three-dimensional engineered constructs. This method revolves around modification of collagen through the addition of azide-L-proline to cell culture media. The incorporation of azide-L-proline into the neocollagen produced by cells can then be detected by reaction with 10 mM of a Click-IT Alexa Fluor 488 DIBO Alkyne. The reaction was shown as being specific to the collagen as little background staining was observed in cultures, which did not contain the modified proline, and the staining was also depleted after treatment with collagenase and colocalization of collagen type I staining by immunochemistry assay. Real-time online staining of collagen deposition was observed under different culture conditions without affecting proliferation. Collagen deposition was observed to be increased under mechanical stimulation; however, the localization varied across stimulation regimes. This is a new technique for real-time monitoring of cell-produced collagen and will be a valuable addition to the tissue engineering field.

  17. The role of Aspergillus flavus veA in the production of extracellular proteins during growth on starch substrates.

    Science.gov (United States)

    Duran, Rocio M; Gregersen, Scott; Smith, Timothy D; Bhetariya, Preetida J; Cary, Jeffrey W; Harris-Coward, Pamela Y; Mattison, Christopher P; Grimm, Casey; Calvo, Ana M

    2014-06-01

    The aflatoxin-producer and opportunistic plant pathogenic, filamentous fungus Aspergillus flavus is responsible for the contamination of corn and other important agricultural commodities. In order to obtain nutrients from the host A. flavus produces a variety of extracellular hydrolytic enzymes. Interestingly, A. flavus amylase and protease activity are dependent on the global regulator veA, a gene known to regulate morphogenesis and secondary metabolism in numerous fungi. Analysis of starch degradation by fungal enzymes secreted into broths of starch- or corn kernel-based media showed a notable accumulation of glucose in samples of the A. flavus control strain while the deletion veA sample accumulated high levels of maltose and maltotriose and only a small amount of glucose. Furthermore, SDS-PAGE and proteomics analysis of culture broths from starch- or corn kernel-based media demonstrated differential production of a number of proteins that included a reduction in the amount of a glucoamylase protein in the veA mutant compared to the control strain, while an alpha-amylase was produced in greater quantities in the veA mutant. Quantitative real-time PCR and western blot analyses using anti-glucoamylase or alpha-amylase antisera supported the proteomics results. Additionally, an overall reduction in protease activity was observed in the veA mutant including production of the alkaline protease, oryzin, compared to the control strain. These findings contribute to our knowledge of mechanisms controlling production of hydrolases and other extracellular proteins during growth of A. flavus on natural starch-based substrates.

  18. Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives: compatibility study with detergent ingredients and washing performance.

    Science.gov (United States)

    Grbavčić, Sanja; Bezbradica, Dejan; Izrael-Živković, Lidija; Avramović, Nataša; Milosavić, Nenad; Karadžić, Ivanka; Knežević-Jugović, Zorica

    2011-12-01

    An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compatibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol® XP80 and Triton® X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1h) while the protease activity was enhanced by the addition of Triton® WR1339 and Tween® 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Carboxymethyl-cellulase from Erwina chrysanthemi. I. Production and regulation of extracellular carboxymethyl-cellulase

    Energy Technology Data Exchange (ETDEWEB)

    Boyer, M.H.; Chambost, J.P.; Magnan, M.; Cattaneo, J.

    1984-01-01

    Erwinia chrysanthemi strain 3665 growing aerobically in a mineral salts medium containing various carbon sources constitutively secreted low levels of carboxymethyl-cellulase activity. Increased production of this activity was triggered by conditions which reduced the growth rate. The results obtained with continuous culture suggested that this production was controlled by a mechanism similar to catabolite repression. However, other factors might be implicated in the regulation of cellulase production.

  20. Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity.

    Science.gov (United States)

    Hassan, Mohamed M

    2014-11-02

    mycoparasitism effect of the extracellular enzymes.

  1. The characteristics of extracellular polymeric substances and soluble microbial products in moving bed biofilm reactor-membrane bioreactor.

    Science.gov (United States)

    Duan, Liang; Jiang, Wei; Song, Yonghui; Xia, Siqing; Hermanowicz, Slawomir W

    2013-11-01

    The characteristics of extracellular polymeric substances (EPS) and soluble microbial products (SMP) in conventional membrane bioreactor (MBR) and in moving bed biofilm reactor-membrane bioreactors (MBBR-MBR) were investigated in long-term (170 days) experiments. The results showed that all reactors had high removal efficiency of ammonium and COD, despite very different fouling conditions. The MBBR-MBR with media fill ratio of 26.7% had much lower total membrane resistance and no obvious fouling were detected during the whole operation. In contrast, MBR and MBBR-MBR with lower and higher media fill experienced more significant fouling. Low fouling at optimum fill ratio may be due to the higher percentage of small molecular size (100 kDa) of EPS and SMP in the reactor. The composition of EPS and SMP affected fouling due to different O-H bonds in hydroxyl functional groups, and less polysaccharides and lipids. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Influence of solids retention time on membrane fouling: characterization of extracellular polymeric substances and soluble microbial products.

    Science.gov (United States)

    Duan, Liang; Tian, Zhiyong; Song, Yonghui; Jiang, Wei; Tian, Yuan; Li, Shan

    2015-01-01

    The objective of this study was to investigate the influence of solids retention time (SRT) on membrane fouling and the characteristics of biomacromolecules. Four identical laboratory-scale membrane bioreactors (MBRs) were operated with SRTs for 10, 20, 40 and 80 days. The results indicated that membrane fouling occurred faster and more readily under short SRTs. Fouling resistance was the primary source of filtration resistance. The modified fouling index (MFI) results suggested that the more ready fouling at short SRTs could be attributed to higher concentrations of soluble microbial products (SMP). Fourier transform infrared (FTIR) spectra indicated that the SRT had a weak influence on the functional groups of the total extracellular polymeric substances (TEPS) and SMP. However, the MBR under a short SRT had more low-molecular-weight (MW) compounds (100 kDa). Aromatic protein and tryptophan protein-like substances were the dominant groups in the TEPS and SMP, respectively.

  3. KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms.

    Science.gov (United States)

    Aguilar, Claudio; Vlamakis, Hera; Guzman, Alejandra; Losick, Richard; Kolter, Roberto

    2010-05-18

    Bacillus subtilis cells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion of kinD suppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed.

  4. The potential for hydrocarbon biodegradation and production of extracellular polymeric substances by aerobic bacteria isolated from a Brazilian petroleum reservoir.

    Science.gov (United States)

    Vasconcellos, S P; Dellagnezze, B M; Wieland, A; Klock, J-H; Santos Neto, E V; Marsaioli, A J; Oliveira, V M; Michaelis, W

    2011-06-01

    Extracellular polymeric substances (EPS) can contribute to the cellular degradation of hydrocarbons and have a huge potential for application in biotechnological processes, such as bioremediation and microbial enhanced oil recovery (MEOR). Four bacterial strains from a Brazilian petroleum reservoir were investigated for EPS production, emulsification ability and biodegradation activity when hydrocarbons were supplied as substrates for microbial growth. Two strains of Bacillus species had the highest EPS production when phenanthrene and n-octadecane were offered as carbon sources, either individually or in a mixture. While Pseudomonas sp. and Dietzia sp., the other two evaluated strains, had the highest hydrocarbon biodegradation indices, EPS production was not detected. Low EPS production may not necessarily be indicative of an absence of emulsifier activity, as indicated by the results of a surface tension reduction assay and emulsification indices for the strain of Dietzia sp. The combined results gathered in this work suggest that a microbial consortium consisting of bacteria with interdependent metabolisms could thrive in petroleum reservoirs, thus overcoming the limitations imposed on each individual species by the harsh conditions found in such environments.

  5. A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

    Directory of Open Access Journals (Sweden)

    Ulrike Resch

    2016-11-01

    Full Text Available Export of macromolecules via extracellular membrane-derived vesicles (MVs plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS, the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to “anchorless surface proteins.” Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response.

  6. Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

    Directory of Open Access Journals (Sweden)

    Deepali Bisht

    2013-01-01

    Full Text Available Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100. The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L-1; starch, 15.0 g.L-1; triton-X-100, 0.93 mL.L-1; incubation temperature, 34.12 ºC and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL-1. The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R² value (0.9987. The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization.

  7. Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

    Science.gov (United States)

    Bisht, Deepali; Yadav, Santosh Kumar; Darmwal, Nandan Singh

    2013-01-01

    Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L−1; starch, 15.0 g.L−1; triton-X-100, 0.93 mL.L−1; incubation temperature, 34.12 °C and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL−1). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R2) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization. PMID:24159311

  8. OPTIMIZATION OF EXTRACELLULAR TANNASE PRODUCTION BY ASPERGILLUS NIGER VAN TIEGHEM USING RESPONSE SURFACE METHODOLOGY

    Directory of Open Access Journals (Sweden)

    Hamada Abou-Bakr

    2013-12-01

    Full Text Available Response surface methodology (RSM was used to optimize the production of tannase by a newly isolate of Aspergillus niger Van Tieghem using rotatable central composite design (RCCD. This statistical optimization process was carried out involving four of quantitative growth parameters (variables, namely tannic acid concentration, nitrogen source concentration, initial pH of the medium and inoculum size. A mathematical model expressing the production process of tannase by submerged fermentation (SmF technique was generated statistically in the form of a second order polynomial equation. The model indicated the presence of significant linear, quadratic and interaction effects of the studied variables on tannase production by the fungal isolate. The results showed maximum tannase production (580 U/50 ml medium at 2% tannic acid, 4 g/l sodium nitrate, pH 4 and inoculum size of 5×107 spores/50 ml medium, which was also verified by experimental data.

  9. Pomelo peels as alternative substrate for extracellular pectinase production by Aspergillus niger HFM-8

    Directory of Open Access Journals (Sweden)

    Ibrahim, D.

    2013-12-01

    Full Text Available Aims: The aim of this work was to develop an effective bioprocess to enhance the pectinase production by solid-state cultures of Aspergillus niger HFM-8. Methodology and results: The pectinase production produced by A. niger HFM-8 was studied under solid state fermentation using Malaysian pomelo (Citrus grandis peel as the substrate. This local agricultural waste is rich with lignocellulolytic material including pectin acts as the inducer of pectinase production. Under optimized conditions, 5 g of 0.75 mm pomelo peel size, moisture content of 60% (v/w sterile distilled water pH 5.0, inoculums size of 1x10^4 spores/mL, cultivation temperature of room temperature (30 ± 2 °C, no mixing incurred and with the addition of 1% (w/w citrus pectin and 0.1% (w/w urea has produced pectinase production of 306.89 U/g substrate and 0.78 mg glucosamine/g substrate of fungal growth on the 8th day of cultivation. Conclusion, significance and impact of study: There was 48.82% increment in enzyme production after the improvement of parameters. It was found that pomelo peel is a suitable feedstock for pectinase production.

  10. Detection of Tox A Gene in Pseudomonas aeruginosa Strains Isolated from Dairy Products Using PCR and Determining the Antibiotic Resistance Pattern

    Directory of Open Access Journals (Sweden)

    Faeze Zadsafar

    2017-08-01

    Conclusion: Due to high presence of Pseudomonas aeruginosa in raw milk and existence of antibiotic resistance genes in this bacterium, applying appropriate strategies for hygiene control in animal husbandries, is necessary to prevent the spread of bacteria.

  11. The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing.

    Directory of Open Access Journals (Sweden)

    Andreas Dötsch

    Full Text Available In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS. By the use of rapid amplification of cDNA ends (5'-RACE we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms.

  12. Application of a novel enzymatic pretreatment using crude hydrolytic extracellular enzyme solution to microalgal biomass for dark fermentative hydrogen production.

    Science.gov (United States)

    Yun, Yeo-Myeong; Kim, Dong-Hoon; Oh, You-Kwan; Shin, Hang-Sik; Jung, Kyung-Won

    2014-05-01

    In this study, a novel enzymatic pretreatment of Chlorella vulgaris for dark fermentative hydrogen production (DFHP) was performed using crude hydrolytic extracellular enzyme solution (CHEES) extracted from the H2 fermented effluent of food waste. It was found that the enzyme extracted at 52 h had the highest hydrolysis efficiency of microalgal biomass, resulting in the highest H2 yield of 43.1 mL H2/g dry cell weight along with shorter lag periods. Even though a high amount of VFAs was accumulated in CHEES, especially butyrate, the fermentative bacteria on the DFHP was not affected from product inhibition. It also appears that the presence of organic acids, especially lactate and acetate, contained in the CHEES facilitated enhancement of H2 production acted as a co-substrate. Therefore, all of the experimental results suggest that the enhancement of DFHP performance caused by CHEES has a dual role as the hydrolysis enhancer and the co-substrate supplier. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Co-culture of chondrons and mesenchymal stromal cells reduces the loss of collagen VI and improves extracellular matrix production.

    Science.gov (United States)

    Owida, H A; De Las Heras Ruiz, T; Dhillon, A; Yang, Y; Kuiper, N J

    2017-12-01

    Adult articular chondrocytes are surrounded by a pericellular matrix (PCM) to form a chondron. The PCM is rich in hyaluronan, proteoglycans, and collagen II, and it is the exclusive location of collagen VI in articular cartilage. Collagen VI anchors the chondrocyte to the PCM. It has been suggested that co-culture of chondrons with mesenchymal stromal cells (MSCs) might enhance extracellular matrix (ECM) production. This co-culture study investigates whether MSCs help to preserve the PCM and increase ECM production. Primary bovine chondrons or chondrocytes or rat MSCs were cultured alone to establish a baseline level for ECM production. A xenogeneic co-culture monolayer model using rat MSCs (20, 50, and 80%) was established. PCM maintenance and ECM production were assessed by biochemical assays, immunofluorescence, and histological staining. Co-culture of MSCs with chondrons enhanced ECM matrix production, as compared to chondrocyte or chondron only cultures. The ratio 50:50 co-culture of MSCs and chondrons resulted in the highest increase in GAG production (18.5 ± 0.54 pg/cell at day 1 and 11 ± 0.38 pg/cell at day 7 in 50:50 co-culture versus 16.8 ± 0.61 pg/cell at day 1 and 10 ± 0.45 pg/cell at day 7 in chondron monoculture). The co-culture of MSCs with chondrons appeared to decelerate the loss of the PCM as determined by collagen VI expression, whilst the expression of high-temperature requirement serine protease A1 (HtrA1) demonstrated an inverse relationship to that of the collagen VI. Together, this implies that MSCs directly or indirectly inhibited HtrA1 activity and the co-culture of MSCs with chondrons enhanced ECM synthesis and the preservation of the PCM.

  14. Pulsed-low intensity ultrasound enhances extracellular matrix production by fibroblasts encapsulated in alginate

    Directory of Open Access Journals (Sweden)

    Siti PM Bohari

    2012-12-01

    Full Text Available In this study, the effect of pulsed-low intensity ultrasound on cell proliferation, collagen production and glycosaminoglycan deposition by 3T3 fibroblasts encapsulated in alginate was evaluated. Hoechst 33258 assay for cell number, hydroxyproline assay for collagen content and dimethylamine blue assay for glycosaminoglycan content were performed on samples from cell cultures treated with pulsed-low intensity ultrasound and a control group. Pulsed-low intensity ultrasound shows no effect on cell proliferation, while collagen and glycosaminoglycan contents were consistently higher in the samples treated with pulsed-low intensity ultrasound, showing a statistically significant difference (p < 0.05 on day 10. Alcian blue staining showed that glycosaminoglycans were deposited around the cells in both groups. These results suggest that pulsed-low intensity ultrasound shows no effect on cell proliferation but has potential for inducing collagen and glycosaminoglycan production in cells cultured in alginate gels.

  15. Hydrogen production of Enterobacter aerogenes altered by extracellular and intracellular redox states

    Energy Technology Data Exchange (ETDEWEB)

    Nakashimada, Y.; Rachman, M.A.; Kakizono, T.; Nishio, N. [Hiroshima University, Higashi-Hiroshima (Japan). Graduate School of Advanced Sciences of Matter, Department of Molecular Biotechnology

    2002-12-01

    Enterobacter aerogenes HU-101, tested for its hydrogen production in batch cultures on various substrates, produced the highest amount of hydrogen when the substrate was glycerol. The yield of hydrogen is a function of the degree to which the substrates are reduced. To examine the effect of intracellular redox state on hydrogen yield, glucose-limiting chemostat cultures were carried out at various pH using strain HU-101 and its mutant AY-2. For both strains, the molar yield and the production rate of hydrogen and the hydrogenase activity in the cell-free extract were optimal at the culture pH of 6.3. The highest NADH/NAD ratio in both strains was also observed at pH 6.3, at which the ratio in AY-2 was more than two-fold that of HU-101. Furthermore, NAD(P)H-dependent hydrogen formation was observed in the cell-free extract of AY-2, and hydrogenase activity was found not in the cytoplasmic but in the cell membrane fraction, suggesting that a high intracellular redox state, that is a high NADH/NAD ratio, would accelerate hydrogen production by driving membrane-bound NAD(P)H-dependent hydrogenase. (author)

  16. Process optimization by response surface methodology for extracellular alkaline protease production from bacillus subtilis

    International Nuclear Information System (INIS)

    Mushtaq, Z.; Adnan, A.; Mehmood, Z.

    2014-01-01

    Three microbial cultures Bacillus subtilis DSM 1970, Bacillus subtilis GCU-8 and Bacillus licheniformis DSM 1969 were screened for protease production by casein agar plate method. Among these Bacillus subtilis GCU-8 was found to be the most potent protease producer in wide pH range (5.0 to 8.0). Fermentation conditions were optimized for the production of alkaline protease using two statistical tools: Placket Burmen Model for linear regression study and Response Surface Model for interactive effects of significant factors on production. The alkaline protease was optimally produced after 48 hours of incubation at 37 degree C in fermentation media containing equal amounts of substrates (soybean meal and wheat bran, 7.5 g), MgSO/sub 4/ 7H/sub 2/O, 0.10 g and yeast extract 0.55 g. The protease was purified to homogeneity by salt precipitation, ion-exchange chromatography and size exclusion chromatography. The homogeneity and molecular weights were checked by SDS-PAGE. The protease was 45 KDa protein, predominantly alkaline and optimally active at pH 8.0. (author)

  17. Complement activation by Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Jensen, E T; Kharazmi, A; Garred, P

    1993-01-01

    In chronic infections, such as the bronchopulmonary Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients, bacteria persist despite an intact host immune defense and frequent antibiotic treatment. An important reason for the persistence of the bacteria is their capacity for the biofilm...... mode of growth. In this study we investigated the role of biofilms in activation of complement, a major contributor to the inflammatory process. Complement activation by P. aeruginosa was examined in a complement consumption assay, production of C3 and factor B conversion products assessed by crossed...... immuno-electrophoresis, C5a generation tested by a PMN chemotactic assay, and terminal complement complex formation measured by ELISA. Two of the four assays showed that P. aeruginosa grown in biofilm activated complement less than planktonic bacteria, and all assays showed that activation by intact...

  18. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase

    Directory of Open Access Journals (Sweden)

    Biplab Kumar Dash

    2015-01-01

    Full Text Available A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25% as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L and sodium lauryl sulfate (0.2 g/L resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.

  19. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase.

    Science.gov (United States)

    Dash, Biplab Kumar; Rahman, M Mizanur; Sarker, Palash Kumar

    2015-01-01

    A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37 °C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50 °C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.

  20. Identifying metabolic pathways for production of extracellular polymeric substances by the diatom Fragilariopsis cylindrus inhabiting sea ice.

    Science.gov (United States)

    Aslam, Shazia N; Strauss, Jan; Thomas, David N; Mock, Thomas; Underwood, Graham J C

    2018-05-01

    Diatoms are significant primary producers in sea ice, an ephemeral habitat with steep vertical gradients of temperature and salinity characterizing the ice matrix environment. To cope with the variable and challenging conditions, sea ice diatoms produce polysaccharide-rich extracellular polymeric substances (EPS) that play important roles in adhesion, cell protection, ligand binding and as organic carbon sources. Significant differences in EPS concentrations and chemical composition corresponding to temperature and salinity gradients were present in sea ice from the Weddell Sea and Eastern Antarctic regions of the Southern Ocean. To reconstruct the first metabolic pathway for EPS production in diatoms, we exposed Fragilariopsis cylindrus, a key bi-polar diatom species, to simulated sea ice formation. Transcriptome profiling under varying conditions of EPS production identified a significant number of genes and divergent alleles. Their complex differential expression patterns under simulated sea ice formation was aligned with physiological and biochemical properties of the cells, and with field measurements of sea ice EPS characteristics. Thus, the molecular complexity of the EPS pathway suggests metabolic plasticity in F. cylindrus is required to cope with the challenging conditions of the highly variable and extreme sea ice habitat.

  1. Simultaneous production of intracellular triacylglycerols and extracellular polyol esters of fatty acids by Rhodotorula babjevae and Rhodotorula aff. paludigena.

    Science.gov (United States)

    Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Cathcart, Erin; Fiehn, Oliver; German, J Bruce; Block, David E; Boundy-Mills, Kyria L

    2017-10-01

    Microbial oils have been analyzed as alternatives to petroleum. However, just a handful of microbes have been successfully adapted to produce chemicals that can compete with their petroleum counterparts. One of the reasons behind the low success rate is the overall economic inefficiency of valorizing a single product. This study presents a lab-scale analysis of two yeast species that simultaneously produce multiple high-value bioproducts: intracellular triacylglycerols (TG) and extracellular polyol esters of fatty acids (PEFA), two lipid classes with immediate applications in the biofuels and surfactant industries. At harvest, the yeast strain Rhodotorula aff. paludigena UCDFST 81-84 secreted 20.9 ± 0.2 g L -1 PEFA and produced 8.8 ± 1.0 g L -1 TG, while the yeast strain Rhodotorula babjevae UCDFST 04-877 secreted 11.2 ± 1.6 g L -1 PEFA and 18.5 ± 1.7 g L -1 TG. The overall glucose conversion was 0.24 and 0.22 g (total lipid) g (glucose) -1 , respectively. The results present a stable and scalable microbial growth platform yielding multiple co-products.

  2. A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms

    DEFF Research Database (Denmark)

    Allesen-Holm, Marie; Barken, Kim Bundvig; Yang, Liang

    2006-01-01

    -type P. aeruginosa biofilms stained with different DNA stains suggested that the extracellular DNA is located primarily in the stalks of mushroom-shaped multicellular structures, with a high concentration especially in the outer part of the stalks forming a border between the stalk-forming bacteria...... to whole-genome DNA. Evidence that the extracellular DNA in P. aeruginosa biofilms and cultures is generated via lysis of a subpopulation of the bacteria was obtained through experiments where extracellular beta-galactosidase released from lacZ-containing P. aeruginosa strains was assessed. Experiments...... and the cap-forming bacteria. Biofilms formed by lasIrhlI, pqsA and fliMpilA mutants contained less extracellular DNA than biofilms formed by the wild type, and the mutant biofilms were more susceptible to treatment with sodium dodecyl sulphate than the wild-type biofilm....

  3. Optimization Conditions of Extracellular Proteases Production from a Newly Isolated Streptomyces Pseudogrisiolus NRC-15

    Directory of Open Access Journals (Sweden)

    El-Sayed E. Mostafa

    2012-01-01

    Full Text Available Microbial protease represents the most important industrial enzymes, which have an active role in biotechnological processes. The objective of this study was to isolate new strain of Streptomyces that produce proteolytic enzymes with novel properties and the development of the low-cost medium. An alkaline protease producer strain NRC-15 was isolated from Egyptian soil sample. The cultural, morphological, physiological characters and chemotaxonomic evidence strongly indicated that the NRC-15 strain represents a novel species of the genus Streptomyces, hence the name Strptomyces pseudogrisiolus NRC-15. The culture conditions for higher protease production by NRC-15 were optimized with respect to carbon and nitrogen sources, metal ions, pH and temperature. Maximum protease production was obtained in the medium supplemented with 1% glucose, 1% yeast extract, 6% NaCl and 100 μmol/L of Tween 20, initial pH 9.0 at 50 °C for 96 h. The current results confirm that for this strain, a great ability to produce alkaline proteases, which supports the use of applications in industry.

  4. Simvastatin attenuates acrolein-induced mucin production in rats: involvement of the Ras/extracellular signal-regulated kinase pathway.

    Science.gov (United States)

    Chen, Ya-Juan; Chen, Peng; Wang, Hai-Xia; Wang, Tao; Chen, Lei; Wang, Xun; Sun, Bei-Bei; Liu, Dai-Shun; Xu, Dan; An, Jing; Wen, Fu-Qiang

    2010-06-01

    Airway mucus overproduction is a cardinal feature of airway inflammatory diseases, such as chronic obstructive pulmonary disease and cystic fibrosis. Since the small G-protein Ras is known to modulate cellular functions in the lung, we sought to investigate whether the Ras inhibitor simvastatin could attenuate acrolein-induced mucin production in rat airways. Rats were exposed to acrolein for 12 days, after first being pretreated intragastrically for 24 h with either simvastatin alone or simvastatin in combination with mevalonate, which prevents the isoprenylation needed for Ras activation. Lung tissue was analyzed for extracellular signal-regulated kinase (ERK) activity, goblet cell metaplasia and mucin production. To analyze the effect of simvastatin on mucin production in more detail, acrolein-exposed human airway epithelial NCI-H292 cells were pretreated with simvastatin alone or together with mevalonate. Culture medium was collected to detect mucin secretion, and cell lysates were examined for Ras-GTPase activity and epidermal growth factor receptor (EGFR)/ERK phosphorylation. In vivo, simvastatin treatment dose-dependently suppressed acrolein-induced goblet cell hyperplasia and metaplasia in bronchial epithelium and inhibited ERK phosphorylation in rat lung homogenates. Moreover, simvastatin inhibited Muc5AC mucin synthesis at both the mRNA and protein levels in the lung. In vitro, simvastatin pretreatment attenuated the acrolein-induced significant increase in MUC5AC mucin expression, Ras-GTPase activity and EGFR/ERK phosphorylation. These inhibitory effects of simvastatin were neutralized by mevalonate administration both in vitro and in vivo. Our results suggest that simvastatin may attenuate acrolein-induced mucin protein synthesis in the airway and airway inflammation, possibly by blocking ERK activation mediated by Ras protein isoprenylation. Thus, the evidence from the experiment suggests that human trials are warranted to determine the potential

  5. CHANGES IN THE MORPHOLOGY AND POLYSACCHARIDE CONTENT OF MICROCYSTIS AERUGINOSA (CYANOBACTERIA) DURING FLAGELLATE GRAZING(1).

    Science.gov (United States)

    Yang, Zhou; Kong, Fanxiang; Shi, Xiaoli; Zhang, Min; Xing, Peng; Cao, Huansheng

    2008-06-01

    To investigate the changes in the morphology and polysaccharide content of Microcystis aeruginosa (Kütz.) Kütz. during flagellate grazing, cultures of M. aeruginosa were exposed to grazing Ochromonas sp. for a period of 9 d under controlled laboratory conditions. M. aeruginosa responded actively to flagellate grazing and formed colonies, most of which were made up of several or dozens of cells, suggesting that flagellate grazing may be one of the biotic factors responsible for colony formation in M. aeruginosa. When colonies were formed, the cell surface ultrastructure changed, and the polysaccharide layer on the surface of the cell wall became thicker. This change indicated that synthesis and secretion of extracellular polysaccharide (EPS) of M. aeruginosa cells increased under flagellate grazing pressure. The contents of soluble extracellular polysaccharide (sEPS), bound extracellular polysaccharide (bEPS), and total polysaccharide (TPS) in colonial cells of M. aeruginosa increased significantly compared with those in single cells. This finding suggested that the increased amount of EPS on the cell surface may play a role in keeping M. aeruginosa cells together to form colonies. © 2008 Phycological Society of America.

  6. Pseudomonas aeruginosa inhibits the growth of Cryptococcus species.

    Science.gov (United States)

    Rella, Antonella; Yang, Mo Wei; Gruber, Jordon; Montagna, Maria Teresa; Luberto, Chiara; Zhang, Yong-Mei; Del Poeta, Maurizio

    2012-06-01

    Pseudomonas aeruginosa is a ubiquitous and opportunistic bacterium that inhibits the growth of different microorganisms, including Gram-positive bacteria and fungi such as Candida spp. and Aspergillus fumigatus. In this study, we investigated the interaction between P. aeruginosa and Cryptococcus spp. We found that P. aeruginosa PA14 and, to a lesser extent, PAO1 significantly inhibited the growth of Cryptococcus spp. The inhibition of growth was observed on solid medium by the visualization of a zone of inhibition of yeast growth and in liquid culture by viable cell counting. Interestingly, such inhibition was only observed when P. aeruginosa and Cryptococcus were co-cultured. Minimal inhibition was observed when cell-cell contact was prevented using a separation membrane, suggesting that cell contact is required for inhibition. Using mutant strains of Pseudomonas quinoline signaling, we showed that P. aeruginosa inhibited the growth of Cryptococcus spp. by producing antifungal molecules pyocyanin, a redox-active phenazine, and 2-heptyl-3,4-dihydroxyquinoline (PQS), an extracellular quorum-sensing signal. Because both P. aeruginosa and Cryptococcus neoformans are commonly found in lung infections of immunocompromised patients, this study may have important implication for the interaction of these microbes in both an ecological and a clinical point of view.

  7. Production of Extracellular Lipase from Aspergillus niger by Solid-State Fermentation

    Directory of Open Access Journals (Sweden)

    Janny Coca Armas

    2006-01-01

    Full Text Available Lipase production in Aspergillus niger J-1 was tested using both submerged fermentation (SmF and solid-state fermentation (SSF on a mineral culture medium and wheat bran, respectively. The optimization of the culture medium was carried out for both SmF and SSF. The maximum lipase activity, 1.46 IU/mL, was obtained during the submerged fermentation in a medium containing glucose at 2 % and olive oil at 2 % under conditions of 1 vvm and 450 m–1. However, 9.14 IU/g of dry solid substrate equivalent to 4.8 IU/mL of lipase activity was reached using solid-state fermentation process with a medium containing 0.75 % of ammonium sulphate and 0.34 % of urea. The optimum pH and temperature for enzymatic activity were pH=6 and 40 °C, respectively. The enzyme also exhibited 80 % of its initial activity in neutral and mildly acid media and at temperatures between 20 and 30 °C for a period of 24 hours.

  8. Extracellular Phytase Production by the Wine Yeast S. cerevisiae (Finarome Strain) during Submerged Fermentation.

    Science.gov (United States)

    Kłosowski, Grzegorz; Mikulski, Dawid; Jankowiak, Oliwia

    2018-04-08

    One of the key steps in the production of phytases of microbial origin is selection of culture parameters, followed by isolation of the enzyme and evaluation of its catalytic activity. It was found that conditions for S. cerevisiae yeast culture, strain Finarome, giving the reduction in phytic acid concentration of more than 98% within 24 h of incubation were as follows: pH 5.5, 32 °C, continuous stirring at 80 rpm, the use of mannose as a carbon source and aspartic acid as a source of nitrogen. The highest catalytic activity of the isolated phytase was observed at 37 °C, pH 4.0 and using phytate as substrate at concentration of 5.0 mM. The presence of ethanol in the medium at a concentration of 12% v / v reduces the catalytic activity to above 60%. Properties of phytase derived from S. cerevisiae yeast culture, strain Finarome, indicate the possibility of its application in the form of a cell's free crude protein isolate for the hydrolysis of phytic acid to improve the efficiency of alcoholic fermentation processes. Our results also suggest a possibility to use the strain under study to obtain a fusant derived with specialized distillery strains, capable of carrying out a highly efficient fermentation process combined with the utilization of phytates.

  9. Extracellular production of avicelase by the thermophilic soil bacterium Bacillus sp. SMIA-2

    Directory of Open Access Journals (Sweden)

    Luciana Ribeiro Coutinho Oliveira

    2014-05-01

    Full Text Available Nowadays, the isolation of new bacterial strains that produce enzymes with novel properties is a subject of great relevance to the scientific community. This study, in order to search for producers of new cellulase strains, investigated the avicelase production by thermophilic Bacillus sp. strain SMIA-2. The best avicelase activity was observed in a culture medium containing 0.5% (w v-1 avicel and 0.5% (w v-1 corn steep liquor with initial pH 7.5-8.0 incubated at 50oC. When avicel was replaced in the medium by the treated sugarcane bagasse (0.5%, w v-1 the avicelase activity levels were not affected. Studies on the avicelase characterization revealed that the optimum pH of the enzyme was found to be 8.5 and the enzyme retained more than 80% of its activity after incubation at room temperature for 2h at pH 6.5-8.5. The optimum temperature of this enzyme was 70oC and the enzyme retained 67% of the original activity after 20 min. of heat treatment at 70oC. Avicelase was stimulated by Mn2+ and Co2+, whereas Hg2+ greatly inhibited the enzyme activity

  10. Identification, recombinant production and partial biochemical characterization of an extracellular cold-active serine-metalloprotease from an Antarctic Pseudomonas isolate

    Directory of Open Access Journals (Sweden)

    Natalia Fullana

    2017-08-01

    Full Text Available Cold-adapted enzymes are generally derived from psychrophilic microorganisms and have features that make them very attractive for industrial and biotechnological purposes. In this work, we identified a 50 kDa extracellular protease (MP10 from the Antarctic isolate Pseudomonas sp. AU10. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and partially characterized. MP10 is an alkaline thermosensitive serine-metallo protease with optimal activity at pH 8.0 and 40 ℃, in the presence of 1.5 mM Ca2+. MP10 showed 100% residual activity and stability (up to 60 min when incubated with 7% of non-ionic surfactants (Triton X-100, Tween-80 and Tween-20 and 1.5% of the oxidizing agent hydrogen peroxide. The 3D MP10 structure was predicted and compared with the crystal structure of mesophilic homologous protease produced by Pseudomonas aeruginosa PA01 (reference strain and other proteases, showing similarity in surface area and volume of proteins, but a significantly higher surface pocket area and volume of MP10. The observed differences presumably may explain the enhanced activity of MP10 for substrate binding at low temperatures. These results give insight to the potential use of MP10 in developing new biotechnologically processes active at low to moderate temperatures, probably with focus in the detergent industry.

  11. Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy.

    Science.gov (United States)

    Feng, Yue; Liu, Song; Jiao, Yun; Gao, Hui; Wang, Miao; Du, Guocheng; Chen, Jian

    2017-02-01

    L-asparaginase (EC 3.5.1.1, ASN) exhibits great commercial value due to its uses in the food and medicine industry. In this study, we reported the enhanced expression of type II ASN from Bacillus subtilis 168 in B. subtilis WB600 through a combined strategy. First, eight signal peptides (the signal peptide of the ASN, ywbN, yvgO, amyE, oppA, vpr, lipA, and wapA) were used for ASN secretion in B. subtilis by using Hpa II promoter, respectively. The signal peptide wapA achieved the highest extracellular ASN activity (28.91 U/mL). Second, Hpa II promoter was replaced by a strong promoter, P43 promoter, resulting in 38.1 % enhanced ASN activity. By two rounds of error-prone PCR mutation, the P43 promoter variants with remarkably enhanced strength (D7, E2, H6, B2, and F3) were identified. B2 (-28: A → G, -13: A → G) achieved ASN activity up to 51.13 U/mL. Third, after deletion of the N-terminal 25-residues, ASN activity reached 102.41 U/mL, which was 100 % higher than that of the intact ASN. At last, the extracellular ASN of the B. subtilis arrived at 407.6 U/mL (2.5 g/L of ASN protein) in a 3-L bioreactor by using a fed-batch strategy. The purified ASN showed maximal activity at 65 °C and its half-life at 65 °C was 61 min. The K m and k cat of the ASN were 5.29 mM and 54.4 s -1 , respectively. To the best of our knowledge, we obtained the highest yield of ASN in a food-grade host ever reported, which may benefit the industrial production and application of ASN.

  12. One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm

    Directory of Open Access Journals (Sweden)

    Parinaz Ghadam

    2017-05-01

    Full Text Available Objective(s: Pseudomonas aeruginosais a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Alginate lyase gene (algl is a member of alginate producing operon which by glycosidase activity produces primer for other enzymes in this cluster. Also this activity can destroy the extracellular alginate; therefore this enzyme participates in alginate production and destruction pathway. Alginate lyase causes detachment of a biofilm by reducing its adhesion to the surfaces, and increases phagocytosis and antibiotic susceptibility. In this study, alginate lyase was purified in just one step and its properties were investigated. Materials and Methods: The purification was done by affinity chromatography, analysed by SDS-PAGE, and its effect on P. aeruginosa biofilms was surveyed by micro titer plate assay and SEM. The substrate specificity of the enzyme was determined by PCR. Results: Alginate lyase from isolate 48 was purified in one step. It is more thermally resistant than alginate lyase from Pseudomonas aeruginosa PAO1 and poly M, poly G and poly MG alginate were the substrate of this enzyme. Moreover, it has an eradication effect on biofilms from P. aeruginosa 48 and PAO1. Conclusion: In this study an alginate lyase with many characteristics suitable in medicine such as thermal stability, effective on poly M alginate, and bacterial biofilm destructive was introduced and purified.

  13. Biofilm formation and disinfectant resistance of Salmonella sp. in mono- and dual-species with Pseudomonas aeruginosa.

    Science.gov (United States)

    Pang, X Y; Yang, Y S; Yuk, H G

    2017-09-01

    This study aimed to evaluate the biofilm formation and disinfectant resistance of Salmonella cells in mono- and dual-species biofilms with Pseudomonas aeruginosa, and to investigate the role of extracellular polymeric substances (EPS) in the protection of biofilms against disinfection treatment. The populations of Salmonella in mono- or dual-species biofilms with P. aeruginosa on stainless steel (SS) coupons were determined before and after exposure to commercial disinfectant, 50 μg ml -1 chlorine or 200 μg ml -1 Ecolab ® Whisper™ V (a blend of four effective quaternary ammonium compounds (QAC)). In addition, EPS amount from biofilms was quantified and biofilm structures were observed using scanning electron microscopy (SEM). Antagonistic interactions between Salmonella and P. aeruginosa resulted in lower planktonic population level of Salmonella, and lower density in dual-species biofilms compared to mono-species biofilms. The presence of P. aeruginosa significantly enhanced disinfectant resistance of S. Typhimurium and S. Enteritidis biofilm cells for 2 days, and led to an average of 50% increase in polysaccharides amount in dual-species biofilms than mono-species biofilms of Salmonella. Microscopy observation showed the presence of large microcolonies covered by EPS in dual-species biofilms but not in mono-species ones. The presence of P. aeruginosa in dual-species culture inhibited the growth of Salmonella cells in planktonic phase and in biofilms, but protected Salmonella cells in biofilms from disinfection treatment, by providing more production of EPS in dual-species biofilms than mono-species ones. This study provides insights into inter-species interaction, with regard to biofilm population dynamics and disinfectant resistance. Thus, a sanitation protocol should be designed considering the protective role of secondary species to pathogens in biofilms on SS surface which has been widely used at food surfaces and manufacturers. © 2017 The Society

  14. Membrane-anchored MucR mediates nitrate-dependent regulation of alginate production in Pseudomonas aeruginosa

    KAUST Repository

    Wang, Yajie; Hay, Iain D.; Rehman, Zahid Ur; Rehm, Bernd H A

    2015-01-01

    of MucR impaired alginate promoter activity and global c-di-GMP levels, alginate yields were not directly correlated with alginate promoter activity or c-di-GMP levels, suggesting that nitrate and MucR modulate alginate production at a post

  15. Standardized chemical synthesis of Pseudomonas aeruginosa pyocyanin

    Directory of Open Access Journals (Sweden)

    Rajkumar Cheluvappa

    2014-01-01

    As we have extracted pyocyanin both from P. aeruginosa cultures, and via chemical synthesis; we know the procedural and product-quality differences. We endorse the relative ease, safety, and convenience of using the chemical synthesis described here. Crucially, our “naturally endotoxin-free” pyocyanin can be extracted easily without using infectious bacteria.

  16. Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.

    Science.gov (United States)

    Dagar, Vikas Kumar; Adivitiya; Devi, Nirmala; Khasa, Yogender Pal

    2016-10-01

    Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni-NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % β-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.

  17. Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles.

    Science.gov (United States)

    Mota, Rita; Pereira, Sara B; Meazzini, Marianna; Fernandes, Rui; Santos, Arlete; Evans, Caroline A; De Philippis, Roberto; Wright, Phillip C; Tamagnini, Paula

    2015-04-29

    The effects of several heavy metals on the growth/survival, EPS production, ultrastructure and protein profiles of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110 were evaluated. Our results clearly show that each heavy metal affects the cells in a particular manner, triggering distinctive responses. Concerning chronic exposure, cells were more affected by Cu(2+) followed by Pb(2+), Cd(2+), and Li(+). The presence of metal leads to remarkable ultrastructural changes, mainly at the thylakoid level. The comparison of the proteomes (iTRAQ) allowed to follow the stress responses and to distinguish specific effects related to the time of exposure and/or the concentration of an essential (Cu(2+)) and a non-essential (Cd(2+)) metal. The majority of the proteins identified and with fold changes were associated with photosynthesis, CO2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism. Moreover, our results indicate that during chronic exposure to sub-lethal concentrations of Cu(2+), the cells tune down their metabolic rate to invest energy in the activation of detoxification mechanisms, which eventually result in a remarkable recovery. In contrast, the toxic effects of Cd(2+) are cumulative. Unexpectedly, the amount of released polysaccharides (RPS) was not enhanced by the presence of heavy metals. This work shows the holistic effects of different heavy metals on the cells of the highly efficient EPS-producer the cyanobacterium Cyanothece sp. CCY 0110. The growth/survival, EPS production, ultrastructure, protein profiles and stress response were evaluated. The knowledge generated by this study will contribute to the implementation of heavy-metal removal systems based on cyanobacteria EPS or their isolated polymers. Copyright © 2015. Published by Elsevier B.V.

  18. Invasive lobular carcinoma with extracellular mucin production-a novel pattern of lobular carcinomas of the breast. Clinico-pathological description of eight cases.

    Science.gov (United States)

    Cserni, Gábor; Floris, Giuseppe; Koufopoulos, Nektarios; Kovács, Anikó; Nonni, Afroditi; Regitnig, Peter; Stahls, Anders; Varga, Zsuzsanna

    2017-07-01

    Invasive lobular carcinoma of the breast is known to produce intracellular mucin and has been recognized in single-case reports to show extracellular mucin production, as well. This latter morphology is not only rare but must also be under- or misdiagnosed. The aim was to better characterize this entity. Cases of lobular cancers demonstrating extracellular mucin formation were identified in a multi-institutional effort and their clinical and morphologic features were assessed. Immunohistochemistry was used to characterize the E-cadherin-membrane complex, neuroendocrine differentiation, and to some extent, mucin formation. All but one of the eight cases occurred in postmenopausal patients. Extracellular mucin production was present in 5 to 50% of the tumour samples and rarely also appeared in nodal and distant metastases. The tumours were completely E-cadherin negative and showed cytoplasmic p120 positivity. The majority (n = 6/8) was also completely negative for β-catenin, but two tumours displayed focal β-catenin positivity in the mucinous area. MUC1 and MUC2 expression was observed in all and 7/8 tumours, respectively; neuroendocrine differentiation was present in only one. Invasive lobular carcinoma with extracellular mucin formation is a rare morphologic variant of lobular carcinoma prone to be misdiagnosed and warranting further studies.

  19. Pseudomonas aeruginosa Biofilm, a Programmed Bacterial Life for Fitness.

    Science.gov (United States)

    Lee, Keehoon; Yoon, Sang Sun

    2017-06-28

    A biofilm is a community of microbes that typically inhabit on surfaces and are encased in an extracellular matrix. Biofilms display very dissimilar characteristics to their planktonic counterparts. Biofilms are ubiquitous in the environment and influence our lives tremendously in both positive and negative ways. Pseudomonas aeruginosa is a bacterium known to produce robust biofilms. P. aeruginosa biofilms cause severe problems in immunocompromised patients, including those with cystic fibrosis or wound infection. Moreover, the unique biofilm properties further complicate the eradication of the biofilm infection, leading to the development of chronic infections. In this review, we discuss the history of biofilm research and general characteristics of bacterial biofilms. Then, distinct features pertaining to each stage of P. aeruginosa biofilm development are highlighted. Furthermore, infections caused by biofilms on their own or in association with other bacterial species ( i.e. , multispecies biofilms) are discussed in detail.

  20. The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes.

    Science.gov (United States)

    Azzolina, B A; Yuan, X; Anderson, M S; El-Sherbeini, M

    2001-04-01

    We have cloned the Pseudomonas aeruginosa cell wall biosynthesis and cell division gene cluster that corresponds to the mra operon in the 2-min region of the Escherichia coli chromosome. The organization of the two chromosomal regions in P. aeruginosa and E. coli is remarkably similar with the following gene order: pbp3/pbpB, murE, murF, mraY, murD, ftsW, murG, murC, ddlB, ftsQ, ftsA, ftsZ, and envA/LpxC. All of the above P. aeruginosa genes are transcribed from the same strand of DNA with very small, if any, intragenic regions, indicating that these genes may constitute a single operon. All five amino acid ligases, MurC, MurD, MurE, MurF, and DdlB, in addition to MurG and MraY were cloned in expression vectors. The four recombinant P. aeruginosa Mur ligases, MurC, MurD, MurE, and MurF were overproduced in E. coli and purified as active enzymes. Copyright 2001 Academic Press.

  1. Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor β.

    Science.gov (United States)

    Rawal, S Y; Dabbous, M Kh; Tipton, D A

    2012-06-01

    Marijuana (Cannabis sativa) use may be associated with gingival enlargement, resembling that caused by phenytoin. Cannabidiol (CBD), a nonpsychotropic Cannabis derivative, is structurally similar to phenytoin. While there are many reports on effects of phenytoin on human gingival fibroblasts, there is no information on effects of Cannabis components on these cells. The objective of this study was to determine effects of CBD on human gingival fibroblast fibrogenic and matrix-degrading activities. Fibroblasts were incubated with CBD in serum-free medium for 1-6 d. The effect of CBD on cell viability was determined by measuring activity of a mitochondrial enzyme. The fibrogenic molecule transforming growth factor β and the extracellular matrix molecule fibronectin were measured by ELISA. Pro-MMP-1 and total MMP-2 were measured by ELISA. Activity of MMP-2 was determined via a colorimetric assay in which a detection enzyme is activated by active MMP-2. Data were analysed using ANOVA and Scheffe's F procedure for post hoc comparisons. Cannabidiol had little or no significant effect on cell viability. Low CBD concentrations increased transforming growth factor β production by as much as 40% (p Cannabidiol increased fibronectin production by as much as approximately 100% (p < 0.001). Lower CBD concentrations increased MMP production, but the highest concentrations decreased production of both MMPs (p < 0.05) and decreased MMP-2 activity (p < 0.02). The data suggest that the CBD may promote fibrotic gingival enlargement by increasing gingival fibroblast production of transforming growth factor β and fibronectin, while decreasing MMP production and activity. © 2011 John Wiley & Sons A/S.

  2. Purification, characterization, molecular cloning and extracellular production of a phospholipase A1 from Streptomyces albidoflavus NA297.

    Science.gov (United States)

    Sugimori, Daisuke; Kano, Kota; Matsumoto, Yusaku

    2012-01-01

    A novel metal ion-independent phospholipase A1 of Streptomyces albidoflavus isolated from Japanese soil has been purified and characterized. The enzyme consists of a 33-residue N-terminal signal secretion sequence and a 269-residue mature protein with a deduced molecular weight of 27,199. Efficient and extracellular production of the recombinant enzyme was successfully achieved using Streptomyces lividans cells and an expression vector. A large amount (25 mg protein, 14.7 kU) of recombinant enzyme with high specific activity (588 U/mg protein) was purified by simple purification steps. The maximum activity was found at pH 7.2 and 50 °C. At pH 7.2, the enzyme preferably hydrolyzed phosphatidic acid and phosphatidylserine; however, the substrate specificity was dependent on the reaction pH. The enzyme hydrolyzed lysophosphatidylcholine and not triglyceride and the p-nitrophenyl ester of fatty acids. At the reaction equilibrium, the molar ratio of released free fatty acids (sn-1:sn-2) was 63:37. The hydrolysis of phosphatidic acid at 50 °C and pH 7.2 gave apparent V max and k cat values of 1389 μmol min(-1) mg protein(-1) and 630 s(-1), respectively. The apparent K m and k cat/K m values were 2.38 mM and 265 mM(-1) s(-1), respectively. Mutagenesis analysis showed that Ser11 is essential for the catalytic function of the enzyme and the active site may include residues Ser216 and His218.

  3. Flavonoids Suppress Pseudomonas aeruginosa Virulence through Allosteric Inhibition of Quorum-sensing Receptors*

    Science.gov (United States)

    Paczkowski, Jon E.; Mukherjee, Sampriti; McCready, Amelia R.; Cong, Jian-Ping; Aquino, Christopher J.; Kim, Hahn; Henke, Brad R.; Smith, Chari D.; Bassler, Bonnie L.

    2017-01-01

    Quorum sensing is a process of cell-cell communication that bacteria use to regulate collective behaviors. Quorum sensing depends on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. In many bacterial species, quorum sensing controls virulence factor production. Thus, disrupting quorum sensing is considered a promising strategy to combat bacterial pathogenicity. Several members of a family of naturally produced plant metabolites called flavonoids inhibit Pseudomonas aeruginosa biofilm formation by an unknown mechanism. Here, we explore this family of molecules further, and we demonstrate that flavonoids specifically inhibit quorum sensing via antagonism of the autoinducer-binding receptors, LasR and RhlR. Structure-activity relationship analyses demonstrate that the presence of two hydroxyl moieties in the flavone A-ring backbone are essential for potent inhibition of LasR/RhlR. Biochemical analyses reveal that the flavonoids function non-competitively to prevent LasR/RhlR DNA binding. Administration of the flavonoids to P. aeruginosa alters transcription of quorum sensing-controlled target promoters and suppresses virulence factor production, confirming their potential as anti-infectives that do not function by traditional bacteriocidal or bacteriostatic mechanisms. PMID:28119451

  4. Moderate extracellular acidification inhibits capsaicin-induced cell death through regulating calcium mobilization, NF-{kappa}B translocation and ROS production in synoviocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Fen; Yang, Shuang; Zhao, Dan; Zhu, Shuyan; Wang, Yuxiang [Department of Biophysics, School of Physics and Key Laboratory of Bioactive Materials of Education Ministry, Nankai University, Tianjin 300071 (China); Li, Junying, E-mail: jyli04@nankai.edu.cn [Department of Biophysics, School of Physics and Key Laboratory of Bioactive Materials of Education Ministry, Nankai University, Tianjin 300071 (China)

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Moderate extracellular acidification regulates intracellular Ca{sup 2+} mobilization. Black-Right-Pointing-Pointer Moderate acidification activates NF-{kappa}B nuclear translocation in synoviocytes. Black-Right-Pointing-Pointer Moderate acidification depresses the ROS production induced by capsaicin. Black-Right-Pointing-Pointer Moderate acidification inhibits capsaicin-caused synoviocyte death. -- Abstract: We previously show the expression of transient receptor potential vanilloid 1 (TRPV1) in primary synoviocytes from collagen-induced arthritis (CIA) rats. Capsaicin and lowered extracellular pH from 7.4 to 5.5 induce cell death through TRPV1-mediated Ca{sup 2+} entry and reactive oxygen species (ROS) production. However, under the pathological condition in rheumatoid arthritis, the synovial fluid is acidified to a moderate level (about pH 6.8). In the present study, we examined the effects of pH 6.8 on the TRPV1-mediated cell death. Our finding is different or even opposite from what was observed at pH 5.5. We found that the moderate extracellular acidification (from pH 7.4 to 6.8) inhibited the capsaicin-induced Ca{sup 2+} entry through attenuating the activity of TRPV1. In the mean time, it triggered a phospholipse C (PLC)-related Ca{sup 2+} release from intracellular stores. The nuclear translocation of NF-{kappa}B was found at pH 6.8, and this also depends on PLC activation. Moreover, the capsaicin-evoked massive ROS production and cell death were depressed at pH 6.8, both of which are dependent on the activation of PLC and NF-{kappa}B. Taken together, these results suggested that the moderate extracellular acidification inhibited the capsaicin-induced synoviocyte death through regulating Ca{sup 2+} mobilization, activating NF-{kappa}B nuclear translocation and depressing ROS production.

  5. Moderate extracellular acidification inhibits capsaicin-induced cell death through regulating calcium mobilization, NF-κB translocation and ROS production in synoviocytes

    International Nuclear Information System (INIS)

    Hu, Fen; Yang, Shuang; Zhao, Dan; Zhu, Shuyan; Wang, Yuxiang; Li, Junying

    2012-01-01

    Highlights: ► Moderate extracellular acidification regulates intracellular Ca 2+ mobilization. ► Moderate acidification activates NF-κB nuclear translocation in synoviocytes. ► Moderate acidification depresses the ROS production induced by capsaicin. ► Moderate acidification inhibits capsaicin-caused synoviocyte death. -- Abstract: We previously show the expression of transient receptor potential vanilloid 1 (TRPV1) in primary synoviocytes from collagen-induced arthritis (CIA) rats. Capsaicin and lowered extracellular pH from 7.4 to 5.5 induce cell death through TRPV1-mediated Ca 2+ entry and reactive oxygen species (ROS) production. However, under the pathological condition in rheumatoid arthritis, the synovial fluid is acidified to a moderate level (about pH 6.8). In the present study, we examined the effects of pH 6.8 on the TRPV1-mediated cell death. Our finding is different or even opposite from what was observed at pH 5.5. We found that the moderate extracellular acidification (from pH 7.4 to 6.8) inhibited the capsaicin-induced Ca 2+ entry through attenuating the activity of TRPV1. In the mean time, it triggered a phospholipse C (PLC)-related Ca 2+ release from intracellular stores. The nuclear translocation of NF-κB was found at pH 6.8, and this also depends on PLC activation. Moreover, the capsaicin-evoked massive ROS production and cell death were depressed at pH 6.8, both of which are dependent on the activation of PLC and NF-κB. Taken together, these results suggested that the moderate extracellular acidification inhibited the capsaicin-induced synoviocyte death through regulating Ca 2+ mobilization, activating NF-κB nuclear translocation and depressing ROS production.

  6. Interactions between the antimicrobial agent triclosan and the bloom-forming cyanobacteria Microcystis aeruginosa

    International Nuclear Information System (INIS)

    Huang, Xiaolong; Tu, Yenan; Song, Chaofeng; Li, Tiancui; Lin, Juan; Wu, Yonghong; Liu, Jiantong; Wu, Chenxi

    2016-01-01

    Highlights: • Triclosan inhibit the growth and photosynthesis of M. aeruginosa at environmental relevant level. • TEM imaging showed destruction of M. aeruginosa cell ultrastructure during triclosan exposure. • Triclosan can be biotransformed by M. aeruginosa with methylation as a major pathway. • Presence of M. aeruginosa enhanced the photodegradation of triclosan. - Abstract: Cyanobacteria can co-exist in eutrophic waters with chemicals or other substances derived from personal care products discharged in wastewater. In this work, we investigate the interactions between the antimicrobial agent triclosan (TCS) and the bloom-forming cyanobacteria Microcystis aeruginosa. M. aeruginosa was very sensitive to TCS with the 96 h lowest observed effect concentration of 1.0 and 10 μg/L for inhibition of growth and photosynthetic activity, respectively. Exposure to TCS at environmentally relevant levels (0.1–2.0 μg/L) also affected the activities of superoxide dismutase (SOD) and the generation of reduced glutathione (GSH), while microcystin production was not affected. Transmission electron microscope (TEM) examination showed the destruction of M. aeruginosa cell ultrastructure during TCS exposure. TCS however, can be biotransformed by M. aeruginosa with methylation as a major biotransformation pathway. Furthermore, the presence of M. aeruginosa in solution promoted the photodegradation of TCS. Overall, our results demonstrate that M. aeruginosa plays an important role in the dissipation of TCS in aquatic environments but high residual TCS can exert toxic effects on M. aeruginosa.

  7. Interactions between the antimicrobial agent triclosan and the bloom-forming cyanobacteria Microcystis aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xiaolong [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Wuhan Zhongke Hydrobiological Environment Engineering Co., Ltd, Wuhan 430071 (China); Tu, Yenan; Song, Chaofeng; Li, Tiancui; Lin, Juan [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Graduate University of the Chinese Academy of Sciences, Beijing 100049 (China); Wu, Yonghong [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Sciences, Chinese Academy of Sciences, Nanjing 210008 (China); Liu, Jiantong [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Wu, Chenxi, E-mail: chenxi.wu@ihb.ac.cn [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China)

    2016-03-15

    Highlights: • Triclosan inhibit the growth and photosynthesis of M. aeruginosa at environmental relevant level. • TEM imaging showed destruction of M. aeruginosa cell ultrastructure during triclosan exposure. • Triclosan can be biotransformed by M. aeruginosa with methylation as a major pathway. • Presence of M. aeruginosa enhanced the photodegradation of triclosan. - Abstract: Cyanobacteria can co-exist in eutrophic waters with chemicals or other substances derived from personal care products discharged in wastewater. In this work, we investigate the interactions between the antimicrobial agent triclosan (TCS) and the bloom-forming cyanobacteria Microcystis aeruginosa. M. aeruginosa was very sensitive to TCS with the 96 h lowest observed effect concentration of 1.0 and 10 μg/L for inhibition of growth and photosynthetic activity, respectively. Exposure to TCS at environmentally relevant levels (0.1–2.0 μg/L) also affected the activities of superoxide dismutase (SOD) and the generation of reduced glutathione (GSH), while microcystin production was not affected. Transmission electron microscope (TEM) examination showed the destruction of M. aeruginosa cell ultrastructure during TCS exposure. TCS however, can be biotransformed by M. aeruginosa with methylation as a major biotransformation pathway. Furthermore, the presence of M. aeruginosa in solution promoted the photodegradation of TCS. Overall, our results demonstrate that M. aeruginosa plays an important role in the dissipation of TCS in aquatic environments but high residual TCS can exert toxic effects on M. aeruginosa.

  8. Ginger Extract Inhibits Biofilm Formation by Pseudomonas aeruginosa PA14

    Science.gov (United States)

    Kim, Han-Shin; Park, Hee-Deung

    2013-01-01

    Bacterial biofilm formation can cause serious problems in clinical and industrial settings, which drives the development or screening of biofilm inhibitors. Some biofilm inhibitors have been screened from natural products or modified from natural compounds. Ginger has been used as a medicinal herb to treat infectious diseases for thousands of years, which leads to the hypothesis that it may contain chemicals inhibiting biofilm formation. To test this hypothesis, we evaluated ginger’s ability to inhibit Pseudomonas aeruginosa PA14 biofilm formation. A static biofilm assay demonstrated that biofilm development was reduced by 39–56% when ginger extract was added to the culture. In addition, various phenotypes were altered after ginger addition of PA14. Ginger extract decreased production of extracellular polymeric substances. This finding was confirmed by chemical analysis and confocal laser scanning microscopy. Furthermore, ginger extract formed noticeably less rugose colonies on agar plates containing Congo red and facilitated swarming motility on soft agar plates. The inhibition of biofilm formation and the altered phenotypes appear to be linked to a reduced level of a second messenger, bis-(3′-5′)-cyclic dimeric guanosine monophosphate. Importantly, ginger extract inhibited biofilm formation in both Gram-positive and Gram-negative bacteria. Also, surface biofilm cells formed with ginger extract detached more easily with surfactant than did those without ginger extract. Taken together, these findings provide a foundation for the possible discovery of a broad spectrum biofilm inhibitor. PMID:24086697

  9. Extracellular polysaccharide production by a strain of Pleurotus djamor isolated in the south of Brazil and antitumor activity on Sarcoma 180

    Directory of Open Access Journals (Sweden)

    Gisele Martini Borges

    2013-12-01

    Full Text Available Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L and pH values (3.0-4.0 to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0 led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (Y P/S = 0.072 and maximum extracellular polysaccharide productivity (Q Pmax = 11.26 mg/L.h were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (Y X/S = 0.24 and maximal biomass productivity (Q Xmax = 32.2 mg/L.h were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm³ in untreated group and 1.6 cm³ in treated group (p = 0.05 with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells.

  10. Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase.

    Science.gov (United States)

    El-Ghonemy, Dina Helmy; Ali, Thanaa Hamed; El-Bondkly, Ahmed Mohamed; Moharam, Maysa El-Sayed; Talkhan, Fatma Nabeeh

    2014-11-01

    Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH4Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.

  11. Oxalate metabolism in liquid cultures of Ceriporiopsis subvermispora : a possible pathway for extracellular H2O2 production

    Science.gov (United States)

    Ulises. Urzua; Claudio. Aguilar; Philip J. Kersten; Rafael. Vicuna

    1998-01-01

    In this work, the source of extracellular hydrogen peroxide in cultures of Ceriporiopsis subvermispora was investigated. A thorough search for the presence in the growth medium of oxidases known to be produced by other fungi gave negative results. We therefore explored the prospect that H2O2 might arise from the oxidation of organic acids by MnP. Both oxalate and...

  12. Levels of Circulating MMCN-151, a Degradation Product of Mimecan, Reflect Pathological Extracellular Matrix Remodeling in Apolipoprotein E Knockout Mice

    DEFF Research Database (Denmark)

    Barascuk, N; Vassiliadis, E; Zheng, Qiuju

    2011-01-01

    Arterial extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) is one of the major hallmarks of atherosclerosis. Mimecan, also known as osteoglycin has been implicated in the integrity of the ECM. This study assessed the validity of an enzyme-linked immunosorbent assay (ELISA...

  13. Investigation of Pseudomonas aeruginosa quorum-sensing signaling system for identifying multiple inhibitors using molecular docking and structural analysis methodology.

    Science.gov (United States)

    Soheili, Vahid; Bazzaz, Bibi Sedigheh Fazly; Abdollahpour, Nooshin; Hadizadeh, Farzin

    2015-12-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and a common Gram-negative bacterium in hospital-acquired infections. It causes death in many burn victims, cystic-fibrosis and neutropenic-cancer patients. It is known that P. aeruginosa biofilm maturation and production of cell-associated and extracellular virulence factors such as pyocyanin, elastase and rhamnolipids are under the control of a quorum-sensing (QS) system. Among several proteins involved in the Pseudomonas QS mechanism, LasR and PqsE play an important role in its cascade signaling system. They can cause increases in QS factors, biofilm maturation, and the production of virulence factors. Therefore, inhibition of these proteins can reduce the pathogenicity of P. aeruginosa. According to the structure of corresponding auto-inducers bound to these proteins, in silico calculations were performed with some non-steroidal anti-inflammatory drugs (NSAIDs) to estimate possible interactions and find the co-inhibitors of LasR and PqsE. The results showed that oxicams (Piroxicam and Meloxicam) can interact well with active sites of both proteins with the Ki of 119.43 nM and 4.0 μM for Meloxicam and 201.39 nM and 4.88 μM against LasR and PqsE, respectively. These findings suggested that Piroxicam and Meloxicam can be used as potential inhibitors for control of the P. aeruginosa QS signaling system and biofilm formation, and may be used in the design of multiple inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. PAMDB: a comprehensive Pseudomonas aeruginosa metabolome database.

    Science.gov (United States)

    Huang, Weiliang; Brewer, Luke K; Jones, Jace W; Nguyen, Angela T; Marcu, Ana; Wishart, David S; Oglesby-Sherrouse, Amanda G; Kane, Maureen A; Wilks, Angela

    2018-01-04

    The Pseudomonas aeruginosaMetabolome Database (PAMDB, http://pseudomonas.umaryland.edu) is a searchable, richly annotated metabolite database specific to P. aeruginosa. P. aeruginosa is a soil organism and significant opportunistic pathogen that adapts to its environment through a versatile energy metabolism network. Furthermore, P. aeruginosa is a model organism for the study of biofilm formation, quorum sensing, and bioremediation processes, each of which are dependent on unique pathways and metabolites. The PAMDB is modelled on the Escherichia coli (ECMDB), yeast (YMDB) and human (HMDB) metabolome databases and contains >4370 metabolites and 938 pathways with links to over 1260 genes and proteins. The database information was compiled from electronic databases, journal articles and mass spectrometry (MS) metabolomic data obtained in our laboratories. For each metabolite entered, we provide detailed compound descriptions, names and synonyms, structural and physiochemical information, nuclear magnetic resonance (NMR) and MS spectra, enzymes and pathway information, as well as gene and protein sequences. The database allows extensive searching via chemical names, structure and molecular weight, together with gene, protein and pathway relationships. The PAMBD and its future iterations will provide a valuable resource to biologists, natural product chemists and clinicians in identifying active compounds, potential biomarkers and clinical diagnostics. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Cylindrospermopsin induced changes in growth, toxin production and antioxidant response of Acutodesmus acuminatus and Microcystis aeruginosa under differing light and nitrogen conditions.

    Science.gov (United States)

    Chia, Mathias Ahii; Cordeiro-Araújo, Micheline Kézia; Lorenzi, Adriana Sturion; Bittencourt-Oliveira, Maria do Carmo

    2017-08-01

    Growing evidence suggests that some bioactive metabolites (e.g. cyanotoxins) produced by cyanobacteria have allelopathic potential, due to their inhibitory or stimulatory effects on competing species. Although a number of studies have shown that the cyanotoxin cylindrospermopsin (CYN) has variable effects on phytoplankton species, the impact of changing physicochemical conditions on its allelopathic potential is yet to be investigated. We investigated the physiological response of Microcystis aeruginosa (Cyanobacteria) and Acutodesmus acuminatus (Chlorophyta) to CYN under varying nitrogen and light conditions. At 24h, higher microcystins content of M. aeruginosa was recorded under limited light in the presence of CYN, while at 120h the lower levels of the toxins were observed in the presence of CYN under optimum light. Total MCs concentration was significantly (pnitrogen conditions. On the other hand, there were no significant (p>0.05) changes in total MCs concentrations after exposure to CYN under high nitrogen conditions. As expected, limited light and limited nitrogen conditions resulted in lower cell density of both species, while CYN only significantly (pnitrogen condition, the presence of CYN increased internal H 2 O 2 content of both species, which resulted in significant (pnitrogen. These results showed that M. aeruginosa and A. acuminatus have variable response to CYN under changing light and nitrogen conditions, and demonstrate that need to consider changes in physicochemical conditions during ecotoxicological and ecophysiological investigations. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Screening of Coprinus species for the production of extracellular peroxidase and evaluation of its applicability to the treatment of aqueous phenol

    International Nuclear Information System (INIS)

    Ikehata, K.; Buchanan, I.D.

    2002-01-01

    Twenty-nine strains of Coprinus species comprising 16 strains from 12 identified species and 13 unidentified strains as well as one Arthromyces ramosus strain were screened for the production of extracellular peroxidase. Among the fungi examined, three strains of C. cinereus, UAMH 4103, UAMH 7907 and IFO 30116, as well as one Coprinus sp., UAMH 10067, which was isolated from urea treated soil, were shown to produce large amounts of extracellular peroxidase. The performance of crude peroxidase, obtained from liquid culture of C. cinereus, (CIP) on phenol removal from synthetic wastewater was evaluated and compared with that of purified horseradish peroxidase and A. ramosus peroxidase. Although crude CIP performed better than both purified enzymes, its superiority vanished in the presence of poly(ethylene glycol), a known protective agent of peroxidase. This suggests that the residual soluble substances present in crude CIP have protective effects similar to those of poly(ethylene glycol). (author)

  17. Flow environment and matrix structure interact to determine spatial competition in Pseudomonas aeruginosa biofilms.

    Science.gov (United States)

    Nadell, Carey D; Ricaurte, Deirdre; Yan, Jing; Drescher, Knut; Bassler, Bonnie L

    2017-01-13

    Bacteria often live in biofilms, which are microbial communities surrounded by a secreted extracellular matrix. Here, we demonstrate that hydrodynamic flow and matrix organization interact to shape competitive dynamics in Pseudomonas aeruginosa biofilms. Irrespective of initial frequency, in competition with matrix mutants, wild-type cells always increase in relative abundance in planar microfluidic devices under simple flow regimes. By contrast, in microenvironments with complex, irregular flow profiles - which are common in natural environments - wild-type matrix-producing and isogenic non-producing strains can coexist. This result stems from local obstruction of flow by wild-type matrix producers, which generates regions of near-zero shear that allow matrix mutants to locally accumulate. Our findings connect the evolutionary stability of matrix production with the hydrodynamics and spatial structure of the surrounding environment, providing a potential explanation for the variation in biofilm matrix secretion observed among bacteria in natural environments.

  18. Effect of a pesticide on the extracellular slime production and pathogenicity of a non-target phytopathogen

    Energy Technology Data Exchange (ETDEWEB)

    Balasubramanian, A [Tamil Nadu Agricultural Univ., Coimbatore (India); Nilakantan, Gita [University of Agricultural Sciences, Bangalore (India)

    1978-12-01

    Aldicarb (2 methyl thio) propionaldehyde-0-(methyl carbamoyl oxime), a systemic insecticide treatment altered the quantity and the quality of the extracellular polysaccharides (slime) produced by Pseudomonas solanacearum. Although 5 ppm (normal dose) aldicarb treatment reduced the quality of polysaccharides produced by the cells, the incorporation of /sup 14/C (glucose) label and the reducing sugar contents was higher than the other treatments. Chromatographic analysis of the hydrolysed polysaccharides showed that aldicarb treatment altered their qualitative composition also. The extracellular polysaccharides produced by the pathogen treated with 5 ppm aldicarb caused wilting of tomato seedlings earlier than others, indicating thereby, that the wilt inducing factor in the slime was altered by the pesticide treatment. The limited translocation of the /sup 14/C labelled polysaccharides in the wilted seedlings indicated mechanical blocking of the vascular system of the plants.

  19. Effect of a pesticide on the extracellular slime production and pathogenicity of a non-target phytopathogen

    International Nuclear Information System (INIS)

    Balasubramanian, A.; Nilakantan, Gita

    1978-01-01

    Aldicarb (2 methyl thio) propionaldehyde-0-(methyl carbamoyl oxime), a systemic insecticide treatment altered the quantity and the quality of the extracellular polysaccharides (slime) produced by Pseudomonas solanacearum. Although 5 ppm (normal dose) aldicarb treatment reduced the quality of polysaccharides produced by the cells, the incorporation of 14 C (glucose) label and the reducing sugar contents was higher than the other treatments. Chromatographic analysis of the hydrolysed polysaccharides showed that aldicarb treatment altered their qualitative composition also. The extracellular polysaccharides produced by the pathogen treated with 5 ppm aldicarb caused wilting of tomato seedlings earlier than others, indicating thereby, that the wilt inducing factor in the slime was altered by the pesticide treatment. The limited translocation of the 14 C labelled polysaccharides in the wilted seedlings indicated mechanical blocking of the vascular system of the plants. (author)

  20. In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR.

    Science.gov (United States)

    Li, R; Rajan, R; Wong, W C V; Reid, D G; Duer, M J; Somovilla, V J; Martinez-Saez, N; Bernardes, G J L; Hayward, R; Shanahan, C M

    2017-12-14

    Non-enzymatic glycation of extracellular matrix with (U- 13 C 5 )-d-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and hemiaminal (CH 3 -CH(OH)NHR) substructures. Changes in charged residue proportions and distribution may be as important as crosslinking in provoking and understanding harmful tissue changes.

  1. The Pseudomonas aeruginosa PSL Polysaccharide Is a Social but Noncheatable Trait in Biofilms.

    Science.gov (United States)

    Irie, Yasuhiko; Roberts, Aled E L; Kragh, Kasper N; Gordon, Vernita D; Hutchison, Jaime; Allen, Rosalind J; Melaugh, Gavin; Bjarnsholt, Thomas; West, Stuart A; Diggle, Stephen P

    2017-06-20

    Extracellular polysaccharides are compounds secreted by microorganisms into the surrounding environment, and they are important for surface attachment and maintaining structural integrity within biofilms. The social nature of many extracellular polysaccharides remains unclear, and it has been suggested that they could function as either cooperative public goods or as traits that provide a competitive advantage. Here, we empirically tested the cooperative nature of the PSL polysaccharide, which is crucial for the formation of biofilms in Pseudomonas aeruginosa We show that (i) PSL is not metabolically costly to produce; (ii) PSL provides population-level benefits in biofilms, for both growth and antibiotic tolerance; (iii) the benefits of PSL production are social and are shared with other cells; (iv) the benefits of PSL production appear to be preferentially directed toward cells which produce PSL; (v) cells which do not produce PSL are unable to successfully exploit cells which produce PSL. Taken together, this suggests that PSL is a social but relatively nonexploitable trait and that growth within biofilms selects for PSL-producing strains, even when multiple strains are on a patch (low relatedness at the patch level). IMPORTANCE Many studies have shown that bacterial traits, such as siderophores and quorum sensing, are social in nature. This has led to an impression that secreted traits act as public goods, which are costly to produce but benefit both the producing cell and its surrounding neighbors. Theories and subsequent experiments have shown that such traits are exploitable by asocial cheats, but we show here that this does not always hold true. We demonstrate that the Pseudomonas aeruginosa exopolysaccharide PSL provides social benefits to populations but that it is nonexploitable, because most of the fitness benefits accrue to PSL-producing cells. Our work builds on an increasing body of work showing that secreted traits can have both private and public

  2. Transcription factor σB plays an important role in the production of extracellular membrane-derived vesicles in Listeria monocytogenes.

    Directory of Open Access Journals (Sweden)

    Jung Hwa Lee

    Full Text Available Gram-negative bacteria produce extracellular outer membrane vesicles (OMVs that interact with host cells. Unlike Gram-negative bacteria, less is known about the production and role of extracellular membrane vesicles (MVs in Gram-positive bacteria. The food-borne pathogen Listeria monocytogenes can survive under extreme environmental and energy stress conditions and the transcription factor σ(B is involved in this survival ability. Here, we first determined the production of MVs from L. monocytogenes and evaluated whether general stress transcription factor σ(B affected production of MVs in L. monocytogenes. L. monocytogenes secreted MVs during in vitro broth culture. The wild-type strain actively produced MVs approximately nine times more and also produced more intact shapes of MVs than those of the isogenic ΔsigB mutant. A proteomic analysis showed that 130 and 89 MV proteins were identified in the wild-type and ΔsigB mutant strains, respectively. Wild-type strain-derived MVs contained proteins regulated by σ(B such as transporters (OpuCA and OpuCC, stress response (Kat, metabolism (LacD, translation (InfC, and cell division protein (FtsZ. Gene Ontology (GO enrichment analysis showed that wild-type-derived MV proteins corresponded to several GO terms, including response to stress (heat, acid, and bile resistance and extracellular polysaccharide biosynthetic process, but not the ΔsigB mutant. Internalin B (InlB was almost three times more contained in MVs derived from the wild-type strain than in MVs derived from the ΔsigB mutant. Taken together, these results suggest that σ(B plays a pivotal role in the production of MVs and protein profiles contained in MVs. L. monocytogenes MVs may contribute to host infection and survival ability under various stressful conditions.

  3. Regulatory and metabolic networks for the adaptation of Pseudomonas aeruginosa biofilms to urinary tract-like conditions.

    Directory of Open Access Journals (Sweden)

    Petra Tielen

    Full Text Available Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM. Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections.

  4. Regulatory and Metabolic Networks for the Adaptation of Pseudomonas aeruginosa Biofilms to Urinary Tract-Like Conditions

    Science.gov (United States)

    Dohnt, Katrin; Haddad, Isam; Jänsch, Lothar; Klein, Johannes; Narten, Maike; Pommerenke, Claudia; Scheer, Maurice; Schobert, Max; Schomburg, Dietmar; Thielen, Bernhard; Jahn, Dieter

    2013-01-01

    Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections. PMID:23967252

  5. Regulatory and metabolic networks for the adaptation of Pseudomonas aeruginosa biofilms to urinary tract-like conditions.

    Science.gov (United States)

    Tielen, Petra; Rosin, Nathalie; Meyer, Ann-Kathrin; Dohnt, Katrin; Haddad, Isam; Jänsch, Lothar; Klein, Johannes; Narten, Maike; Pommerenke, Claudia; Scheer, Maurice; Schobert, Max; Schomburg, Dietmar; Thielen, Bernhard; Jahn, Dieter

    2013-01-01

    Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections.

  6. In Vitro Analysis of Pseudomonas aeruginosa Virulence Using Conditions That Mimic the Environment at Specific Infection Sites.

    Science.gov (United States)

    Colmer-Hamood, J A; Dzvova, N; Kruczek, C; Hamood, A N

    2016-01-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes chronic lung infection in patients with cystic fibrosis (CF) and acute systemic infections in severely burned patients and immunocompromised patients including cancer patients undergoing chemotherapy and HIV infected individuals. In response to the environmental conditions at specific infection sites, P. aeruginosa expresses certain sets of cell-associated and extracellular virulence factors that produce tissue damage. Analyzing the mechanisms that govern the production of these virulence factors in vitro requires media that closely mimic the environmental conditions within the infection sites. In this chapter, we review studies based on media that closely resemble three in vivo conditions, the thick mucus accumulated within the lung alveoli of CF patients, the serum-rich wound bed and the bloodstream. Media resembling the CF alveolar mucus include standard laboratory media supplemented with sputum obtained from CF patients as well as prepared synthetic mucus media formulated to contain the individual components of CF sputum. Media supplemented with serum or individual serum components have served as surrogates for the soluble host components of wound infections, while whole blood has been used to investigate the adaptation of pathogens to the bloodstream. Studies using these media have provided valuable information regarding P. aeruginosa gene expression in different host environments as varying sets of genes were differentially regulated during growth in each medium. The unique effects observed indicate the essential role of these in vitro media that closely mimic the in vivo conditions in providing accurate information regarding the pathogenesis of P. aeruginosa infections. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Development of a Pseudomonas aeruginosa Agmatine Biosensor

    OpenAIRE

    Gilbertsen, Adam; Williams, Bryan

    2014-01-01

    Agmatine, decarboxylated arginine, is an important intermediary in polyamine production for many prokaryotes, but serves higher functions in eukaryotes such as nitric oxide inhibition and roles in neurotransmission. Pseudomonas aeruginosa relies on the arginine decarboxylase and agmatine deiminase pathways to convert arginine into putrescine. One of the two known agmatine deiminase operons, aguBA, contains an agmatine sensitive TetR promoter controlled by AguR. We have discovered that this pr...

  8. Acquisition and Role of Molybdate in Pseudomonas aeruginosa

    Science.gov (United States)

    Pederick, Victoria G.; Eijkelkamp, Bart A.; Ween, Miranda P.; Begg, Stephanie L.; Paton, James C.

    2014-01-01

    In microaerophilic or anaerobic environments, Pseudomonas aeruginosa utilizes nitrate reduction for energy production, a process dependent on the availability of the oxyanionic form of molybdenum, molybdate (MoO42−). Here, we show that molybdate acquisition in P. aeruginosa occurs via a high-affinity ATP-binding cassette permease (ModABC). ModA is a cluster D-III solute binding protein capable of interacting with molybdate or tungstate oxyanions. Deletion of the modA gene reduces cellular molybdate concentrations and results in inhibition of anaerobic growth and nitrate reduction. Further, we show that conditions that permit nitrate reduction also cause inhibition of biofilm formation and an alteration in fatty acid composition of P. aeruginosa. Collectively, these data highlight the importance of molybdate for anaerobic growth of P. aeruginosa and reveal novel consequences of nitrate reduction on biofilm formation and cell membrane composition. PMID:25172858

  9. Detection of extracellular proteases from microorganisms on agar plates

    Directory of Open Access Journals (Sweden)

    Alane Beatriz Vermelho

    1996-12-01

    Full Text Available We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

  10. Ellagic acid derivatives from Terminalia chebula Retz. downregulate the expression of quorum sensing genes to attenuate Pseudomonas aeruginosa PAO1 virulence.

    Directory of Open Access Journals (Sweden)

    Sajal Sarabhai

    Full Text Available BACKGROUND: Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors. METHODS AND RESULTS: Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7, obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001 in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05 reduced with enhanced (20% susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI and their cognate receptor (lasR and rhlR genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C(12HSL and C(4HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C(4HSL. F7 also showed antagonistic activity against 3-oxo-C(12HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract. CONCLUSIONS: This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors

  11. Inhibition of Pseudomonas aeruginosa elastase and Pseudomonas keratitis using a thiol-based peptide.

    OpenAIRE

    Burns, F R; Paterson, C A; Gray, R D; Wells, J T

    1990-01-01

    Pseudomonas aeruginosa elastase is a zinc metalloproteinase which is released during P. aeruginosa infections. Pseudomonas keratitis, which occurs following contact lens-induced corneal trauma, can lead to rapid, liquefactive necrosis of the cornea. This destruction has been attributed to the release of both host-derived enzymes and the bacterial products P. aeruginosa elastase, alkaline protease, exotoxin A, and lipopolysaccharide endotoxin. A synthetic metalloproteinase inhibitor, HSCH2 (DL...

  12. Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM).

    Science.gov (United States)

    Dinarvand, Mojdeh; Rezaee, Malahat; Foroughi, Majid

    The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R 2 ) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30°C, 6% (v/v), inoculum size and 150rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  13. High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching.

    Science.gov (United States)

    Yu, Zhenxiao; Zheng, Hongchen; Zhao, Xingya; Li, Shufang; Xu, Jianyong; Song, Hui

    2016-08-01

    The effects of induction parameters, osmolytes and ethanol stress on the productivity of the recombinant alkaline catalase (KatA) in Escherichia coli BL21 (pET26b-KatA) were investigated. The yield of soluble KatA was significantly enhanced by 2% ethanol stress. And a certain amount of Triton X-100 supplementation could markedly improved extracellular ratio of KatA. A total soluble catalase activity of 78,762U/mL with the extracellular ratio of 92.5% was achieved by fed-batch fermentation in a 10L fermentor, which was the highest yield so far. The purified KatA showed high stability at 50°C and pH 6-10. Application of KatA for elimination of H2O2 after cotton fabrics bleaching led to less consumption of water, steam and electric power by 25%, 12% and 16.7% respectively without productivity and quality losing of cotton fabrics. Thus, the recombinant KatA is a promising candidate for industrial production and applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase from Aspergillus niger ATCC 20611

    Science.gov (United States)

    Dinarvand, Mojdeh; Rezaee, Malahat; Masomian, Malihe; Jazayeri, Seyed Davoud; Zareian, Mohsen; Abbasi, Sahar; Ariff, Arbakariya B.

    2013-01-01

    The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO3, Zn+2, and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R 2) more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO3, 1.5 mM (v/v) Zn+2, and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry. PMID:24151605

  15. Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase from Aspergillus niger ATCC 20611

    Directory of Open Access Journals (Sweden)

    Mojdeh Dinarvand

    2013-01-01

    Full Text Available The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM with a five-variable and three-level central composite design (CCD was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO3, Zn+2, and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R2 more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v sucrose, 2.5% (w/v yeast extract, 2% (w/v NaNO3, 1.5 mM (v/v Zn+2, and 1% (v/v Triton X-100 by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry.

  16. Activation of the lectin pathway of complement in experimental human keratitis with Pseudomonas aeruginosa.

    Science.gov (United States)

    Osthoff, Michael; Brown, Karl D; Kong, David C M; Daniell, Mark; Eisen, Damon P

    2014-01-01

    Pseudomonas aeruginosa (P. aeruginosa) microbial keratitis (MK) is a sight-threatening disease. Previous animal studies have identified an important contribution of the complement system to the clearance of P. aeruginosa infection of the cornea. Mannose-binding lectin (MBL), a pattern recognition receptor of the lectin pathway of complement, has been implicated in the host defense against P. aeruginosa. However, studies addressing the role of the lectin pathway in P. aeruginosa MK are lacking. Hence, we sought to determine the activity of the lectin pathway in human MK caused by P. aeruginosa. Primary human corneal epithelial cells (HCECs) from cadaveric donors were exposed to two different P. aeruginosa strains. Gene expression of interleukin (IL)-6, IL-8, MBL, and other complement proteins was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MBL synthesis by enzyme-linked immunosorbent assay and intracellular flow cytometry. MBL gene expression was not detected in unchallenged HCECs. Exposure of HCECs to P. aeruginosa resulted in rapid induction of the transcriptional expression of MBL, IL-6, and IL-8. In addition, expression of several complement proteins of the classical and lectin pathways, but not the alternative pathway, were upregulated after 5 h of challenge, including MBL-associated serine protease 1. However, MBL protein secretion was not detectable 18 h after challenge with P. aeruginosa. MK due to P. aeruginosa triggers activation of MBL and the lectin pathway of complement. However, the physiologic relevance of this finding is unclear, as corresponding MBL oligomer production was not observed.

  17. In vitro production of biofilm in a flow cell system in a strain of Pseudomonas aeruginosa and Staphylococcus aureus and determination of efficiency of ciprofloxacin against them

    Directory of Open Access Journals (Sweden)

    Soham Gupta

    2011-01-01

    Full Text Available Background: Microorganisms develop biofilm on various medical devices. The process is particularly relevant in public health since biofilm associated organisms are much more resistant to antibiotics and have a potential to cause infections in patients with indwelling medical devices. Materials and Methods: To determine the efficiency of an antibiotic against the biofilm it is inappropriate to use traditional technique of determining Minimum Inhibitory Concentration (MIC on the free floating laboratory phenotype. Thus we have induced formation of biofilm in two strains (Pseudomonas aeruginosa and Staphylococcus aureus, which showed heavy growth of biofilm in screening by Tube method in a flow cell system and determined their antibiotic susceptibility against ciprofloxacin by agar dilution method in the range (0.25 mg/ml to 8 mg/ml. The MIC value of ciprofloxacin for the biofilm produced organism was compared with its free form and a standard strain as control on the same plates. Observations: Both the biofilm produced strains showed a higher resistance (MIC > 8 mg/ml than its free form, which were 2 μg/ml for Pseudomonas aeruginosa and 4 mg/ml for Staphylococcus aureus. Thus biofilm can pose a threat in the patient treatment.

  18. Magnesium limitation is an environmental trigger of the Pseudomonas aeruginosa biofilm lifestyle.

    Directory of Open Access Journals (Sweden)

    Heidi Mulcahy

    Full Text Available Biofilm formation is a conserved strategy for long-term bacterial survival in nature and during infections. Biofilms are multicellular aggregates of cells enmeshed in an extracellular matrix. The RetS, GacS and LadS sensors control the switch from a planktonic to a biofilm mode of growth in Pseudomonas aeruginosa. Here we detail our approach to identify environmental triggers of biofilm formation by investigating environmental conditions that repress expression of the biofilm repressor RetS. Mg(2+ limitation repressed the expression of retS leading to increased aggregation, exopolysaccharide (EPS production and biofilm formation. Repression of retS expression under Mg(2+ limitation corresponded with induced expression of the GacA-controlled small regulatory RNAs rsmZ and rsmY and the EPS biosynthesis operons pel and psl. We recently demonstrated that extracellular DNA sequesters Mg(2+ cations and activates the cation-sensing PhoPQ two-component system, which leads to increased antimicrobial peptide resistance in biofilms. Here we show that exogenous DNA and EDTA, through their ability to chelate Mg(2+, promoted biofilm formation. The repression of retS in low Mg(2+ was directly controlled by PhoPQ. PhoP also directly controlled expression of rsmZ but not rsmY suggesting that PhoPQ controls the equilibrium of the small regulatory RNAs and thus fine-tunes the expression of genes in the RetS pathway. In summary, Mg(2+ limitation is a biologically relevant environmental condition and the first bonafide environmental signal identified that results in transcriptional repression of retS and promotes P. aeruginosa biofilm formation.

  19. Development of a Pseudomonas aeruginosa Agmatine Biosensor

    Directory of Open Access Journals (Sweden)

    Adam Gilbertsen

    2014-10-01

    Full Text Available Agmatine, decarboxylated arginine, is an important intermediary in polyamine production for many prokaryotes, but serves higher functions in eukaryotes such as nitric oxide inhibition and roles in neurotransmission. Pseudomonas aeruginosa relies on the arginine decarboxylase and agmatine deiminase pathways to convert arginine into putrescine. One of the two known agmatine deiminase operons, aguBA, contains an agmatine sensitive TetR promoter controlled by AguR. We have discovered that this promoter element can produce a titratable induction of its gene products in response to agmatine, and utilized this discovery to make a luminescent agmatine biosensor in P. aeruginosa. The genome of the P. aeruginosa lab strain UCBPP-PA14 was altered to remove both its ability to synthesize or destroy agmatine, and insertion of the luminescent reporter construct allows it to produce light in proportion to the amount of exogenous agmatine applied from ~100 nM to 1mM. Furthermore it does not respond to related compounds including arginine or putrescine. To demonstrate potential applications the biosensor was used to detect agmatine in spent supernatants, to monitor the development of arginine decarboxylase over time, and to detect agmatine in the spinal cords of live mice.

  20. Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01

    NARCIS (Netherlands)

    Nieuwenhuizen, W.F.; Leeuwen, S. van; Jack, R.W.; Egmond, M.R.; Götz, F.

    2003-01-01

    Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 μg of extracellular alkaline, Ca2+-dependent CDase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was

  1. Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors.

    Science.gov (United States)

    El Harane, Nadia; Kervadec, Anaïs; Bellamy, Valérie; Pidial, Laetitia; Neametalla, Hany J; Perier, Marie-Cécile; Lima Correa, Bruna; Thiébault, Léa; Cagnard, Nicolas; Duché, Angéline; Brunaud, Camille; Lemitre, Mathilde; Gauthier, Jeanne; Bourdillon, Alexandra T; Renault, Marc P; Hovhannisyan, Yeranuhi; Paiva, Solenne; Colas, Alexandre R; Agbulut, Onnik; Hagège, Albert; Silvestre, Jean-Sébastien; Menasché, Philippe; Renault, Nisa K E

    2018-05-21

    We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left

  2. Extracellular vesicles from Leishmania-infected macrophages confer an anti-infection cytokine-production profile to naïve macrophages.

    Directory of Open Access Journals (Sweden)

    André Cronemberger-Andrade

    2014-09-01

    Full Text Available Extracellular vesicles (EVs are structures with phospholipid bilayer membranes and 100-1000 nm diameters. These vesicles are released from cells upon activation of surface receptors and/or apoptosis. The production of EVs by dendritic cells, mast cells, macrophages, and B and T lymphocytes has been extensively reported in the literature. EVs may express MHC class II and other membrane surface molecules and carry antigens. The aim of this study was to investigate the role of EVs from Leishmania-infected macrophages as immune modulatory particles.In this work it was shown that BALB/c mouse bone marrow-derived macrophages, either infected in vitro with Leishmania amazonensis or left uninfected, release comparable amounts of 50-300 nm-diameter extracellular vesicles (EVs. The EVs were characterized by flow cytometry and electron microscopy. The incubation of naïve macrophages with these EVs for 48 hours led to a statistically significant increase in the production of the cytokines IL-12, IL-1β, and TNF-α.EVs derived from macrophages infected with L. amazonensis induce other macrophages, which in vivo could be bystander cells, to produce the proinflammatory cytokines IL-12, IL-1β and TNF-α. This could contribute both to modulate the immune system in favor of a Th1 immune response and to the elimination of the Leishmania, leading, therefore, to the control the infection.

  3. Extracellular Production of Silver Nanoparticles by Using Three Common Species of Dermatophytes: Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis

    International Nuclear Information System (INIS)

    Moazeni, M.; Rashidi, N.; Shahverdi, Ahmad R.; Noorbakhsh, F.; Rezaie, S.

    2012-01-01

    To develop a new green approach for biosynthesis of silver nanoparticles, myconanotechnology has been represented as a novel field of study in nano technology. In this study, we have reported the extracellular synthesis of highly stable silver nanoparticles using three species of dermatophytes: Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. Methods: Clinical strains of these species were grown in a liquid medium containing mineral salt and incubated at 25 d egree C for 5-7 days. The cell-free filtrate of each culture was obtained and subjected to synthesize silver nanoparticles in the presence of 1 m M AgNO 3 . Results: The reduction of Ag + ions in metal nanoparticles was investigated virtually by tracing the solution color which was switched into reddish-light brown after 72 h. For T. mentagrophytes, a UV-visible spectra demonstrating a strong, quite narrow peak located between 422 and 425 nm was obtained. For M. canis, a fairly wide peak centering at 441 nm and for T. rubrum, a weak spectrum to decipher were observed. According to transmission electron microscopy results, fairly uniform, spherical, and small in size with almost less than 50 nm particles were forms in case of T. mentagrophytes. For the other two species, transmission electron microscopy images showed existence of small spherical nano silvers but not as small as nanoparticles synthesized by T. mentagrophytes. Conclusion: We observed that species belong to a single genus of the fungi have variable ability to synthesize silver nanoparticles extracellulary with different efficiency. Furthermore, the extracellular synthesis may make the process simpler and easier for following processes.

  4. A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and AlgT/U activity results in the loss of alginate production

    DEFF Research Database (Denmark)

    Sautter, Robert; Ramos, Damaris; Schneper, Lisa

    2012-01-01

    Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF...... lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg+) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching...... complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism...

  5. The effect of solids retention times on the characterization of extracellular polymeric substances and soluble microbial products in a submerged membrane bioreactor.

    Science.gov (United States)

    Duan, Liang; Song, Yonghui; Yu, Huibin; Xia, Siqing; Hermanowicz, Slawomir W

    2014-07-01

    In this study, the effect of solids retention times (SRTs) on extracellular polymeric substances (EPS) and soluble microbial products (SMPs) were investigated in a membrane bioreactor (MBR) at SRTs of 10, 5 and 3 days. The results showed that more carbohydrates and proteins were accumulated at short SRT, which can due to the higher biomass activity in the reactor. The molecular weight (MW) distribution analysis suggested that macromolecules (MW>30 kDa) and small molecules (MW<1 kDa) were the dominant fraction of EPS and SMP, respectively. The reactor at shorter SRT had more small molecules and less macromolecules of carbohydrates. The MW distribution of total organic carbon (TOC) suggested that other organic moieties were exuded by microbes into the solution. The shorter SRT had more undefined microbial by-product-like substances and different O − H bonds in hydroxyl functional groups. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. OPTIMIZATION OF FERMENTATION PARAMETERS FOR THE PRODUCTION OF EXTRACELLULAR ENDOGLUCANASE, β –GLUCOSIDASE AND ENDOXYLANASE BY A CHROMIUM RESISTANT STRAIN OF TRICHODERMA PSEUDOKONINGII

    Directory of Open Access Journals (Sweden)

    Rina Rani Ray

    2013-08-01

    Full Text Available Trichoderma pseudokoningii, a chromate reducing fungal strain, was isolated from the tannery-effluents. The present Cr (VI resistant strain was found to produce good amount of various extracellular enzymes that included cellulases (endoglucanase and β–glucosidase and hemicellulase (endoxylanase in submerged fermentation (SmF. The titre of β–glucosidase was found to be higher than that of endoglucanase. Cellulases were best induced in presence of 1% of respective substrates whereas only 0.5% xylan could induce endoxylanase production in this strain. Although the optimum temperature for all three enzymes was found to be 27oC, the pH optimum of cellulases (pH 5 were different from that of endoxylanase (pH 6. Under optimized conditions, maximum of production of all these enzymes was achieved within 48 hours of cultivation. Among nitrogen sources tested, potassium nitrate was found to be the most effective followed by gelatin.

  7. An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal

    DEFF Research Database (Denmark)

    Harmsen, Morten; Yang, Liang; Pamp, Sünje Johanna

    2010-01-01

    We review the recent advances in the understanding of the Pseudomonas aeruginosa biofilm lifestyle from studies using in vitro laboratory setups such as flow chambers and microtiter trays. Recent work sheds light on the role of nutrients, motility, and quorum sensing in structure formation in P....... aeruginosa biofilms. The second messenger, c-di-GMP, is established as an important regulator of the synthesis of polysaccharide and protein components of the biofilm matrix. Extracellular DNA is shown to be an essential component of the biofilm matrix. It has become apparent that biofilm formation involves...... interactions between different subpopulations. The molecular mechanisms underlying the tolerance of biofilm bacteria to antimicrobial agents are beginning to be unraveled, and new knowledge has been obtained regarding the environmental cues and regulatory mechanisms involved in biofilm dispersal....

  8. The implication of Pseudomonas aeruginosa biofilms in infections

    DEFF Research Database (Denmark)

    Rybtke, Morten T; Jensen, Peter Østrup; Høiby, Niels

    2011-01-01

    Biofilm formation by bacteria is recognized as a major problem in chronic infections due to their recalcitrance against the immune defense and available antibiotic treatment schemes. The opportunistic pathogen Pseudomonas aeruginosa has drawn special attention in this regard due to its severity o...... treatment strategies where the underlying targets are less prone for resistance development as bacteria, in retrospect, have a unique ability to evade the actions of classic antibiotics.......Biofilm formation by bacteria is recognized as a major problem in chronic infections due to their recalcitrance against the immune defense and available antibiotic treatment schemes. The opportunistic pathogen Pseudomonas aeruginosa has drawn special attention in this regard due to its severity......-up of the extracellular matrix encasing the biofilm-associated bacteria as well as the elaborate signaling mechanisms employed by the bacterium enables it to withstand the continuous stresses imposed by the immune defense and administered antibiotics resulting in a state of chronic inflammation that damages the host...

  9. Raffinose Induces Biofilm Formation by Streptococcus mutans in Low Concentrations of Sucrose by Increasing Production of Extracellular DNA and Fructan.

    Science.gov (United States)

    Nagasawa, Ryo; Sato, Tsutomu; Senpuku, Hidenobu

    2017-08-01

    Streptococcus mutans is the primary etiological agent of dental caries and causes tooth decay by forming a firmly attached biofilm on tooth surfaces. Biofilm formation is induced by the presence of sucrose, which is a substrate for the synthesis of extracellular polysaccharides but not in the presence of oligosaccharides. Nonetheless, in this study, we found that raffinose, which is an oligosaccharide with an intestinal regulatory function and antiallergic effect, induced biofilm formation by S. mutans in a mixed culture with sucrose, which was at concentrations less than those required to induce biofilm formation directly. We analyzed the possible mechanism behind the small requirement for sucrose for biofilm formation in the presence of raffinose. Our results suggested that sucrose contributed to an increase in bacterial cell surface hydrophobicity and biofilm formation. Next, we examined how the effects of raffinose interacted with the effects of sucrose for biofilm formation. We showed that the presence of raffinose induced fructan synthesis by fructosyltransferase and aggregated extracellular DNA (eDNA, which is probably genomic DNA released from dead cells) into the biofilm. eDNA seemed to be important for biofilm formation, because the degradation of DNA by DNase I resulted in a significant reduction in biofilm formation. When assessing the role of fructan in biofilm formation, we found that fructan enhanced eDNA-dependent cell aggregation. Therefore, our results show that raffinose and sucrose have cooperative effects and that this induction of biofilm formation depends on supportive elements that mainly consist of eDNA and fructan. IMPORTANCE The sucrose-dependent mechanism of biofilm formation in Streptococcus mutans has been studied extensively. Nonetheless, the effects of carbohydrates other than sucrose are inadequately understood. Our findings concerning raffinose advance the understanding of the mechanism underlying the joint effects of sucrose and

  10. Dynamics of Phylogenetic Diversity and Its Influence on the Production of Extracellular Protease by Moderately Halotolerant Alkaliphilic Bacteria Acinetobacter Baumannii GTCR407 Nov

    Directory of Open Access Journals (Sweden)

    Thiyagarajan Gurunathan

    2010-07-01

    Full Text Available New characters emerge in the population of microorganisms living in the extreme environments due to its adaptation to ecological association. The microorganisms living in saline habitat utilize complex nutrients by adopting different strategies in Deoxyribonucleic Acid (DNA and Ribonucleic Acid (RNA, which are related to their metabolic and ecological diversities. Isolation and characterization of the organisms producing extracellular protease from such environment were the prime focus of this investigation, which can indicate the importance of metabolic diversity in phylogeny. Norberg medium was used to isolate halotolerant microorganisms from salt-cured skin. The isolates were screened for high activity of protease and the strain showing maximum activity of protease was taken for further studies. The biochemical characterization and 16s ribosomal RNA sequencing studies confirm that the isolate is Acinetobacter baumannii. Moreover, hydrolysis positive for starch and casein, negative for gelatin shows that the organism is a variant form of A. baumannii. Cell growth parameters such as pH and temperature were optimized and their values are 8 and 37oC respectively. The extracellular production of protease was optimized in the suitable medium and its enzyme activity was 165μg/ml/min. The results imply that the isolate had acquired operational genes through lateral gene transfer (LGT probably from unrelated species in the environment. This indicates that the isolate identified possesses metabolic and ecological diversities with values of phylogenetic delineation

  11. Antinociceptive principle from Curcuma aeruginosa

    OpenAIRE

    Hossain, Chowdhury Faiz; Al-Amin, Mohammad; Sayem, Abu Sadat Md.; Siragee, Ismail Hossain; Tunan, Asif Mahmud; Hassan, Fahima; Kabir, Md. Mohiuddin; Sultana, Gazi Nurun Nahar

    2015-01-01

    Background The rhizome of Curcuma aeruginosa Roxb (Zingiberaceae) has been used as a traditional folk medicine for the treatment of rheumatic disorders in Bangladesh. The aim of the current study was the bioassay-guided isolation and purification of an antinociceptive principle from the methanol extract of C. aeruginosa rhizomes. Methods The antinociceptive activity was determined using acetic acid induced writhing and formalin induced licking in the Swiss albino mice to investigate central a...

  12. Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies.

    Science.gov (United States)

    Laperle, Alex; Masters, Kristyn S; Palecek, Sean P

    2015-01-01

    Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers.

  13. Extracellular adenosine production by ecto-5′-nucleotidase (CD73) enhances radiation-induced lung fibrosis

    Science.gov (United States)

    Wirsdörfer, Florian; de Leve, Simone; Cappuccini, Federica; Eldh, Therese; Meyer, Alina V.; Gau, Eva; Thompson, Linda F.; Chen, Ning-Yuan; Karmouty-Quintana, Harry; Fischer, Ute; Kasper, Michael; Klein, Diana; Ritchey, Jerry W.; Blackburn, Michael R.; Westendorf, Astrid M.; Stuschke, Martin; Jendrossek, Verena

    2016-01-01

    Radiation-induced pulmonary fibrosis is a severe side effect of thoracic irradiation, but its pathogenesis remains poorly understood and no effective treatment is available. In this study, we investigated the role of the extracellular adenosine as generated by the ecto-5'-nucleotidase CD73 in fibrosis development after thoracic irradiation. Exposure of wild-type C57BL/6 mice to a single dose (15 Gray) of whole thorax irradiation triggered a progressive increase in CD73 activity in the lung between 3 and 30 weeks post-irradiation. In parallel, adenosine levels in bronchoalveolar lavage fluid (BALF) were increased by approximately three-fold. Histological evidence of lung fibrosis was observed by 25 weeks after irradiation. Conversely, CD73-deficient mice failed to accumulate adenosine in BALF and exhibited significantly less radiation-induced lung fibrosis (P<0.010). Furthermore, treatment of wild-type mice with pegylated adenosine deaminase (PEG-ADA) or CD73 antibodies also significantly reduced radiation-induced lung fibrosis. Taken together, our findings demonstrate that CD73 potentiates radiation-induced lung fibrosis, suggesting that existing pharmacological strategies for modulating adenosine may be effective in limiting lung toxicities associated with the treatment of thoracic malignancies. PMID:26921334

  14. Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus.

    Science.gov (United States)

    Irazoqui, Javier E; Troemel, Emily R; Feinbaum, Rhonda L; Luhachack, Lyly G; Cezairliyan, Brent O; Ausubel, Frederick M

    2010-07-01

    The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus-triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with

  15. Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus.

    Directory of Open Access Journals (Sweden)

    Javier E Irazoqui

    2010-07-01

    Full Text Available The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs. Because our data suggest that neither the P. aeruginosa nor the S. aureus-triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C

  16. Hydrogen sulfide potentiates interleukin-1β-induced nitric oxide production via enhancement of extracellular signal-regulated kinase activation in rat vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Jeong, Sun-Oh; Pae, Hyun-Ock; Oh, Gi-Su; Jeong, Gil-Saeng; Lee, Bok-Soo; Lee, Seoul; Kim, Du Yong; Rhew, Hyun Yul; Lee, Kang-Min; Chung, Hun-Taeg

    2006-01-01

    Hydrogen sulfide (H 2 S) and nitric oxide (NO) are endogenously synthesized from L-cysteine and L-arginine, respectively. They might constitute a cooperative network to regulate their effects. In this study, we investigated whether H 2 S could affect NO production in rat vascular smooth muscle cells (VSMCs) stimulated with interleukin-1β (IL-1β). Although H 2 S by itself showed no effect on NO production, it augmented IL-β-induced NO production and this effect was associated with increased expression of inducible NO synthase (iNOS) and activation of nuclear factor (NF)-κB. IL-1β activated the extracellular signal-regulated kinase 1/2 (ERK1/2), and this activation was also enhanced by H 2 S. Inhibition of ERK1/2 activation by the selective inhibitor U0126 inhibited IL-1β-induced NF-κB activation, iNOS expression, and NO production either in the absence or presence of H 2 S. Our findings suggest that H 2 S enhances NO production and iNOS expression by potentiating IL-1β-induced NF-κB activation through a mechanism involving ERK1/2 signaling cascade in rat VSMCs

  17. Effects of limonene stress on the growth of and microcystin release by the freshwater cyanobacterium Microcystis aeruginosa FACHB-905.

    Science.gov (United States)

    Hu, Xi; Liu, Yunguo; Zeng, Guangming; Hu, Xinjiang; Wang, Yaqin; Zeng, Xiaoxia

    2014-07-01

    The effects of limonene exposure on the growth of Microcystisaeruginosa and the release of toxic intracellular microcystin (MCY) were tested by evaluating the results obtained from the batch culture experiments with M. aeruginosa FACHB-905. The time series of cell as well as intracellular and extracellular MCY concentrations were evaluated during 5d of the incubation. After exposure to limonene, the number of cells gradually diminished; the net log cell reduction after 5d of the exposure was 3.0, 3.6, and 3.8log when the initial cell densities were set at 1.6×10(7), 1.1×10(6) and 4.1×10(5)cell/mL, respectively. Limonene was found to significantly influence the production and release of MCY. As the limonene exposure could inhibit the increase in the number of cells, the increase in the total MCY concentration in the medium was also inhibited. In the presence of limonene, the intracellular MCY was gradually released into the medium through a gradual reduction in the number of cells. The extracellular MCY concentration in the medium was significantly higher in the limonene-exposed samples than in the control samples, which confirmed that limonene cannot decompose the extracellular MCY. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Serotyping and analysis of produced pigments kinds by Pseudomonas aeruginosa clinical isolates

    Directory of Open Access Journals (Sweden)

    Stanković-Nedeljković Nataša

    2011-01-01

    Full Text Available Background/Aim. Pseudomonas aeruginosa (P. aeruginosa is devided into 20 serotypes on the base of the International Antigenic Typing Scheme. P. aeruginosa serotyping is important because of few reasons but epidemiological is the most important. The aim of the study was serotyping of P. aeruginosa clinical isolates, analysing of single clinical isolates P. aeruginosa present in the particular samples, and analysing of pyocianin and fluorescin production in different isolates of P. aeruginosa. Methods. A total of 223 isolates of P. aeruginosa, isolated in the microbiological laboratory of the Health Center “Aleksinac”, Aleksinac, were examinated. P. aeruginosa isolates were put on the pseudomonas isolation agar, pseudomonas agar base, acetamid agar, asparagin prolin broth, pseudomonas asparagin broth, Bushnnell-Haas agar, cetrimid agar base, King A and King B plates, plates for pyocianin production, plates for fluorescin production and tripticasa soya agar (Himedia. Polyvalent and monovalent serums were used in the agglutination (Biorad. Pigment production was analysed on the bases of growth on the plates for pyocianin and fluorescin production. Results. Serologically, we identificated the serovars as follows: O1, O3, O4, O5, O6, O7, O8, O10, O11 and O12. O1 (38% was the most often serovar, then O11 (19% and O6 (8.6%. A total of 18.6% (42 isolates did not agglutinate with any serum, whereas 21 isolates agglutinated only with polyvalent serum. The majority of P. aeruginosa isolates produced fluorescin, 129 (58.54%, 53 (22.94% produced pyocianin whereas 49 (21.21% isolates produced both pigments. Conclusion. P. aeruginosa was isolated most of the from urine, sputum and other materials. The majority often serovars were O1, O6 and O11. The most of isolates produced fluorescin (58.54%, while 22.94% producted pyocianin and 21.21% both pigments.

  19. Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA.

    Directory of Open Access Journals (Sweden)

    Sandra Bruns

    2010-04-01

    Full Text Available Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A

  20. Effects of Light Intensity and Color on the Biomass, Extracellular Red Pigment, and Citrinin Production of Monascus ruber.

    Science.gov (United States)

    Wang, Liling; Dai, Yang; Chen, Wanping; Shao, Yanchun; Chen, Fusheng

    2016-12-21

    Light is a crucial environmental signal for fungi. In this work, the effects of different light intensities and colors on biomass, Monascus pigments (MPs) and citrinin production of Monascus ruber M7 were investigated. We have demonstrated that low intensity of blue light (500 lx) decreased Monascus biomass, increased MPs accumulation via upregulation of MpigA, MpigB, and MpigJ genes expression, but had no significant influence on citrinin production. High intensity of blue light (1500 lx) decreased citrinin accumulation but had no significant influence on biomass and MPs production after 14 days cultivation. Low intensity of green light (500 lx) stimulated citrinin production via upregulation of pksCT, mrl1, mrl2, and ctnA genes expression. One putative red light photoreceptor and two putative green light photoreceptors were identified in M. ruber M7. These observations will not only guide the practical production of Monascus but also contribute to our understanding light effects on Monascus.

  1. Extracellular matrix glycation and receptor for advanced glycation end-products activation: a missing piece in the puzzle of the association between diabetes and cancer.

    Science.gov (United States)

    Rojas, Armando; Añazco, Carolina; González, Ileana; Araya, Paulina

    2018-04-05

    A growing body of epidemiologic evidence suggests that people with diabetes are at a significantly higher risk of many forms of cancer. However, the molecular mechanisms underlying this association are not fully understood. Cancer cells are surrounded by a complex milieu, also known as tumor microenvironment, which contributes to the development and metastasis of tumors. Of note, one of the major components of this niche is the extracellular matrix (ECM), which becomes highly disorganized during neoplastic progression, thereby stimulating cancer cell transformation, growth and spread. One of the consequences of chronic hyperglycemia, the most frequently observed sign of diabetes and the etiological source of diabetes complications, is the irreversible glycation and oxidation of proteins and lipids leading to the formation of the advanced glycation end-products (AGEs). These compounds may covalently crosslink and biochemically modify structure and functions of many proteins, and AGEs accumulation is particularly high in long-living proteins with low biological turnover, features that are shared by most, if not all, ECM proteins. AGEs-modified proteins are recognized by AGE-binding proteins, and thus glycated ECM components have the potential to trigger Receptor for advanced glycation end-products-dependent mechanisms. The biological consequence of receptor for advanced glycation end-products activation mechanisms seems to be connected, in different ways, to drive some hallmarks of cancer onset and tumor growth. The present review intends to highlight the potential impact of ECM glycation on tumor progression by triggering receptor for advanced glycation end-products-mediated mechanisms.

  2. Drosophila melanogaster as an animal model for the study of Pseudomonas aeruginosa biofilm infections in vivo.

    Directory of Open Access Journals (Sweden)

    Heidi Mulcahy

    2011-10-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3 demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.

  3. Extracellular polysaccharide production by a novel osmotolerant marine strain of Alteromonas macleodii and its application towards biomineralization of silver.

    Directory of Open Access Journals (Sweden)

    Ananya Mehta

    Full Text Available The present study demonstrates exopolysaccharide production by an osmotolerant marine isolate and also describes further application of the purified polysaccharide for production of colloidal suspension of silver nanoparticles with narrow size distribution. Phylogenetic analysis based on 16S r RNA gene sequencing revealed close affinity of the isolate to Alteromonas macleodii. Unlike earlier reports, where glucose was used as the carbon source, lactose was found to be the most suitable substrate for polysaccharide production. The strain was capable of producing 23.4 gl(-1 exopolysaccharide with a productivity of 7.8 gl(-1 day(-1 when 15% (w/v lactose was used as carbon source. Furthermore, the purified polysaccharide was able to produce spherical shaped silver nanoparticles of around 70 nm size as characterized by Uv-vis spectroscopy, Dynamic light scattering and Transmission electron microscopy. These observations suggested possible commercial potential of the isolated strain for production of a polysaccharide which has the capability of synthesizing biocompatible metal nanoparticle.

  4. Retinoic Acid Modulates Interferon-γ Production by Hepatic Natural Killer T Cells via Phosphatase 2A and the Extracellular Signal-Regulated Kinase Pathway

    Science.gov (United States)

    Chang, Heng-Kwei

    2015-01-01

    Retinoic acid (RA), an active metabolite converted from vitamin A, plays an active role in immune function, such as defending against infections and immune regulation. Although RA affects various types of immune cells, including antigen-presenting cells, B lymphocytes, and T lymphocytes, whether it affects natural killer T (NKT) cells remain unknown. In this study, we found that RA decreased interferon (IFN)-γ production by activated NKT cells through T-cell receptor (TCR) and CD28. We also found that RA reduced extracellular signal-regulated kinase (ERK) phosphorylation, but increased phosphatase 2A (PP2A) activity in TCR/CD28-stimulated NKT cells. The increased PP2A activity, at least partly, contributed to the reduction of ERK phosphorylation. Since inhibition of ERK activation decreases IFN-γ production by TCR/CD28-stimulated NKT cells, RA may downregulate IFN-γ production by TCR/CD28-stimulated NKT cells through the PP2A-ERK pathway. Our results demonstrated a novel function of RA in modulating the IFN-γ expression by activated NKT cells. PMID:25343668

  5. CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum.

    Science.gov (United States)

    Kersey, Caleb M; Agyemang, Paul A; Dumenyo, C Korsi

    2012-01-01

    Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

  6. Effects of antibiotics on quorum sensing in pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Skindersø, Mette Elena; Alhede, Morten; Phipps, Richard Kerry

    2008-01-01

    in animal infection models. Treatment of cystic fibrosis (CF) patients chronically infected with P. aeruginosa with the macrolide antibiotic azithromycin (AZM) has been demonstrated to improve the clinical outcome. Several studies indicate that AZM may accomplish its beneficial action in CF patients....... Three of the antibiotics tested, AZM, ceftazidime (CFT), and ciprofloxacin (CPR), were very active in the assay and were further examined for their effects on QS-regulated virulence factor production in P. aeruginosa. The effects of the three antibiotics administered at subinhibitory concentrations were...... by impeding QS, thereby reducing the pathogenicity of P. aeruginosa. This led us to investigate whether QS inhibition is a common feature of antibiotics. We present the results of a screening of 12 antibiotics for their QS-inhibitory activities using a previously described QS inhibitor selector 1 strain...

  7. Stigmatellin Y - An anti-biofilm compound from Bacillus subtilis BR4 possibly interferes in PQS-PqsR mediated quorum sensing system in Pseudomonas aeruginosa.

    Science.gov (United States)

    Boopathi, Seenivasan; Vashisth, Rajesh; Manoharan, Prabu; Kandasamy, Ruckmani; Sivakumar, Natesan

    2017-05-15

    Hitherto this is the first report pertaining to production of biofilm inhibitory compound(s) (BIC) from Bacillus subtilis BR4 against Pseudomonas aeruginosa (ATCC 27853) coupled with production optimization. In order to achieve this, combinations of media components were formulated by employing statistical tools such as Plackett-Burman analysis and central composite rotatable design (CCRD). It was evident that at 35mlL -1 glycerol and 3.8gL -1 casamino acid, anti-biofilm activity and production of extracellular protein significantly increased by 1.5-fold and 1.2-fold, respectively. These results corroborate that the combination of glycerol and casamino acid plays a key role in the production of BIC. Further, metabolic profiling of BIC was carried out using liquid chromatography/tandem mass spectrometry (LC-MS/MS) based on m/z value. The presence of Stigmatellin Y was predicted with monoisotopic neutral mass of 484.2825Da. In support of optimization study, higher production of BIC was confirmed in the optimized-media-grown BR4 (OPT-BR4) than in the ideal-media-grown BR4 (ID-BR4) by LC-MS/MS analysis. PqsR in P. aeruginosa is a potential target for anti-virulent therapy. Molecular docking study has revealed that Stigmatellin Y interacts with PqsR in the similar orientation like a cognate signal (PQS) and synthetic inhibitor. In addition, Stigmatellin Y was found to exhibit interaction with four more amino acid residues of PqsR to establish strong affinity. Stigmatellin Y thus might play a role of competitor for PQS to distract PQS-PqsR mediated communication in P. aeruginosa. The present investigation thus paves new avenues to develop anti-Pseudomonas virulent therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases.

    LENUS (Irish Health Repository)

    Guyot, Nicolas

    2010-06-01

    Elafin is a 6-kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases [neutrophil elastase (NE) and proteinase 3] with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect of Pseudomonas aeruginosa proteases on elafin function. We found that P. aeruginosa PAO1-conditioned medium and two purified Pseudomonas metalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), are able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaves elafin at the amino-terminal Lys6-Gly7 peptide bond, resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidence that elafin is susceptible to proteolytic cleavage at alternative sites by P. aeruginosa metalloproteinases, which can affect different biological functions of elafin.

  9. Potential use of Algae Microcystis aeruginosa (Chroococaceae) in ...

    African Journals Online (AJOL)

    A comparison of growth response of algal species in varying concentrations of petroleum products to assess their bioremediation potentials was carried out using Microcystis aeruginosa as test organism. The modified Chu #10 standard medium for algal culture was used for the experimental set up which lasted for ...

  10. Current therapies for pseudomonas aeruginosa.

    Science.gov (United States)

    Giamarellou, Helen; Kanellakopoulou, Kyriaki

    2008-04-01

    Based on the worldwide prevalence of multidrug-resistant strains of Pseudomas aeruginosa and the fact that no newer antipseudomonal agents are available, this article aims to investigate therapeutic solutions for combating infections caused by P aeruginosa, including multidrug-resistant strains. The article focuses mainly on colistin, the re-emerging old antibiotic that possesses prominent antipseudomonal activity in vitro and on doripenem, a newer carbapenem that seems to be close to its global marketing. Regarding older antipseudomonal antibiotics that have been reviewed extensively, only newer aspects on their use are considered in this article.

  11. Extracellular nucleotide derivatives protect cardiomyctes against hypoxic stress

    DEFF Research Database (Denmark)

    Golan, O; Issan, Y; Isak, A

    2011-01-01

    assures cardioprotection. Treatment with extracellular nucleotides, or with tri/di-phosphate, administered under normoxic conditions or during hypoxic conditions, led to a decrease in reactive oxygen species production. CONCLUSIONS: Extracellular tri/di-phosphates are apparently the molecule responsible...

  12. Evaluation of a FRET-peptide substrate to predict virulence in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Wendy E Kaman

    Full Text Available Pseudomonas aeruginosa produces a number of proteases that are associated with virulence and disease progression. A substrate able to detect P. aeruginosa-specific proteolytic activity could help to rapidly alert clinicians to the virulence potential of individual P. aeruginosa strains. For this purpose we designed a set of P. aeruginosa-specific fluorogenic substrates, comprising fluorescence resonance energy transfer (FRET-labeled peptides, and evaluated their applicability to P. aeruginosa virulence in a range of clinical isolates. A FRET-peptide comprising three glycines (3xGly was found to be specific for the detection of P. aeruginosa proteases. Further screening of 97 P. aeruginosa clinical isolates showed a wide variation in 3xGly cleavage activity. The absence of 3xGly degradation by a lasI knock out strain indicated that 3xGly cleavage by P. aeruginosa could be quorum sensing (QS-related, a hypothesis strengthened by the observation of a strong correlation between 3xGly cleavage, LasA staphylolytic activity and pyocyanin production. Additionally, isolates able to cleave 3xGly were more susceptible to the QS inhibiting antibiotic azithromycin (AZM. In conclusion, we designed and evaluated a 3xGly substrate possibly useful as a simple tool to predict virulence and AZM susceptibility.

  13. Extracellular phospholipase production of oral Candida albicans isolates from smokers, diabetics, asthmatics, denture wearers and healthy individuals following brief exposure to polyene, echinocandin and azole antimycotics

    Directory of Open Access Journals (Sweden)

    Arjuna N.B. Ellepola

    Full Text Available Abstract Objective Candida albicans is the primary causative agent of oral candidosis, and one of its key virulent attributes is considered to be its ability to produce extracellular phospholipases that facilitate cellular invasion. Oral candidosis can be treated with polyenes, and azoles, and the more recently introduced echinocandins. However, once administered, the intraoral concentration of these drugs tend to be sub-therapeutic and rather transient due to factors such as the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, the pathogenic yeasts may undergo a brief exposure to antifungal drugs. We, therefore, evaluated the phospholipase production of oral C. albicans isolates following brief exposure to sub-therapeutic concentrations of the foregoing antifungals. Materials and methods Fifty C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sub-therapeutic concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for one hour. Thereafter the drugs were removed and the phospholipase production was determined by a plate assay using an egg yolk-agar medium. Results The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 10.65, 12.14, 11.45 and 6.40% following exposure to nystatin, amphotericin B, caspofungin and ketoconazole, respectively. This suppression was not significant following exposure to fluconazole. Conclusions Despite the sub-therapeutic, intra oral, bioavailability of polyenes, echinocandins and ketoconazole, they are likely to produce a persistent antifungal effect by suppressing phospholipase production, which is a key virulent attribute of this common pathogenic yeast.

  14. Nitric oxide production by Biomphalaria glabrata haemocytes: effects of Schistosoma mansoni ESPs and regulation through the extracellular signal-regulated kinase pathway

    Directory of Open Access Journals (Sweden)

    Kirk Ruth S

    2009-04-01

    Full Text Available Abstract Background Schistosoma mansoni uses Biomphalaria glabrata as an intermediate host during its complex life cycle. In the snail, the parasite initially transforms from a miracidium into a mother sporocyst and during this process excretory-secretory products (ESPs are released. Nitric oxide (NO and its reactive intermediates play an important role in host defence responses against pathogens. This study therefore aimed to determine the effects of S. mansoni ESPs on NO production in defence cells (haemocytes from schistosome-susceptible and schistosome-resistant B. glabrata strains. As S. mansoni ESPs have previously been shown to inhibit extracellular signal-regulated kinase (ERK phosphorylation (activation in haemocytes from susceptible, but not resistant, B. glabrata the regulation of NO output by ERK in these cells was also investigated. Results Haemocytes from resistant snails challenged with S. mansoni ESPs (20 μg/ml over 5 h displayed an increase in NO production that was 3.3 times greater than that observed for unchallenged haemocytes; lower concentrations of ESPs (0.1–10 μg/ml did not significantly increase NO output. In contrast, haemocytes from susceptible snails showed no significant change in NO output following challenge with ESPs at any concentration used (0.1–20 μg/ml. Western blotting revealed that U0126 (1 μM or 10 μM blocked the phosphorylation (activation status of ERK in haemocytes from both snail strains. Inhibition of ERK signalling by U0126 attenuated considerably intracellular NO production in haemocytes from both susceptible and resistant B. glabrata strains, identifying ERK as a key regulator of NO output in these cells. Conclusion S. mansoni ESPs differentially influence intracellular NO levels in susceptible and resistant B. glabrata haemocytes, possibly through modulation of the ERK signalling pathway. Such effects might facilitate survival of S. mansoni in its intermediate host.

  15. Intracellular phase for an extracellular bacterial pathogen: MgtC shows the way

    Directory of Open Access Journals (Sweden)

    Audrey Bernut

    2015-08-01

    Full Text Available Pseudomonas aeruginosa is an extracellular pathogen known to impair host phagocytic functions. However, our recent results identify MgtC as a novel actor in P. aeruginosa virulence, which plays a role in an intramacrophage phase of this pathogen. In agreement with its intracellular function, P. aeruginosa mgtC gene expression is strongly induced when the bacteria reside within macrophages. MgtC was previously known as a horizontally-acquired virulence factor important for multiplication inside macrophages in several intracellular bacterial pathogens. MgtC thus provides a singular example of a virulence determinant that subverts macrophages both in intracellular and extracellular pathogens. Moreover, we demonstrate that P. aeru-ginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosaMgtC prevents biofilm formation. We propose that MgtC has a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to the host in relation to the different bacterial lifestyles. MgtC thus appears as an attractive target for antivirulence strategies and our work provides a natural peptide as MgtC antagonist, which paves the way for the development of MgtC inhibitors.

  16. Biotransformation of myrcene by Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Hashemi Elham

    2011-05-01

    Full Text Available Abstract Background Dihydrolinalool and terpineol are sources of fragrances that provide a unique volatile terpenoid alcohol of low toxicity and thus are widely used in the perfumery industry, in folk medicine, and in aromatherapy. They are important chemical constituents of the essential oil of many plants. Previous studies have concerned the biotransformation of limonene by Pseudomonas putida. The objective of this research was to study biotransformation of myrcene by Pseudomonas aeruginosa. The culture preparation was done using such variables as different microbial methods and incubation periods to obtain maximum cells of P. aeruginosa for myrcene biotransformation. Results It was found that myrcene was converted to dihydrolinalool and 2,6-dimethyloctane in high percentages. The biotransformation products were identified by Fourier-transform infrared spectroscopy (FT-IR, ultraviolet (UV analysis, gas chromatography (GC, and gas chromatography-mass spectroscopy (GC-MS. Comparison of the different incubation times showed that 3 days was more effective, the major products being 2,6-dimethyloctane (90.0% and α-terpineol (7.7% and comprising 97.7%. In contrast, the main compounds derived for an incubation time of 1.5 days were dihydrolinalool (79.5% and 2,6-dimethyloctane (9.3%, with a total yield of 88.8%.

  17. Only Acyl Carrier Protein 1 (AcpP1 Functions in Pseudomonas aeruginosa Fatty Acid Synthesis

    Directory of Open Access Journals (Sweden)

    Jin-Cheng Ma

    2017-11-01

    Full Text Available The genome of Pseudomonas aeruginosa contains three open reading frames, PA2966, PA1869, and PA3334, which encode putative acyl carrier proteins, AcpP1, AcpP2, and AcpP3, respectively. In this study, we found that, although these apo-ACPs were successfully phosphopantetheinylated by P. aeruginosa phosphopantetheinyl transferase (PcpS and all holo-forms of these proteins could be acylated by Vibrio harveyi acyl-ACP synthetase (AasS, only AcpP1 could be used as a substrate for the synthesis of fatty acids, catalyzed by P. aeruginosa cell free extracts in vitro, and only acpP1 gene could restore growth in the Escherichia coliacpP mutant strain CY1877. And P. aeruginosaacpP1 could not be deleted, while disruption of acpP2 or acpP3 in the P. aeruginosa genome allowed mutant strains to grow as well as the wild type strain. These findings confirmed that only P. aeruginosa AcpP1 functions in fatty acid biosynthesis, and that acpP2 and acpP3 do not play roles in the fatty acid synthetic pathway. Moreover, disruption of acpP2 and acpP3 did not affect the ability of P. aeruginosa to produce N-acylhomoserine lactones (AHL, but replacement of P. aeruginosaacpP1 with E. coliacpP caused P. aeruginosa to reduce the production of AHL molecules, which indicated that neither P. aeruginosa AcpP2 nor AcpP3 can act as a substrate for synthesis of AHL molecules in vivo. Furthermore, replacement of acpP1 with E. coliacpP reduced the ability of P. aeruginosa to produce some exo-products and abolished swarming motility in P. aeruginosa.

  18. Dialogue inter-règne entre Pseudomonas aeruginosa et les cellules de l'immunité innée. Rôle de la production de L-kynurénine par les bactéries

    OpenAIRE

    Genestet , Charlotte

    2014-01-01

    Pseudomonas aeruginosa is responsible for persistent infections in cystic fibrosis patients, suggesting an ability to circumvent innate immune defenses. Many host cells produce kynurenine, which is known to control immune system homeostasis. Interestingly this bacterium uses the kynurenine pathway to catabolize tryptophan. In addition, preliminary results of our laboratory showed that during acute pulmonary infection in mice with a strain of P. aeruginosa which does not produce kynurenine, th...

  19. Extracellular matrix production by nucleus pulposus and bone marrow stem cells in response to altered oxygen and glucose microenvironments.

    Science.gov (United States)

    Naqvi, Syeda M; Buckley, Conor T

    2015-12-01

    Bone marrow (BM) stem cells may be an ideal source of cells for intervertebral disc (IVD) regeneration. However, the harsh biochemical microenvironment of the IVD may significantly influence the biological and metabolic vitality of injected stem cells and impair their repair potential. This study investigated the viability and production of key matrix proteins by nucleus pulposus (NP) and BM stem cells cultured in the typical biochemical microenvironment of the IVD consisting of altered oxygen and glucose concentrations. Culture-expanded NP cells and BM stem cells were encapsulated in 1.5% alginate and ionically crosslinked to form cylindrical hydrogel constructs. Hydrogel constructs were maintained under different glucose concentrations (1, 5 and 25 mM) and external oxygen concentrations (5 and 20%). Cell viability was measured using the Live/Dead® assay and the production of sulphated glycosaminoglycans (sGAG), and collagen was quantified biochemically and histologically. For BM stem cells, IVD-like micro-environmental conditions (5 mM glucose and 5% oxygen) increased the accumulation of sGAG and collagen. In contrast, low glucose conditions (1 mM glucose) combined with 5% external oxygen concentration promoted cell death, inhibiting proliferation and the accumulation of sGAG and collagen. NP-encapsulated alginate constructs were relatively insensitive to oxygen concentration or glucose condition in that they accumulated similar amounts of sGAG under all conditions. Under IVD-like microenvironmental conditions, NP cells were found to have a lower glucose consumption rate compared with BM cells and may in fact be more suitable to adapt and sustain the harsh microenvironmental conditions. Considering the highly specialised microenvironment of the central NP, these results indicate that IVD-like concentrations of low glucose and low oxygen are critical and influential for the survival and biological behaviour of stem cells. Such findings may promote and accelerate

  20. Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide-enzyme complex

    NARCIS (Netherlands)

    Braun, P; Bitter, W; Tommassen, J

    2000-01-01

    The propeptide of Pseudomonas aeruginosa elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium. Since expression of the lasB gene, which encodes

  1. Lysophosphatidic acid inhibition of the accumulation of Pseudomonas aeruginosa PAO1 alginate, pyoverdin, elastase and LasA

    DEFF Research Database (Denmark)

    Laux, D.C.; Corson, J.M.; Givskov, Michael Christian

    2002-01-01

    . In the present study, a lysophospholipid, 1-paimitoyl-2-hydroxy-sn-glycero-3-phosphate [also called monopalmitoylphosphatidic acid (MPPA)], which accumulates in inflammatory exudates, was shown to inhibit the extracellular accumulation of P. aeruginosa PAO1 alginate, elastase, LasA protease and the siderophore...

  2. Degradation of Uniquely Glycosylated Secretory Immunoglobulin A in Tears From Patients With Pseudomonas aeruginosa Keratitis

    DEFF Research Database (Denmark)

    Lomholt, Jeanet Andersen; Kilian, Mogens

    2008-01-01

    PURPOSE. To investigate the integrity of secretory IgA (S-IgA) in tear fluid during bacterial keratitis and to evaluate the significance of specific Pseudomonas aeruginosa extracellular proteases in the observed degradation of S-IgA. METHODS. The integrity of component chains of S-IgA in tear fluid...... from patients with keratitis caused by P. aeruginosa, Streptococcus group G, Moraxella catarrhalis, Staphylococcus aureus, coagulase-negative staphylococci, and the IgA1 protease-producing Streptococcus pneumoniae were compared with S-IgA in tear fluid, colostrum, and saliva from healthy individuals......, and with tear S-IgA incubated with clinical isolates and genetically engineered P. aeruginosa strains with different protease profiles. Degradation of S-IgA and the significance of its glycosylation were analyzed in Western blots developed with antibodies against individual chains of S-IgA. RESULTS. Secretory...

  3. Interactions between polymorphonuclear leukocytes and Pseudomonas aeruginosa biofilms on silicone implants in vivo

    DEFF Research Database (Denmark)

    van Gennip, Maria; Hultqvist, Louise Dahl; Alhede, Morten

    2012-01-01

    (PMNs). In contrast, the number of cells of a P. aeruginosa rhlA mutant that cannot produce rhamnolipids was significantly reduced on the implants by day 1, and the bacteria were actively phagocytosed by infiltrating PMNs. In addition, we identified extracellular wire-like structures around the bacteria......Chronic infections with Pseudomonas aeruginosa persist because the bacterium forms biofilms that are tolerant to antibiotic treatment and the host immune response. Scanning electron microscopy and confocal laser scanning microscopy were used to visualize biofilm development in vivo following...... intraperitoneal inoculation of mice with bacteria growing on hollow silicone tubes, as well as to examine the interaction between these bacteria and the host innate immune response. Wild-type P. aeruginosa developed biofilms within 1 day that trapped and caused visible cavities in polymorphonuclear leukocytes...

  4. Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa.

    Science.gov (United States)

    Kirienko, Natalia V; Ausubel, Frederick M; Ruvkun, Gary

    2015-02-10

    In the arms race of bacterial pathogenesis, bacteria produce an array of toxins and virulence factors that disrupt core host processes. Hosts mitigate the ensuing damage by responding with immune countermeasures. The iron-binding siderophore pyoverdin is a key virulence mediator of the human pathogen Pseudomonas aeruginosa, but its pathogenic mechanism has not been established. Here we demonstrate that pyoverdin enters Caenorhabditis elegans and that it is sufficient to mediate host killing. Moreover, we show that iron chelation disrupts mitochondrial homeostasis and triggers mitophagy both in C. elegans and mammalian cells. Finally, we show that mitophagy provides protection both against the extracellular pathogen P. aeruginosa and to treatment with a xenobiotic chelator, phenanthroline, in C. elegans. Although autophagic machinery has been shown to target intracellular bacteria for degradation (a process known as xenophagy), our report establishes a role for authentic mitochondrial autophagy in the innate immune defense against P. aeruginosa.

  5. Killing of Pseudomonas aeruginosa by Chicken Cathelicidin-2 Is Immunogenically Silent, Preventing Lung Inflammation In Vivo

    Science.gov (United States)

    Coorens, Maarten; Banaschewski, Brandon J. H.; Baer, Brandon J.; Yamashita, Cory; van Dijk, Albert; Veldhuizen, Ruud A. W.; Veldhuizen, Edwin J. A.

    2017-01-01

    ABSTRACT The development of antibiotic resistance by Pseudomonas aeruginosa is a major concern in the treatment of bacterial pneumonia. In the search for novel anti-infective therapies, the chicken-derived peptide cathelicidin-2 (CATH-2) has emerged as a potential candidate, with strong broad-spectrum antimicrobial activity and the ability to limit inflammation by inhibiting Toll-like receptor 2 (TLR2) and TLR4 activation. However, as it is unknown how CATH-2 affects inflammation in vivo, we investigated how CATH-2-mediated killing of P. aeruginosa affects lung inflammation in a murine model. First, murine macrophages were used to determine whether CATH-2-mediated killing of P. aeruginosa reduced proinflammatory cytokine production in vitro. Next, a murine lung model was used to analyze how CATH-2-mediated killing of P. aeruginosa affects neutrophil and macrophage recruitment as well as cytokine/chemokine production in the lung. Our results show that CATH-2 kills P. aeruginosa in an immunogenically silent manner both in vitro and in vivo. Treatment with CATH-2-killed P. aeruginosa showed reduced neutrophil recruitment to the lung as well as inhibition of cytokine and chemokine production, compared to treatment with heat- or gentamicin-killed bacteria. Together, these results show the potential for CATH-2 as a dual-activity antibiotic in bacterial pneumonia, which can both kill P. aeruginosa and prevent excessive inflammation. PMID:28947647

  6. Identification of a Potential ISR Determinant from Pseudomonas aeruginosa PM12 against Fusarium Wilt in Tomato

    Directory of Open Access Journals (Sweden)

    Sabin Fatima

    2017-05-01

    Full Text Available Biocontrol of plant diseases through induction of systemic resistance is an environmental friendly substitute to chemicals in crop protection measures. Different biotic and abiotic elicitors can trigger the plant for induced resistance. Present study was designed to explore the potential of Pseudomonas aeruginosa PM12 in inducing systemic resistance in tomato against Fusarium wilt. Initially the bioactive compound, responsible for ISR, was separated and identified from extracellular filtrate of P. aeruginosa PM12. After that purification and characterization of the bacterial crude extracts was carried out through a series of organic solvents. The fractions exhibiting ISR activity were further divided into sub-fractions through column chromatography. Sub fraction showing maximum ISR activity was subjected to Gas chromatography/mass spectrometry for the identification of compounds. Analytical result showed three compounds in the ISR active sub-fraction viz: 3-hydroxy-5-methoxy benzene methanol (HMB, eugenol and tyrosine. Subsequent bioassays proved that HMB is the potential ISR determinant that significantly ameliorated Fusarium wilt of tomato when applied as soil drench method at the rate of 10 mM. In the next step of this study, GC-MS analysis was performed to detect changes induced in primary and secondary metabolites of tomato plants by the ISR determinant. Plants were treated with HMB and Fusarium oxysporum in different combinations showing intensive re- modulations in defense related pathways. This work concludes that HMB is the potential elicitor involved in dynamic reprogramming of plant pathways which functionally contributes in defense responses. Furthermore the use of biocontrol agents as natural enemies of soil borne pathogens besides enhancing production potential of crop can provide a complementary tactic for sustainable integrated pest management.

  7. Purification and Characterization of Extracellular enzyme from Aspergillus fumigatus and Its Application on a pennisetum sp for enhanced glucose production

    Directory of Open Access Journals (Sweden)

    Sonali Mohapatra

    2017-12-01

    Full Text Available Aspergillus species are saprophytic fungi widely distributed in nature and are associated with a number of human diseases. The present study was investigated for production of extracellular cellulase from Aspergillus fumigatus which could be potentially used for degradation of cellulose in lignocellulosic biomass for bioethanol production. In the present work, A. fumigatus were grown in fungal basal medium and preserved at 30 °C for 72 h. The cellulase enzyme was filtered (using Whatman filter paper, precipitated (using ammonium sulphate, dialysed and then purified on a Sepharose 6B ion exchange column. The cellulase enzyme showed a purification of 0.4 fold and the molecular weight was determined as 100 kDa by SDS-PAGE. The optimum pH, temperature, incubation time of the enzyme was determined to be pH 7.0, 35 °C and 24 h respectively. The presence of metal ion Mn2+, followed by Ca2+ and Co2+ was found to increase the cellulase activity. Notably, the cellulase activity was not significantly affected in the presence of additives like EDTA, and Triton X-100 and β-mercaptoethanol. Response surface methodology was used to design optimisation experiments for saccharification of lignocellulosic biomass (hybrid napier grass and the response i.e. glucose yield was considered as the product. The glucose yield was considerably increased from 101.4 mg/g to 856.5 mg/g in the optimised conditions of 35°C, pH 5.2 with substrate concentration (ultrasono assisted alkali pretreated biomass of 3.5 g, with enzyme concentration of 3 ml was incubated for 24 h. Further, the statistical analysis using ANNOVA demonstrated a p- value of less than 0.005 and the R2 value of 90.18.

  8. Development of antibiotic resistance in Pseudomonas aeruginosa during two decades of antipseudomonal treatment at the Danish CF Center

    DEFF Research Database (Denmark)

    Ciofu, O; Giwercman, B; Pedersen, S S

    1994-01-01

    resistance in P. aeruginosa strains isolated from Danish CF patients over a period of 18 years by testing the in vitro efficacy of carbenicillin, piperacillin, ceftazidime, tobramycin and ciprofloxacin against P. aeruginosa strains collected in 1973 (51 strains), 1980 (80 strains), 1985 (58 strains...... was found between the MIC and the number of antipseudomonal courses of antibiotics. The proportion of resistant in vivo selected P. aeruginosa strains, presumed to be stably derepressed producers of chromosomal beta-lactamase, also increased significantly during the period studied. Our results confirm...... that the beta-lactamase production is an important mechanism of antibiotic resistance in P. aeruginosa....

  9. Characterization of the rcsA Gene from Pantoea sp. Strain PPE7 and Its Influence on Extracellular Polysaccharide Production and Virulence on Pleurotus eryngii

    Directory of Open Access Journals (Sweden)

    Min Keun Kim

    2017-06-01

    Full Text Available RcsA is a positive activator of extracellular polysaccharide (EPS synthesis in the Enterobacteriaceae. The rcsA gene of the soft rot pathogen Pantoea sp. strain PPE7 in Pleurotus eryngii was cloned by PCR amplification, and its role in EPS synthesis and virulence was investigated. The RcsA protein contains 3 highly conserved domains, and the C-terminal end of the open reading frame shared significant amino acid homology to the helix-turn-helix DNA binding motif of bacterial activator proteins. The inactivation of rcsA by insertional mutagenesis created mutants that had decreased production of EPS compared to the wild-type strain and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. The Pantoea sp. strain PPE7 rcsA gene was shown to strongly affect the formation of the disease symptoms of a mushroom pathogen and to act as the virulence factor to cause soft rot disease in P. eryngii.

  10. Pseudomonas aeruginosa Psl Exopolysaccharide Interacts with the Antimicrobial Peptide LG21

    Directory of Open Access Journals (Sweden)

    Joyce Seow Fong Chin

    2017-09-01

    Full Text Available Biofilm formation by opportunistic pathogens serves as one of the major causes of chronic and persistent infections. Bacterial cells in the biofilms are embedded in their self-generated protective extracellular polymeric substances (EPS, which include exopolysaccharides, large adhesin proteins and extracellular DNA. In this study, we identified an antimicrobial peptide (AMP LG21 that is able to interact specifically with the Psl exopolysaccharide of Pseudomonas aeruginosa, thus it can be used as a diagnostic tool for P. aeruginosa biofilms. Molecular dynamics simulation analysis showed that residues numbered from 15 to 21 (WKRKRFG in LG21 are involved in interacting with Psl. Our study indicates that host immune systems might detect and interact with microbial biofilms through AMPs. Engineering biofilm EPS-targeting AMPs might provide novel strategies for biofilm detection and treatment.

  11. Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis

    DEFF Research Database (Denmark)

    Marvig, Rasmus Lykke; Madsen Sommer, Lea Mette; Molin, Søren

    2015-01-01

    fibrosis. Our analysis of 36 P. aeruginosa lineages identified convergent molecular evolution in 52 genes. This list of genes suggests a role in host adaptation for remodeling of regulatory networks and central metabolism, acquisition of antibiotic resistance and loss of extracellular virulence factors....... Furthermore, we find an ordered succession of mutations in key regulatory networks. Accordingly, mutations in downstream transcriptional regulators were contingent upon mutations in upstream regulators, suggesting that remodeling of regulatory networks might be important in adaptation. The characterization...

  12. Extracellular DNA as matrix component in microbial biofilms

    DEFF Research Database (Denmark)

    Chiang, Wen-Chi; Tolker-Nielsen, Tim

    2010-01-01

    Bacteria in nature primarily live in surface-associated communities commonly known as biofilms. Because bacteria in biofilms, in many cases, display tolerance to host immune systems, antibiotics, and biocides, they are often difficult or impossible to eradicate. Biofilm formation, therefore, leads...... to various persistent infections in humans and animals, and to a variety of complications in industry, where solid–water interfaces occur. Knowledge about the molecular mechanisms involved in biofilm formation is necessary for creating strategies to control biofilms. Recent studies have shown...... that extracellular DNA is an important component of the extracellular matrix of microbial biofilms. The present chapter is focussed on extracellular DNA as matrix component in biofilms formed by Pseudomonas aeruginosa as an example from the Gram-negative bacteria, and Streptococcus and Staphylococcus as examples...

  13. Carbapenem Susceptibility and Multidrug-Resistance in Pseudomonas aeruginosa Isolates in Egypt.

    Science.gov (United States)

    Hashem, Hany; Hanora, Amro; Abdalla, Salah; Shawky, Alaa; Saad, Alaa

    2016-11-01

    Resistant Pseudomonas aeruginosa is a serious concern for antimicrobial therapy, as the common isolates exhibit variable grades of resistance, involving beta-lactamase enzymes, beside native defense mechanisms. The present study was designed to determine the occurrence of Metallo-β- Lactamases (MBL) and Amp C harboring P. aeruginosa isolates from Suez Canal university hospital in Ismailia, Egypt. A total of 147 P. aeruginosa isolates, recovered from 311 patients during a 10-month period, were collected between May 2013 and February 2014; the isolates were collected from urine, wound and sputum. Minimum inhibitory concentration (MIC) determined by agar dilution methods was ≥2 μg/mL for meropenem and imipenem. Identification of P. aeruginosa was confirmed using API 20NE. Metallo-β- Lactamases and Amp C were detected based on different phenotypic methods. Overall, 26.5% of P. aeruginosa isolates (39/147) were carbapenem resistant isolates. Furthermore, 64.1% (25/39) were MBL producers, these isolates were screened by the combined disc and disc diffusion methods to determine the ability of MBL production. Both MBL and Amp C harbored P. aeruginosa isolates were 28% (7/25). Sixty-four percent of P. aeruginosa isolates were multidrug resistant (MDR) (16/25). The sensitivity toward polymyxin, imipenem, norfloxacin, piperacillin-tazobactam and gentamicin was 99%, 91%, 88%, 82% and 78%, respectively. The resistance rate towards cefotaxime, ceftazidime, cefepime, aztreonam and meropenem was 98.6%, 86%, 71.4%, 34% and 30%, respectively. Multidrug resistance was significantly associated with MBL production in P. aeruginosa . Early detection of MBL-producing P. aeruginosa and hospital antibiotic policy prescription helps proper antimicrobial therapy and avoidance of dissemination of these multidrug resistance isolates.

  14. Catheter-related infections caused by Pseudomonas aeruginosa: virulence factors involved and their relationships.

    Science.gov (United States)

    Olejnickova, Katerina; Hola, Veronika; Ruzicka, Filip

    2014-11-01

    The nosocomial pathogen Pseudomonas aeruginosa is equipped with a large arsenal of cell-associated and secreted virulence factors which enhance its invasive potential. The complex relationships among virulence determinants have hitherto not been fully elucidated. In the present study, 175 catheter-related isolates were observed for the presence of selected virulence factors, namely extracellular enzymes and siderophore production, biofilm formation, resistance to antibiotics, and motility. A high percentage of the strains produced most of the tested virulence factors. A positive correlation was identified between the production of several exoproducts, and also between the formation of both types of biofilm. An opposite trend was observed between the two types of biofilm and the production of siderophores. Whereas the relationship between the submerged biofilm production (i.e. the biofilm formed on the solid surface below the water level) and the siderophore secretion was negative, the production of air-liquid interface (A-L) biofilm (i.e. the biofilm floating on the surface of the cultivation medium) and the siderophore secretion were positively correlated. All correlations were statistically significant at the level P = 0.05 with the correlation coefficient γ ≥ 0.50. Our results suggest that: (1) the co-production of the lytic enzymes and siderophores can play an important role in the pathogenesis of the catheter-related infections and should be taken into account when the virulence potential is assessed; (2) biofilm-positive strains are capable of forming both submerged and non-attached A-L biofilms; and (3) the different micro-environment in the submerged biofilm and A-L biofilm layers have opposite consequences for the production of other virulence factors. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  15. Silver against Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Kirketerp-Møller, K.; Kristiansen, S.

    2007-01-01

    bacteria in both the planktonic and biofilm modes of growth. The action of silver on mature in vitro biofilms of Pseudomonas aeruginosa, a primary pathogen of chronic infected wounds, was investigated. The results show that silver is very effective against mature biofilms of P. aeruginosa......, but that the silver concentration is important. A concentration of 5-10 ig/mL silver sulfadiazine eradicated the biofilm whereas a lower concentration (1 ig/mL) had no effect. The bactericidal concentration of silver required to eradicate the bacterial biofilm was 10-100 times higher than that used to eradicate...... planktonic bacteria. These observations strongly indicate that the concentration of silver in currently available wound dressings is much too low for treatment of chronic biofilm wounds. It is suggested that clinicians and manufacturers of the said wound dressings consider whether they are treating wounds...

  16. Effects of hyperbaric oxygen on Pseudomonas aeruginosa susceptibility to imipenem and macrophages.

    Science.gov (United States)

    Lima, Flavia Luna; Joazeiro, Paulo Pinto; Lancellotti, Marcelo; de Hollanda, Luciana Maria; de Araújo Lima, Bruna; Linares, Edlaine; Augusto, Ohara; Brocchi, Marcelo; Giorgio, Selma

    2015-01-01

    The seriousness to treat burn wounds infected with Pseudomonas aeruginosa led us to examine whether the effect of the carbapenem antibiotic imipenem is enhanced by hyperbaric oxygen (HBO). The effects of HBO (100% O2, 3 ATA, 5 h) in combination with imipenen on bacterial counts of six isolates of P. aeruginosa and bacterial ultrastructure were investigated. Infected macrophages were exposed to HBO (100% O2, 3 ATA, 90 min) and the production of reactive oxygen species monitored. HBO enhanced the effects of imipenen. HBO increased superoxide anion production by macrophages and likely kills bacteria by oxidative mechanisms. HBO in combination with imipenem can be used to kill P. aeruginosa in vitro and such treatment may be beneficial for the patients with injuries containing the P. aeruginosa.

  17. Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

    Science.gov (United States)

    Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

    2016-11-01

    To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  18. In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold.

    Science.gov (United States)

    Xie, Lin; Zhang, Nan; Marsano, Anna; Vunjak-Novakovic, Gordana; Zhang, Yanru; Lopez, Mandi J

    2013-12-01

    Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.

  19. Production of extracellular polymeric substances (EPS) by Serratia sp.1 using wastewater sludge as raw material and flocculation activity of the EPS produced.

    Science.gov (United States)

    Bezawada, J; Hoang, N V; More, T T; Yan, S; Tyagi, N; Tyagi, R D; Surampalli, R Y

    2013-10-15

    Growth profile and extracellular polymeric substances (EPS) production of Serratia sp.1 was studied in shake flask fermentation for 72 h using wastewater sludge as raw material. Maximum cell concentration of 6.7 × 10(9) cfu/mL was obtained at 48 h fermentation time. EPS dry weight, flocculation activity and dewaterability of different EPS (tightly bound or TB-EPS, loosely bound or LB-EPS and broth-EPS or B-EPS) were also measured. The highest concentration of LB-EPS (2.45 g/L) and TB-EPS (0.99 g/L) were attained at 48 h of fermentation. Maximum flocculation activity and dewaterability (ΔCST) of TB-EPS (76.4%, 14.5s and 76.5%, 15.5s), LB-EPS (67.8%, 8.1s and 64.7%, 7.6s) and broth EPS (61%, 6.1s and 70.4%, 6.8s) were obtained at 36 and 48 h of growth. Higher flocculation activity and dewaterability were achieved with TB-EPS than with the two other EPS. Characterization of TB-EPS and LB-EPS was done in terms of their protein and carbohydrate content. Protein content was much higher in TB-EPS where as carbohydrate content was only slightly higher in TB-EPS than LB-EPS. Morphology of the Serratia strain after fermentation in sludge and TSB was observed under a scanning electron microscope and the cell size was found to be bigger in the sludge medium than the TSB medium. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Pseudomonas aeruginosa Population Structure Revisited

    Science.gov (United States)

    Pirnay, Jean-Paul; Bilocq, Florence; Pot, Bruno; Cornelis, Pierre; Zizi, Martin; Van Eldere, Johan; Deschaght, Pieter; Vaneechoutte, Mario; Jennes, Serge; Pitt, Tyrone; De Vos, Daniel

    2009-01-01

    At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P

  1. Human galectins induce conversion of dermal fibroblasts into myofibroblasts and production of extracellular matrix: potential application in tissue engineering and wound repair

    Czech Academy of Sciences Publication Activity Database

    Dvořánková, B.; Szabo, P.; Lacina, L.; Gál, P.; Uhrová, J.; Zima, T.; Kaltner, H.; André, S.; Gabius, H. J.; Syková, Eva; Smetana Jr., K.

    2011-01-01

    Roč. 194, č. 6 (2011), s. 469-480 ISSN 1422-6405 Grant - others:GA MŠk(CZ) 1M0538 Program:1M Institutional research plan: CEZ:AV0Z50390703 Keywords : extracellular matrix * fibronectin * keratinocyte Subject RIV: FH - Neurology Impact factor: 2.203, year: 2011

  2. Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents

    DEFF Research Database (Denmark)

    Yang, Liang; Liu, Yang; Sternberg, Claus

    2010-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for a wide range of infections. Production of virulence factors and biofilm formation by P. aeruginosa are partly regulated by cell-to-cell communication quorum-sensing systems. Identification of quorum-quenching reagents...... which block the quorum-sensing process can facilitate development of novel treatment strategies for P. aeruginosa infections. We have used molecular dynamics simulation and experimental studies to elucidate the efficiencies of two potential quorum-quenching reagents, triclosan and green tea...... epigallocatechin gallate (EGCG), which both function as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) from the bacterial type II fatty acid synthesis pathway. Our studies suggest that EGCG has a higher binding affinity towards ENR of P. aeruginosa and is an efficient quorum-quenching reagent...

  3. Urotensin II Induces ER Stress and EMT and Increase Extracellular Matrix Production in Renal Tubular Epithelial Cell in Early Diabetic Mice

    Directory of Open Access Journals (Sweden)

    Xin-Xin Pang

    2016-07-01

    Full Text Available Background/Aims: Urotensin II (UII and its receptor are highly expressed in the kidney tissue of patients with diabetic nephropathy (DN. The aim of this study is to examine the roles of UII in the induction of endoplasmic reticulum stress (ER stress and Epithelial-mesenchymal transition (EMT in DN in vivo and in vitro. Methods: Kidney tissues were collected from patients with DN. C57BL/6 mice and mice with UII receptor knock out were injected with two consecutive doses of streptozotocin to induce diabetes and were sacrificed at 3th week for in vivo study. HK-2 cells in vitro were cultured and treated with UII. Markers of ER stress and EMT, fibronectin and type IV collagen were detected by immunohistochemistry, real time PCR and western blot. Results: We found that the expressions of protein of UII, GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were upregulated while E-cadherin protein was downregulated as shown by immunohistochemistry or western blot analysis in kidney of diabetic mice in comparison to normal control; moreover expressions of GRP78, CHOP, ALPHA-SMA, fibronectin and type IV collagen were inhibited while E-caherin expression was enhanced in kidney in diabetic mice with UII receptor knock out in comparison to C57BL/6 diabetic mice. In HK-2 cells, UII induced upregulation of GRP78, CHOP, ALPHA-SMA, fibroblast-specifc protein 1(FSP-1, fibronectin and type collagen and downregulation of E-cadherin. UII receptor antagonist can block UII-induced ER stress and EMT; moreover, 4-PBA can inhibit the mRNA expression of ALPHA-SMA and FSP1 induced by UII in HK-2 cells. Conclusions: We are the first to verify UII induces ER stress and EMT and increase extracellular matrix production in renal tubular epithelial cell in early diabetic mice. Moreover, UII may induce renal tubular epithelial EMT via triggering ER stress pathway in vitro, which might be the new pathogenic pathway for the development of renal fibrosis in DN.

  4. Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.

    Science.gov (United States)

    Folders, J; Algra, J; Roelofs, M S; van Loon, L C; Tommassen, J; Bitter, W

    2001-12-01

    The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.

  5. Production of extracellular lipase by the phytopathogenic fungus Fusarium solani FS1 Produção de lipase extracelular pelo fungo fitopatogênico Fusarium solani FS1

    OpenAIRE

    Maria de Mascena Diniz Maia; Marcia Maria Camargo de Morais; Marcos Antonio de Morais Jr.; Eduardo Henrique Magalhães Melo; José Luiz de Lima Filho

    1999-01-01

    A Brazilian strain of Fusarium solani was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.l-1 of lipase after 72 hours of cultivation at 25oC in shake-flask at 120 rpm in a medium containing 3% (w/v) peptone plus 0.5% (v/v) olive oil. Glucose (1% w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3% (w/v) resulted in a reduced lipase production while increased olive oil concentration (above 0.5%) did n...

  6. Production of medium-chain volatile flavour esters in Pichia pastoris whole-cell biocatalysts with extracellular expression of Saccharomyces cerevisiae acyl-CoA: ethanol O-acyltransferase Eht1 or Eeb1

    DEFF Research Database (Denmark)

    Zhuang, Shiwen; Fu, Junshu; Powell, Chris

    2015-01-01

    Medium-chain volatile flavour esters are important molecules since they have extensive applications in food, fragrance, cosmetic, paint and coating industries, which determine different characteristics of aroma or taste in commercial products. Biosynthesis of these compounds by alcoholysis...... pastoris yeasts with functional extracellular expression of Eht1 or Eeb1 were constructed. Flavour production was established through an integrated process with coupled enzyme formation and ester biosynthesis in the recombinant yeasts in one pot, leading to the formation of volatile C6-C14 methyl and ethyl...

  7. Ciprofloxacin susceptibility of Pseudomonas aeruginosa isolates from keratitis

    DEFF Research Database (Denmark)

    Lomholt, JA; Kilian, Mogens

    2003-01-01

    AIM: To examine the ciprofloxacin susceptibility of 106 Pseudomonas aeruginosa eye isolates from the United Kingdom, Denmark, India, the United States, and Australia, and to determine the molecular mechanisms of resistance. METHODS: Ciprofloxacin susceptibility was tested by an agar dilution method...... keratitis, endophthalmitis, contact lens associated red eye (CLARE), and contact lens storage cases showed MIC values below 1 mg/l. Several allelic forms of gyrA and a single variation in the mexR gene product were detected in 10 ciprofloxacin susceptible strains. CONCLUSIONS: The vast majority of eye...... isolates of P aeruginosa from European countries are fully susceptible to ciprofloxacin and the concentration of ciprofloxacin eye drops used for local treatment (3000 mg/l) exceeds MIC values for strains recorded as resistant. Mutations in more than one target gene were associated with higher MIC values....

  8. Ciprofloxacin susceptibility of Pseudomonas aeruginosa isolates from keratitis

    DEFF Research Database (Denmark)

    Lomholt, JA; Kilian, Mogens

    2003-01-01

    keratitis, endophthalmitis, contact lens associated red eye (CLARE), and contact lens storage cases showed MIC values below 1 mg/l. Several allelic forms of gyrA and a single variation in the mexR gene product were detected in 10 ciprofloxacin susceptible strains. CONCLUSIONS: The vast majority of eye......AIM: To examine the ciprofloxacin susceptibility of 106 Pseudomonas aeruginosa eye isolates from the United Kingdom, Denmark, India, the United States, and Australia, and to determine the molecular mechanisms of resistance. METHODS: Ciprofloxacin susceptibility was tested by an agar dilution method...... isolates of P aeruginosa from European countries are fully susceptible to ciprofloxacin and the concentration of ciprofloxacin eye drops used for local treatment (3000 mg/l) exceeds MIC values for strains recorded as resistant. Mutations in more than one target gene were associated with higher MIC values....

  9. Trehalose biosynthesis promotes Pseudomonas aeruginosa pathogenicity in plants.

    Science.gov (United States)

    Djonović, Slavica; Urbach, Jonathan M; Drenkard, Eliana; Bush, Jenifer; Feinbaum, Rhonda; Ausubel, Jonathan L; Traficante, David; Risech, Martina; Kocks, Christine; Fischbach, Michael A; Priebe, Gregory P; Ausubel, Frederick M

    2013-03-01

    Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved "house-keeping" anabolic pathway (trehalose

  10. Trehalose biosynthesis promotes Pseudomonas aeruginosa pathogenicity in plants.

    Directory of Open Access Journals (Sweden)

    Slavica Djonović

    2013-03-01

    Full Text Available Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved "house-keeping" anabolic

  11. Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa.

    Science.gov (United States)

    Elamin, Ayssar A; Steinicke, Susanne; Oehlmann, Wulf; Braun, Yvonne; Wanas, Hanaa; Shuralev, Eduard A; Huck, Carmen; Maringer, Marko; Rohde, Manfred; Singh, Mahavir

    2017-01-01

    For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection.

  12. Arsenate biotransformation by Microcystis aeruginosa under different nitrogen and phosphorus levels.

    Science.gov (United States)

    Che, Feifei; Du, Miaomiao; Yan, Changzhou

    2018-04-01

    The arsenate (As(V)) biotransformation by Microcystis aeruginosa in a medium with different concentrations of nitrogen (N) and phosphorus (P) has been studied under laboratory conditions. When 15μg/L As(V) was added, N and P in the medium showed effective regulation on arsenic (As) metabolism in M. aeruginosa, resulting in significant differences in the algal growth among different N and P treatments. Under 0.2mg/L P treatment, increases in N concentration (4-20mg/L) significantly stimulated the cell growth and therefore indirectly enhanced the production of dimethylarsinic acid (DMA), the main As metabolite, accounting for 71%-79% of the total As in the medium. Meanwhile, 10-20mg/L N treatments accelerated the ability of As metabolization by M. aeruginosa, leading to higher contents of DMA per cell. However, As(V) uptake by M. aeruginosa was significantly impeded by 0.5-1.0mg/L P treatment, resulting in smaller rates of As transformation in M. aeruginosa as well as lower contents of As metabolites in the medium. Our data demonstrated that As(V) transformation by M. aeruginosa was significantly accelerated by increasing N levels, while it was inhibited by increasing P levels. Overall, both P and N play key roles in As(V) biotransformation processes. Copyright © 2017. Published by Elsevier B.V.

  13. Emergence of Imipenem-Resistant Pseudomonas aeruginosa Clinical Isolates from Egypt Coharboring VIM and IMP Carbapenemases.

    Science.gov (United States)

    El-Domany, Ramadan Ahmed; Emara, Mohamed; El-Magd, Mohammed A; Moustafa, Walaa H; Abdeltwab, Nesma M

    2017-09-01

    Pseudomonas aeruginosa is an important human pathogen and the leading cause of nosocomial infections. P. aeruginosa is characterized by massive intrinsic resistance to a multiple classes of antibiotics with carbapenems being the most potent inhibitor of P. aeruginosa and considered the first choice for its treatment. Therefore, it is crucial to investigate novel mechanisms of resistance of P. aeruginosa to carbapenems for achieving successful therapy. A total of 114 P. aeruginosa isolates from two university hospitals in Egypt were recruited in this study. Antimicrobial susceptibility testing revealed that 50 isolates (43.8%) exhibited multidrug-resistant (MDR) phenotype, of them 14 isolates (12.2%) were imipenem (IPM)-resistant. Of these 14 isolates, 13 isolates (11.4%) exhibited the metallo-β-lactamase (MBL) phenotype. MBLs encoding genes, VIM and IMP, were identified by PCR. PCR results revealed that four isolates harbored the VIM gene alone, one isolate harbored IMP gene alone, and four isolates harbored both genes. The correct size of PCR products of VIM and IMP genes (390 and 188 bp, respectively) were sequenced to confirm results of PCR and to look for any possible polymorphism among MBL genes of tested isolates. Data analysis of these sequences showed 100% identity of nucleotide sequences of MBL genes among tested Egyptian patients. To our knowledge, this is the first report of IMP carbapenemase-encoding gene in Africa and the first detection of the emergence of P. aeruginosa coproducing VIM and IMP genes in Egypt.

  14. Interactions between Microcystis aeruginosa and coexisting amoxicillin contaminant at different phosphorus levels.

    Science.gov (United States)

    Liu, Ying; Chen, Shi; Chen, Xiao; Zhang, Jian; Gao, Baoyu

    2015-10-30

    Microcystis aeruginosa was cultured with 0.05-5 mg L(-1) of phosphorus and exposed to 200-500 ng L(-1) of amoxicillin for seven days. Amoxicillin presented no significant effect (p>0.05) on the growth of M. aeruginosa at phosphorus levels of 0.05 and 0.2 mg L(-1), but stimulated algal growth as a hormesis effect at phosphorus levels of 1 and 5 mg L(-1). Phosphorus and amoxicillin affected the contents of chlorophyll-a, adenosine triphosphate (ATP) and malondialdehyde, the expression of psbA and rbcL, as well as the activities of adenosinetriphosphatase and glutathione S-transferase in similar manners, but regulated the production and release of microcystins and the activities of superoxide dismutase and peroxidase in different ways. Increased photosynthesis activity was related with the ATP consumption for the stress response to amoxicillin, and the stress response was enhanced as the phosphorus concentration increased. The biodegradation of amoxicillin by M. aeruginosa increased from 11.5% to 28.2% as the phosphorus concentration increased. Coexisting amoxicillin aggravated M. aeruginosa pollution by increasing cell density and concentration of microcystins, while M. aeruginosa alleviated amoxicillin pollution via biodegradation. The interactions between M. aeruginosa and amoxicillin were significantly regulated by phosphorus (p<0.05) and led to a complicated situation of combined pollution. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Anaerobic Corrosion of 304 Stainless Steel Caused by the Pseudomonas aeruginosa Biofilm

    Directory of Open Access Journals (Sweden)

    Ru Jia

    2017-11-01

    Full Text Available Pseudomonas aeruginosa is a ubiquitous bacterium capable of forming problematic biofilms in many environments. They cause biocorrosion of medical implants and industrial equipment and infrastructure. Aerobic corrosion of P. aeruginosa against stainless steels has been reported by some researchers while there is a lack of reports on anaerobic P. aeruginosa corrosion in the literature. In this work, the corrosion by a wild-type P. aeruginosa (strain PAO1 biofilm against 304 stainless steel (304 SS was investigated under strictly anaerobic condition for up to 14 days. The anaerobic corrosion of 304 SS by P. aeruginosa was reported for the first time. Results showed that the average sessile cell counts on 304 SS coupons after 7- and 14-day incubations were 4.8 × 107 and 6.2 × 107 cells/cm2, respectively. Scanning electron microscopy and confocal laser scanning microscopy corroborated the sessile cell counts. The X-ray diffraction analysis identified the corrosion product as iron nitride, confirming that the corrosion was caused by the nitrate reducing biofilm. The largest pit depths on 304 SS surfaces after the 7- and 14-day incubations with P. aeruginosa were 3.9 and 7.4 μm, respectively. Electrochemical tests corroborated the pitting data.

  16. THE FORMATION OF BIOFILMS BY PSEUDOMONAS AERUGINOSA STRAINS, AND METHODS OF ITS CONTROL (REVIEW

    Directory of Open Access Journals (Sweden)

    Sarkis-Ivanova VV

    2017-03-01

    Full Text Available The majority of natural bacterial populations exist in the form of biofilms, highly-structured multicellular communities incorporated in extracellular polymer matrix of own production. For a considerable part of clinically significant species this form of existence provides optimal conditions for reaching of pathogenic and colonizational potential, and also encourages saving of metabolically inactive part of population which is characterized by a low level of sensitivity to antibiotics impact. The latter plays a significant role in formation of chronic persistent infections resistant to antibiotics treatment. Members of microbial group are united basing on the principle which excludes antagonism, determines their nutritional, energetic and another connections between them and environment. Such a connection of microorganisms community behavior received a special definition of “quorum sensing”. Quorum sensing is an ability of some bacteria (probably, also another microorganisms to communicate and coordinate their behavior through secretion of substances which are the signals for coordination of certain behavior or interaction between bacteria of the same type or subtype depending on their growth tightness. When the concentration of excreted signaling agents reaches a borderline value the group of bacteria starts working as a single organism. Furthermore, signaling agents for Gram-negative and Gram-positive microorganisms differ. For instance, for Pseudomonas aeruginosa microorganism a signaling molecule of quorum sensing is acyl homoserine lactone, and biofilm created by this pathogen produces pyocyanin which functions as an electronic transporter in current generation. A large-scale investigation of P.aeruginosa biofilm morphology, mechanisms of its production and degradation, peculiarities of formation under different conditions and impact of various factors are at the infancy stage. According to undivided opinion of scientists who

  17. Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression.

    Science.gov (United States)

    Kasper, Stephen H; Bonocora, Richard P; Wade, Joseph T; Musah, Rabi Ann; Cady, Nathaniel C

    2016-04-15

    The opportunistic pathogen Pseudomonas aeruginosa utilizes multiple quorum sensing (QS) pathways to coordinate an arsenal of virulence factors. We previously identified several cysteine-based compounds inspired by natural products from the plant Petiveria alliacea which are capable of antagonizing multiple QS circuits as well as reducing P. aeruginosa biofilm formation. To understand the global effects of such compounds on virulence factor production and elucidate their mechanism of action, RNA-seq transcriptomic analysis was performed on P. aeruginosa PAO1 exposed to S-phenyl-l-cysteine sulfoxide, the most potent inhibitor from the prior study. Exposure to this inhibitor down-regulated expression of several QS-regulated virulence operons (e.g., phenazine biosynthesis, type VI secretion systems). Interestingly, many genes that were differentially regulated pertain to the related metabolic pathways that yield precursors of pyochelin, tricarboxylic acid cycle intermediates, phenazines, and Pseudomonas quinolone signal (PQS). Activation of the MexT-regulon was also indicated, including the multidrug efflux pump encoded by mexEF-oprN, which has previously been shown to inhibit QS and pathogenicity. Deeper investigation of the metabolites involved in these systems revealed that S-phenyl-l-cysteine sulfoxide has structural similarity to kynurenine, a precursor of anthranilate, which is critical for P. aeruginosa virulence. By supplementing exogenous anthranilate, the QS-inhibitory effect was reversed. Finally, it was shown that S-phenyl-l-cysteine sulfoxide competitively inhibits P. aeruginosa kynureninase (KynU) activity in vitro and reduces PQS production in vivo. The kynurenine pathway has been implicated in P. aeruginosa QS and virulence factor expression; however, this is the first study to show that targeted inhibition of KynU affects P. aeruginosa gene expression and QS, suggesting a potential antivirulence strategy.

  18. Pattern formation in Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Parsek, Matthew R.; Tolker-Nielsen, Tim

    2008-01-01

    Bacteria are capable of forming elaborate multicellular communities called biofilms. Pattern formation in biofilms depends on cell proliferation and cellular migration in response to the available nutrients and other external cues, as well as on self-generated intercellular signal molecules...... and the production of an extracellular matrix that serves as a structural 'scaffolding' for the biofilm cells. Pattern formation in biofilms allows cells to position themselves favorably within nutrient gradients and enables buildup and maintenance of physiologically distinct subpopulations, which facilitates...... survival of one or more subpopulations upon environmental insult, and therefore plays an important role in the innate tolerance displayed by biofilms toward adverse conditions....

  19. Electrical conductivity measurements of bacterial nanowires from Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Maruthupandy, Muthusamy; Anand, Muthusamy; Beevi, Akbar Sait Hameedha; Priya, Radhakrishnan Jeeva; Maduraiveeran, Govindhan

    2015-01-01

    The extracellular appendages of bacteria (flagella) that transfer electrons to electrodes are called bacterial nanowires. This study focuses on the isolation and separation of nanowires that are attached via Pseudomonas aeruginosa bacterial culture. The size and roughness of separated nanowires were measured using transmission electron microscopy (TEM) and atomic force microscopy (AFM), respectively. The obtained bacterial nanowires indicated a clear image of bacterial nanowires measuring 16 nm in diameter. The formation of bacterial nanowires was confirmed by microscopic studies (AFM and TEM) and the conductivity nature of bacterial nanowire was investigated by electrochemical techniques. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), which are nondestructive voltammetry techniques, suggest that bacterial nanowires could be the source of electrons—which may be used in various applications, for example, microbial fuel cells, biosensors, organic solar cells, and bioelectronic devices. Routine analysis of electron transfer between bacterial nanowires and the electrode was performed, providing insight into the extracellular electron transfer (EET) to the electrode. CV revealed the catalytic electron transferability of bacterial nanowires and electrodes and showed excellent redox activities. CV and EIS studies showed that bacterial nanowires can charge the surface by producing and storing sufficient electrons, behave as a capacitor, and have features consistent with EET. Finally, electrochemical studies confirmed the development of bacterial nanowires with EET. This study suggests that bacterial nanowires can be used to fabricate biomolecular sensors and nanoelectronic devices. (paper)

  20. Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis.

    Science.gov (United States)

    Marvig, Rasmus Lykke; Sommer, Lea Mette; Molin, Søren; Johansen, Helle Krogh

    2015-01-01

    Little is known about how within-host evolution compares between genotypically different strains of the same pathogenic species. We sequenced the whole genomes of 474 longitudinally collected clinical isolates of Pseudomonas aeruginosa sampled from 34 children and young individuals with cystic fibrosis. Our analysis of 36 P. aeruginosa lineages identified convergent molecular evolution in 52 genes. This list of genes suggests a role in host adaptation for remodeling of regulatory networks and central metabolism, acquisition of antibiotic resistance and loss of extracellular virulence factors. Furthermore, we find an ordered succession of mutations in key regulatory networks. Accordingly, mutations in downstream transcriptional regulators were contingent upon mutations in upstream regulators, suggesting that remodeling of regulatory networks might be important in adaptation. The characterization of genes involved in host adaptation may help in predicting bacterial evolution in patients with cystic fibrosis and in the design of future intervention strategies.

  1. Microbial cells as biosorbents for heavy metals: accumulation of Uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Strandberg, G.W.; Shumate, S.E. II; Parrott, J.R. Jr.

    1981-01-01

    Uranium accumulated extracellularly on the surfaces of Saccharomyces cerevisiae cells. The rate and extent of accumulation were subject to environmental parameters, such as pH, temperature, and interference by certain anions and cations. Uranium accumulation by Pseudomonas aeruginosa occurred intracellularly and was extremely rapid (<10 s), and no response to environmental parameters could be detected. Metabolism was not required for metal uptake by either organism. Cell-bound uranium reached a concentration of 10 to 15% of the dry cell weight, but only 32% of the S. cerevisiae cells and 44% of the P. aeruginosa cells within a given population possessed visible uranium deposits when examined by electron microscopy. Rates of uranium uptake by S. cerevisiae were increased by chemical pretreatment of the cells. Uranium could be removed chemically from S. cerevisiae cells, and the cells could then be reused as a biosorbent

  2. Management of soybean oil refinery wastes through recycling them for producing biosurfactant using Pseudomonas aeruginosa MR01.

    Science.gov (United States)

    Partovi, Maryam; Lotfabad, Tayebe Bagheri; Roostaazad, Reza; Bahmaei, Manochehr; Tayyebi, Shokoufe

    2013-06-01

    Biosurfactant production through a fermentation process involving the biodegradation of soybean oil refining wastes was studied. Pseudomonas aeruginosa MR01 was able to produce extracellular biosurfactant when it was cultured in three soybean oil refinement wastes; acid oil, deodorizer distillate and soapstock, at different carbon to nitrogen ratios. Subsequent fermentation kinetics in the three types of waste culture were also investigated and compared with kinetic behavior in soybean oil medium. Biodegradation of wastes, biosurfactant production, biomass growth, nitrate consumption and the number of colony forming units were detected in four proposed media, at specified time intervals. Unexpectedly, wastes could stimulate the biodegradation activity of MR01 bacterial cells and thus biosurfactant synthesis beyond that of the refined soybean oil. This is evident from higher yields of biodegradation and production, as revealed in the waste cultures (Ydeg|(Soybean oil) = 53.9 % YP/S|(Soybean oil) = 0.31 g g(-1), respectively). Although production yields were approximately the same in the three waste cultures (YP/S|(wastes) =/~ 0.5 g g(-1)), microbial activity resulted in higher yields of biodegradation (96.5 ± 1.13 %), maximum specific growth rate (μ max = 0.26 ± 0.02 h(-1)), and biosurfactant purity (89.6 %) with a productivity of 14.55 ± 1.10 g l(-1), during the bioconversion of soapstock into biosurfactant. Consequently, applying soybean oil soapstock as a substrate for the production of biosurfactant with commercial value has the potential to provide a combination of economical production with environmental protection through the biosynthesis of an environmentally friendly (green) compound and reduction of waste load entering the environment. Moreover, this work inferred spectrophotometry as an easy method to detect rhamnolipids in the biosurfactant products.

  3. A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack.

    Science.gov (United States)

    Tseng, Boo Shan; Reichhardt, Courtney; Merrihew, Gennifer E; Araujo-Hernandez, Sophia A; Harrison, Joe J; MacCoss, Michael J; Parsek, Matthew R

    2018-04-10

    Pseudomonas aeruginosa produces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix of P. aeruginosa biofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system during P. aeruginosa biofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilm P. aeruginosa cells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response. IMPORTANCE Proteins associated with the extracellular matrix of bacterial aggregates called biofilms have long been suggested to provide many important functions to the community. To date, however, only proteins that provide structural roles have been described, and few matrix-associated proteins have been identified. We developed a method to identify matrix proteins and characterized one. We show that this protein, when associated with the biofilm matrix, can inhibit a bactericidal enzyme produced by the immune system during infection and protect biofilm cells from death induced by the enzyme. This may represent a novel mechanism of protection for biofilms, further increasing their tolerance against the immune response. Together, our results are the first to show a nonstructural function for a confirmed matrix

  4. Pseudomonas aeruginosa (Family Pseudomonadaceae) is an ...

    African Journals Online (AJOL)

    Pseudomonas aeruginosa (Family Pseudomonadaceae) is an obligate aerobic, motile, gram negative bacillus.which is able to grow and survive in almost any environment and resistant to temperature extremes. It is involved in the etiology of several diseases i.

  5. Antibiotics Susceptibility Pattern of Pseudomonas aeruginosa ...

    African Journals Online (AJOL)

    ABSTRACT: This work investigated the prevalence and antibiotics sensitivity of Pseudomonas aeruginosa isolated from ... skin triggers coagulation and an acute inflammatory response ... agents with anti-pseudomonal activity, life-threatening.

  6. Production of Extra-Cellular Proteases from Marine Bacillus Sp. Cultured in Media Containing Ammonium Sulfate as the Sole Nitrogen Source

    Directory of Open Access Journals (Sweden)

    Seri Intan, M.

    2005-01-01

    Full Text Available Useful bacterial strains can be used to increase mineralize activity of an aquatic system. These bacteria can specifically degrade targeted compound by producing extra-cellular enzymes. Three species of Bacillus i.e. B. subtilis, B. pumilus and B. licheniformis acquired from shrimp ponds were tested for their ability to utilize ammonia and produce extracellular enzymes. These bacteria were grown in artificial seawater (30 ppt salinity and pH 7.6 supplemented with decreasing yeast extract concentration but increasing ammonium sulfate concentration. All three bacteria grew in artificial seawater containing only 0.01% yeast extract and 1% ammonium sulfate. However, only B. pumilus and B. licheniformis were able to grow in the medium containing only 1% ammonium sulfate as a sole energy source. Bacterialgrowth reduced when alkaline proteases activities was maximum from culture filtrates of all three bacterial cultures during 24 hour culturing in artificial seawater containing 0.01% yeast extract and 1% ammonium sulfate at 30 C when assayed at pH 9. Bacterial growth increased when acid proteases activities was maximum from culture filtrates of all three bacterial cultures during 48 hour culturing in artificial seawater containing 0.01% yeast extract and 1% ammoniumsulfate at 30 C when assayed at pH 5.

  7. Pseudomonas aeruginosa Trent and zinc homeostasis.

    Science.gov (United States)

    Davies, Corey B; Harrison, Mark D; Huygens, Flavia

    2017-09-01

    Pseudomonas aeruginosa is a Gram-negative pathogen and the major cause of mortality in patients with cystic fibrosis. The mechanisms that P. aeruginosa strains use to regulate intracellular zinc have an effect on infection, antibiotic resistance and the propensity to form biofilms. However, zinc homeostasis in P. aeruginosa strains of variable infectivity has not been compared. In this study, zinc homeostasis in P. aeruginosa Trent, a highly infectious clinical strain, was compared to that of a laboratory P. aeruginosa strain, ATCC27853. Trent was able to tolerate higher concentrations of additional zinc in rich media than ATCC27853. Further, pre-adaptation to additional zinc enhanced the growth of Trent at non-inhibitory concentrations but the impact of pre-adaption on the growth of ATCC27853 under the same conditions was minimal. The results establish clear differences in zinc-induced responses in Trent and ATCC27853, and how zinc homeostasis can be a promising target for the development of novel antimicrobial strategies for P. aeruginosa infection in cystic fibrosis patients. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

    Science.gov (United States)

    Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

    2017-01-01

    Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  9. Detection of VIM-2-, IMP-1- and NDM-1-producing multidrug resistant Pseudomonas aeruginosa in Malaysia.

    Science.gov (United States)

    Liew, Siew Mun; Rajasekaram, Ganeswrei; Puthucheary, Savithri D; Chua, Kek Heng

    2018-02-09

    The increasing incidence of carbapenem-resistant Pseudomonas aeruginosa along with the discovery of novel metallo-β-lactamases (MBLs) is of concern. In this study, the isolation of Malaysian MBL-producing P. aeruginosa clinical strains was investigated. Fifty-three P. aeruginosa clinical strains were isolated from different patients in Sultanah Aminah Hospital, Johor Bahru, Malaysia in 2015. Antimicrobial susceptibility test was conducted. Minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by Etest. The carbapenem-resistant strains were screened for MBL production by IMP-EDTA double disk synergy test (DDST), MBL imipenem/imipenem-inhibitor (IP/IPI) Etest and polymerase chain reaction (PCR). Genotyping was performed by multilocus sequence typing (MLST) analysis. Three (5.7%) clinical strains were identified as MBL producers. Multidrug resistance was observed in the three strains, and two were resistant to all the antimicrobials tested. Sequencing analysis confirmed the three strains to harbour carbapenemase genes: one with bla IMP-1 , one with bla VIM-2 and the other with bla NDM-1 genes. These multidrug resistant strains were identified as sequence type (ST) 235 and ST308. None of the bla IMP-1 and bla NDM-1 genes have been reported in Malaysian P. aeruginosa. The emergence of imipenemase 1 (IMP-1)- and New Delhi metallo-β-lactamase 1 (NDM-1)-producing P. aeruginosa in Malaysia maybe travel-associated. Copyright © 2018. Published by Elsevier Ltd.

  10. Inhibition and dispersal of Agrobacterium tumefaciens biofilms by a small diffusible Pseudomonas aeruginosa exoproduct(s).

    Science.gov (United States)

    Hibbing, Michael E; Fuqua, Clay

    2012-06-01

    Environmental biofilms often contain mixed populations of different species. In these dense communities, competition between biofilm residents for limited nutrients such as iron can be fierce, leading to the evolution of competitive factors that affect the ability of competitors to grow or form biofilms. We have discovered a compound(s) present in the conditioned culture fluids of Pseudomonas aeruginosa that disperses and inhibits the formation of biofilms produced by the facultative plant pathogen Agrobacterium tumefaciens. The inhibitory activity is strongly induced when P. aeruginosa is cultivated in iron-limited conditions, but it does not function through iron sequestration. In addition, the production of the biofilm inhibitory activity is not regulated by the global iron regulatory protein Fur, the iron-responsive extracytoplasmic function σ factor PvdS, or three of the recognized P. aeruginosa quorum-sensing systems. In addition, the compound(s) responsible for the inhibition and dispersal of A. tumefaciens biofilm formation is likely distinct from the recently identified P. aeruginosa dispersal factor, cis-2-decenoic acid (CDA), as dialysis of the culture fluids showed that the inhibitory compound was larger than CDA and culture fluids that dispersed and inhibited biofilm formation by A. tumefaciens had no effect on biofilm formation by P. aeruginosa.

  11. The impact of nosocomially-acquired resistant Pseudomonas aeruginosa infection in a burn unit.

    Science.gov (United States)

    Armour, Alexis D; Shankowsky, Heather A; Swanson, Todd; Lee, Jonathan; Tredget, Edward E

    2007-07-01

    Nosocomially-acquired Pseudomonas aeruginosa remains a serious cause of infection and septic mortality in burn patients. This study was conducted to quantify the impact of nosocomially-transmitted resistant P. aeruginosa in a burn population. Using a TRACS burn database, 48 patients with P. aeruginosa resistant to gentamicin were identified (Pseudomonas group). Thirty-nine were case-matched to controls without resistant P. aeruginosa cultures (control group) for age, total body surface area, admission year, and presence of inhalation injury. Mortality and various morbidity endpoints were examined, as well as antibiotic costs. There was a significantly higher mortality rate in the Pseudomonas group (33% vs. 8%, p products used (packed cells 51.1 +/- 8.0 vs. 21.1 +/- 3.4, p < 0.01; platelets 11.9 +/- 3.0 vs. 1.4 +/- 0.7, p < 0.01) were all significantly higher in the Pseudomonas group. Cost of antibiotics was also significantly higher ($2,658.52 +/- $647.93 vs. $829.22 +/- $152.82, p < 0.01). Nosocomial colonization or infection, or both, of burn patients with aminoglycoside-resistant P. aeruginosa is associated with significantly higher morbidity, mortality, and cost of care. Increased resource consumption did not prevent significantly higher mortality rates when compared with that of control patients. Thus, prevention, identification, and eradication of nosocomial Pseudomonas contamination are critical for cost-effective, successful burn care.

  12. Pseudomonas aeruginosa Dose-Response and Bathing Water Infection

    Science.gov (United States)

    Pseudomonas aeruginosa is the most commonly identified opportunistic pathogen associated with pool acquired bather disease. To better understand why this microorganism poses this protracted problem we recently appraised P. aeruginosa pool risk management. Much is known about the ...

  13. Viscoelastic Properties of Extracellular Polymeric Substances Can Strongly Affect Their Washing Efficiency from Reverse Osmosis Membranes.

    Science.gov (United States)

    Ferrando Chavez, Diana Lila; Nejidat, Ali; Herzberg, Moshe

    2016-09-06

    The role of the viscoelastic properties of biofouling layers in their removal from the membrane was studied. Model fouling layers of extracellular polymeric substances (EPS) originated from microbial biofilms of Pseudomonas aeruginosa PAO1 differentially expressing the Psl polysaccharide were used for controlled washing experiments of fouled RO membranes. In parallel, adsorption experiments and viscoelastic modeling of the EPS layers were conducted in a quartz crystal microbalance with dissipation (QCM-D). During the washing stage, as shear rate was elevated, significant differences in permeate flux recovery between the three different EPS layers were observed. According to the amount of organic carbon remained on the membrane after washing, the magnitude of Psl production provides elevated resistance of the EPS layer to shear stress. The highest flux recovery during the washing stage was observed for the EPS with no Psl. Psl was shown to elevate the layer's shear modulus and shear viscosity but had no effect on the EPS adhesion to the polyamide surface. We conclude that EPS retain on the membrane as a result of the layer viscoelastic properties. These results highlight an important relation between washing efficiency of fouling layers from membranes and their viscoelastic properties, in addition to their adhesion properties.

  14. The immune response to chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is predominantly of the Th2 type

    DEFF Research Database (Denmark)

    Moser, C; Kjaergaard, S; Pressler, T

    2000-01-01

    Most cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa lung infection have a persistent acute type lung inflammation dominated by polymorphonuclear neutrophils (PMN) and a pronounced antibody response against P. aeruginosa. We speculated whether this immune response in CF...... is of the Th2 type and whether a change to a Th1 type immune response could improve the prognosis. Therefore, we studied 14 CF patients with (CF +P) and 14 CF patients without (CF -P) chronic P. aeruginosa lung infection. The specific production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4......: Rho=0.524; ptype immune response is most frequent in CF patients with chronic P. aeruginosa lung infection, and the patients with a Th1-dominated immune response had the best lung function. The clinical implication is that a change to a Th1 type immune response might...

  15. Sequestration and Distribution Characteristics of Cd(II by Microcystis aeruginosa and Its Role in Colony Formation

    Directory of Open Access Journals (Sweden)

    Xiangdong Bi

    2016-01-01

    Full Text Available To investigate the sequestration and distribution characteristics of Cd(II by Microcystis aeruginosa and its role in Microcystis colony formation, M. aeruginosa was exposed to six different Cd(II concentrations for 10 days. Cd(II exposure caused hormesis in the growth of M. aeruginosa. Low concentrations of Cd(II significantly induced formation of small Microcystis colonies (P93% of Cd(II was sequestrated in the groups with lower added concentrations of Cd(II. More than 80% of the sequestrated Cd(II was bioadsorbed by bEPS. The Pearson correlation coefficients of exterior and interior factors related to colony formation of M. aeruginosa revealed that Cd(II could stimulate the production of IPS and bEPS via increasing Cd(II bioaccumulation and bioadsorption. Increased levels of cross-linking between Cd(II and bEPS stimulated algal cell aggregation, which eventually promoted the formation of Microcystis colonies.

  16. Detection of blaCTX-M gene among Pseudomonas aeruginosa isolated from water samples in Baghdad

    Directory of Open Access Journals (Sweden)

    Saba R. Khdair

    2017-11-01

    Full Text Available A total of 50 environmental Pseudomonas aeruginosa isolates were collected from sewage and tap water in Baghdad, Iraq. The MICs of Cefotaxime and Ceftazidime were determined by using agar dilution method, The MIC ranged from 2 to 256 µg/ml.The results of antibiotic sensitivity test showed that among sewage P. aeruginosa isolates, resistance was observed most often to Ticarcillin (92%, Penicillin G (84%, Ceftazidime (12%, (8% for each of Cefotaxime and Ticarcillin. On the other hand, all tap water isolates were sensitive to Ofloxacin and Levofloxacin, Except (5% of isolates were resistant to Cefotaxime (25% to Ceftazidime and (95% to Ticarcillin. All isolates were tested for Extended-Spectrum β-Lactamase (ESBL production. Ten isolates (20% were found to be ESBL producers. All environmental P. aeruginosa isolates were screened for the presence of the blaCTX-M genes by application PCR, Only (30% of them were positive for this test.

  17. Vaccines for preventing infection with Pseudomonas aeruginosa in cystic fibrosis

    DEFF Research Database (Denmark)

    Johansen, Helle Krogh; Gøtzsche, Peter C

    2013-01-01

    Chronic pulmonary infection in cystic fibrosis results in progressive lung damage. Once colonisation of the lungs with Pseudomonas aeruginosa occurs, it is almost impossible to eradicate. Vaccines, aimed at reducing infection with Pseudomonas aeruginosa, have been developed.......Chronic pulmonary infection in cystic fibrosis results in progressive lung damage. Once colonisation of the lungs with Pseudomonas aeruginosa occurs, it is almost impossible to eradicate. Vaccines, aimed at reducing infection with Pseudomonas aeruginosa, have been developed....

  18. Extracellular Gd-CA

    DEFF Research Database (Denmark)

    Thomsen, Henrik S; Marckmann, Peter

    2008-01-01

    Until recently it was believed that extracellular gadolinium-based contrast agents were safe for both the kidneys and all other organs within the dose range up to 0.3 mmol/kg body weight. However, in 2006, it was demonstrated that some gadolinium-based contrast agents may trig the development...... gadolinium-based agent (3-7% versus 0-1% per injection) in patients with reduced renal function. Prevalence after exposure to two gadodiamide injections is as high as 36% in patients with chronic kidney disease (CKD) stage 5. No report of NSF after the most stable agents has been reported in the peer...

  19. Identification of biofilm-associated cluster (bac in Pseudomonas aeruginosa involved in biofilm formation and virulence.

    Directory of Open Access Journals (Sweden)

    Camille Macé

    Full Text Available Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731 that was accumulated by biofilm cells. We report here that a Delta pA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729-pA3732 not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster.

  20. Increased bactericidal activity of colistin on Pseudomonas aeruginosa biofilms in anaerobic conditions

    DEFF Research Database (Denmark)

    Kolpen, Mette; Appeldorff, Cecilie F.; Brandt, Sarah

    2016-01-01

    that production of OH˙may not contribute significantly to the bactericidal activity of colistin on P. aeruginosa biofilm. Thus, we investigated the effect of colistin treatment on biofilm of wild-type PAO1, a catalase-deficient mutant (katA) and a colistin-resistant CF isolate cultured in microtiter plates...

  1. Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression.

    Science.gov (United States)

    Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K

    2008-07-01

    Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA

  2. Regulatory Network Controlling Extracellular Proteins in Erwinia carotovora subsp. carotovora: FlhDC, the Master Regulator of Flagellar Genes, Activates rsmB Regulatory RNA Production by Affecting gacA and hexA (lrhA) Expression▿

    Science.gov (United States)

    Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K.

    2008-01-01

    Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC− mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (HarpinEcc) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC+ plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC− mutant are responsible for the inhibition of rsmB RNA production

  3. Amikacin loaded PLGA nanoparticles against Pseudomonas aeruginosa.

    Science.gov (United States)

    Sabaeifard, Parastoo; Abdi-Ali, Ahya; Soudi, Mohammad Reza; Gamazo, Carlos; Irache, Juan Manuel

    2016-10-10

    Amikacin is a very effective aminoglycoside antibiotic but according to its high toxicity, the use of this antibiotic has been limited. The aim of this study was to formulate and characterize amikacin loaded PLGA nanoparticles. Nanoparticles were synthetized using a solid-in-oil-in-water emulsion technique with different ratio of PLGA 50:50 (Resomer 502H) to drug (100:3.5, 80:3.5 and 60:3.5), two different concentrations of stabilizer (pluronic F68) (0.5% or 1%) and varied g forces to recover the final products. The most efficient formulation based on drug loading (26.0±1.3μg/mg nanoparticle) and encapsulation efficiency (76.8±3.8%) was the one obtained with 100:3.5 PLGA:drug and 0.5% luronic F68, recovered by 20,000×g for 20min. Drug release kinetic study indicated that about 50% of the encapsulated drug was released during the first hour of incubation in phospahte buffer, pH7.4, 37°C, 120rpm. Using different cell viability/cytotoxicity assays, the optimized formulation showed no toxicity against RAW macrophages after 2 and 24h of exposure. Furthermore, released drug was active and maintained its bactericidal activity against Pseudomonas aeruginosa in vitro. These results support the effective utilization of the PLGA nanoparticle formulation for amikacin in further in vivo studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Measurement and evaluation of the effects of pH gradients on the antimicrobial and antivirulence activities of chitosan nanoparticles in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Fadilah Sfouq Aleanizy

    2018-01-01

    Full Text Available Objective: The purpose of this study was to study the antimicrobial activity of chitosan nanoparticles (CSNPs on Pseudomonas aeruginosa with special emphasis on their sensitivity to pH and the effect of pH on their activity. Methodology: Antimicrobial activity of CSNPs against Pseudomonas aeruginosa at different pH was tested using broth dilution method. Further assessment of antivirulence activity and sensitization of CSNPs on Pseudomonas aeruginosa were examined. Results: Significant antimicrobial effects of CSNPs against Pseudomonas aeruginosa were detected at slightly acidic pH 5, whereas the activity was abolished at a pH of greater than 7. The antivirulence activity of CSNPs was then investigated and treatment with CSNPs (1000 ppm resulted in a significant reduction or even complete inhibition of pyocyanin production by P. aeruginosa compared with untreated P. aeruginosa indicating the antivirulence activity of CSNPs. CSNPs also sensitized P. aeruginosa to the lytic effects of sodium dodecyl sulfate (SDS; such sensitization was not blocked by washing chitosan-treated cells prior to SDS exposure revealing that CSNPs disturb the outer membrane leading to irreversible sensitivity to detergent even at low concentration (100 ppm. Conclusions: These findings highlight CSNPs as potentially useful as indirect antimicrobial agents for a variety of applications. Keywords: Chitosan nanoparticles, Pseudomonas aeruginosa, pH, Antimicrobial, Virulence

  5. PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALOPHILIC PROTEASE FROM PSEUDOMONAS AERUGINOSA

    Directory of Open Access Journals (Sweden)

    R. Satheeskumar

    2013-10-01

    Full Text Available Partial purification and characterization of alkalophilic protease production from Pseudomonas aeruginosa was isolated from the gut of marine and coastal waters shrimp Penaeus monodon. The protease production was assayed in submerged fermentation to produce maximum protease activity (423 ± 0.09 U/ml. The enzyme was precipitated with ammonium sulphate and partially purified by ion exchange chromatography through DEAE Sephadex A-50 column. In 10th fraction showed maximum protease activity (734 ± 0.18 U/ml with increase in purification fold. The molecular weight of protease from Pseudomonas aeruginosa was recorded as 60 kDa. The stability of protease was tested at various pH and temperature; it showed maximum protease activity at pH-9 and temperature 50ºC. Among the various surfactants tested for enzyme stability, maximum activity was retained in poly ethylene glycol. The compatibility of protease enzyme with various commercial detergents; the enzyme retained maximum protease activity in tide. The results are indicated that all these properties make the bacterial proteases are most suitable for wide industrial applications.

  6. Identification of Pseudomonas aeruginosa phenazines that kill Caenorhabditis elegans.

    Science.gov (United States)

    Cezairliyan, Brent; Vinayavekhin, Nawaporn; Grenfell-Lee, Daniel; Yuen, Grace J; Saghatelian, Alan; Ausubel, Frederick M

    2013-01-01

    Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches.

  7. Identification of Pseudomonas aeruginosa phenazines that kill Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Brent Cezairliyan

    2013-01-01

    Full Text Available Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches.

  8. Corneal Biofilms: From Planktonic to Microcolony Formation in an Experimental Keratitis Infection with Pseudomonas Aeruginosa.

    Science.gov (United States)

    Saraswathi, Padmanabhan; Beuerman, Roger W

    2015-10-01

    Microbial biofilms commonly comprise part of the infectious scenario, complicating the therapeutic approach. The purpose of this study was to determine in a mouse model of corneal infection if mature biofilms formed and to visualize the stages of biofilm formation. A bacterial keratitis model was established using Pseudomonas aeruginosa ATCC 9027 (1 × 10(8) CFU/ml) to infect the cornea of C57BL/6 black mouse. Eyes were examined post-infection (PI) on days 1, 2, 3, 5, and 7, and imaged by slit lamp microscopy, and light, confocal, and electron microscopy to identify the stages of biofilm formation and the time of appearance. On PI day 1, Gram staining showed rod-shaped bacteria adherent on the corneal surface. On PI days 2 and 3, bacteria were seen within webs of extracellular polymeric substance (EPS) and glycocalyx secretion, imaged by confocal microscopy. Scanning electron microscopy demonstrated microcolonies of active infectious cells bound with thick fibrous material. Transmission electron microscopy substantiated the formation of classical biofilm architecture with P. aeruginosa densely packed within the extracellular polymeric substances on PI days 5 and 7. Direct visual evidence showed that biofilms routinely developed on the biotic surface of the mouse cornea. The mouse model can be used to develop new approaches to deal therapeutically with biofilms in corneal infections. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa.

    Science.gov (United States)

    Oh, Jungmin; Li, Xi-Hui; Kim, Soo-Kyong; Lee, Joon-Hee

    2017-06-30

    Protease IV (PIV), a key virulence factor of Pseudomonas aeruginosa is a secreted lysyl-endopeptidase whose expression is induced by quorum sensing (QS). We found that PIV expressed in QS mutant has severe reduction of activity in culture supernatant (CS), even though it is overexpressed to high level. PIV purified from the QS mutant (M-PIV) had much lower activity than the PIV purified from wild type (P-PIV). We found that the propeptide cleaved from prepro-PIV was co-purified with M-PIV, but never with P-PIV. Since the activity of M-PIV was restored by adding the CS of QS-positive and PIV-deficient strain, we hypothesized that the propeptide binds to and inhibits PIV, and is degraded to activate PIV by a QS-dependent factor. In fact, the CS of the QS-positive and PIV-deficient strain was able to degrade the propeptide. Since the responsible factor should be a QS-dependently expressed extracellular protease, we tested QS-dependent proteases of P. aeruginosa and found that LasB (elastase) can degrade the propeptide and activate M-PIV. We purified the propeptide of PIV and confirmed that the propeptide can bind to and inhibit PIV. We suggest that PIV is post-secretionally activated through the extracellular degradation of the propeptide by LasB, a QS-dependent protease.

  10. A titanium surface with nano-ordered spikes and pores enhances human dermal fibroblastic extracellular matrix production and integration of collagen fibers

    International Nuclear Information System (INIS)

    Yamada, Masahiro; Kato, Eiji; Sakurai, Kaoru; Yamamoto, Akiko

    2016-01-01

    The acquisition of substantial dermal sealing determines the prognosis of percutaneous titanium-based medical devices or prostheses. A nano-topographic titanium surface with ordered nano-spikes and pores has been shown to induce periodontal-like connective tissue attachment and activate gingival fibroblastic functions. This in vitro study aimed to determine whether an alkali-heat (AH) treatment-created nano-topographic titanium surface could enhance human dermal fibroblastic functions and binding strength to the deposited collagen on the titanium surface. The surface topographies of commercially pure titanium machined discs exposed to two different AH treatments were evaluated. Human dermal fibroblastic cultures grown on the discs were evaluated in terms of cellular morphology, proliferation, extracellular matrix (ECM) and proinflammatory cytokine synthesis, and physicochemical binding strength of surface-deposited collagen. An isotropically-patterned, shaggy nano-topography with a sponge-like inner network and numerous well-organized, anisotropically-patterned fine nano-spikes and pores were observed on each nano-topographic surface type via scanning electron microscopy. In contrast to the typical spindle-shaped cells on the machined surfaces, the isotropically- and anisotropically-patterned nano-topographic titanium surfaces had small circular/angular cells containing contractile ring-like structures and elongated, multi-shaped cells with a developed cytoskeletal network and multiple filopodia and lamellipodia, respectively. These nano-topographic surfaces enhanced dermal-related ECM synthesis at both the protein and gene levels, without proinflammatory cytokine synthesis or reduced proliferative activity. Deposited collagen fibers were included in these surfaces and sufficiently bound to the nano-topographies to resist the physical, enzymatic and chemical detachment treatments, in contrast to machined surfaces. Well-organized, isotropically

  11. The interaction between nitrobenzene and Microcystis aeruginosa and its potential to impact water quality.

    Science.gov (United States)

    Liu, Zhiquan; Cui, Fuyi; Ma, Hua; Fan, Zhenqiang; Zhao, Zhiwei; Hou, Zhenling; Liu, Dongmei; Jia, Xuebin

    2013-08-01

    The potential water quality problems caused by the interaction between nitrobezene (NB) and Microcystis aeruginosa was investigated by studying the growth inhibition, the haloacetic acids formation potential (HAAFP) and the secretion of microcystin-LR (MC-LR). The results showed that NB can inhibit the growth of M. aeruginosa, and the value of EC50 increased with the increase of initial algal density. Although NB can hardly react with chlorine to form HAAs, the presence of NB can enhance the HAAFP productivity. The secretion of the intracellular MC-LR is constant under the steady experimental conditions. However, the presence of NB can reduce the MC-LR productivity of M. aeruginosa. Overall, the increased disinfection risk caused by the interaction has more important effect on the safety of drinking water quality than the benefit of the decreased MC-LR productivity, and should be serious considered when the water contained NB and M. aeruginosa is used as drinking water source. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Extracts of Cordia gilletii de wild (Boraginaceae) quench the quorum sensing of Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Okusa, Philippe N; Rasamiravaka, Tsiry; Vandeputte, Olivier; Stévigny, Caroline; Jaziri, Mondher El; Duez, Pierre

    2014-01-01

    The fight against infectious diseases and antimicrobial resistances needs the exploration of new active compounds with new proprieties like disrupting quorum sensing (QS) mechanisms, which is a cell-to-cell communication that regulates bacterial virulence factors. In this work, leaves and root barks extracts of a Congolese medicinal plant, Cordia gilletii, were investigated for their effect on the production of Pseudomonas aeruginosa major virulence factors regulated by QS. The effect of C. gilletii extracts on virulence factors of P. aeruginosa PAO1 was studied by the evaluation of the production of pyocyanine, elastase and biofilm; and by the measurement of the expression of QS-related genes. The dichloromethane extract from root barks was found to quench the production of pyocyanin, a QS-dependent virulence factor in P. aeruginosa PAO1. Moreover, this extract specifically inhibits the expression of several QS-regulated genes (i.e. lasB, rhlA, lasI, lasR, rhlI, and rhlR) and reduces biofilm formation by PAO1. This study contributes to explain the efficacy of C. gilletii in the traditional treatment of infectious diseases caused by P. aeruginosa.

  13. Extracts of Cordia gilletii de wild (Boraginaceae) quench the quorum sensing of Pseudomonas aeruginosa PAO1

    Science.gov (United States)

    Okusa, Philippe N.; Rasamiravaka, Tsiry; Vandeputte, Olivier; Stévigny, Caroline; Jaziri, Mondher El; Duez, Pierre

    2014-01-01

    Aim: The fight against infectious diseases and antimicrobial resistances needs the exploration of new active compounds with new proprieties like disrupting quorum sensing (QS) mechanisms, which is a cell-to-cell communication that regulates bacterial virulence factors. In this work, leaves and root barks extracts of a Congolese medicinal plant, Cordia gilletii, were investigated for their effect on the production of Pseudomonas aeruginosa major virulence factors regulated by QS. Materials and Methods: The effect of C. gilletii extracts on virulence factors of P. aeruginosa PAO1 was studied by the evaluation of the production of pyocyanine, elastase and biofilm; and by the measurement of the expression of QS-related genes. Results: The dichloromethane extract from root barks was found to quench the production of pyocyanin, a QS-dependent virulence factor in P. aeruginosa PAO1. Moreover, this extract specifically inhibits the expression of several QS-regulated genes (i.e. lasB, rhlA, lasI, lasR, rhlI, and rhlR) and reduces biofilm formation by PAO1. Conclusion: This study contributes to explain the efficacy of C. gilletii in the traditional treatment of infectious diseases caused by P. aeruginosa. PMID:26401363

  14. Extracellular Signatures as Indicators of Processing Methods

    Energy Technology Data Exchange (ETDEWEB)

    Wahl, Karen L.

    2012-01-09

    As described in other chapters within this volume, many aspects of microbial cells vary with culture conditions and therefore can potentially be analyzed as forensic signatures of growth conditions. In addition to changes or variations in components of the microbes themselves, extracellular materials indicative of production processes may remain associated with the final bacterial product. It is well recognized that even with considerable effort to make pure products such as fine chemicals or pharmaceuticals, trace impurities from components or synthesis steps associated with production processes can be detected in the final product. These impurities can be used as indicators of production source or methods, such as to help connect drugs of abuse to supply chains. Extracellular residue associated with microbial cells could similarly help to characterize production processes. For successful growth of microorganisms on culture media there must be an available source of carbon, nitrogen, inorganic phosphate and sulfur, trace metals, water and vitamins. The pH, temperature, and a supply of oxygen or other gases must also be appropriate for a given organism for successful culture. The sources of these components and the range in temperature, pH and other variables has adapted over the years with currently a wide range of possible combinations of media components, recipes and parameters to choose from for a given organism. Because of this wide variability in components, mixtures of components, and other parameters, there is the potential for differentiation of cultured organisms based on changes in culture conditions. The challenge remains how to narrow the field of potential combinations and be able to attribute variations in the final bacterial product and extracellular signatures associated with the final product to information about the culture conditions or recipe used in the production of that product.

  15. Expression, purification, crystallization and preliminary crystallographic analysis of PA3885 (TpbA) from Pseudomonas aeruginosa PAO1

    International Nuclear Information System (INIS)

    Yang, Wen; Li, Kan; Bai, Yuwei; Zhou, Ruimin; Zhou, Weihong; Bartlam, Mark

    2010-01-01

    PA3885 (TpbA), a tyrosine phosphatase, may function as a balancing factor between biofilm formation and motility in the opportunistic pathogen P. aeruginosa. Here, the expression, purification, crystallization and preliminary crystallographic analysis of PA3885 from P. aeruginosa PAO1 are reported. Biofilms are important in cell communication and growth in most bacteria and are also responsible for most human clinical infections and diseases. Quorum-sensing systems have been identified to be crucial for biofilm formation and regulation. PA3885 (TpbA), a tyrosine phosphatase, is reported to convert extracellular quorum-sensing signals into internal gene-cascade reactions that result in reduced biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. Here, PA3885 from P. aeruginosa PAO1 was expressed, purified and crystallized. Single crystals were studied by X-ray crystallography and native diffraction data were collected to 2.8 Å resolution. These crystals were determined to belong to space group C2. It was not possible to conclusively determine the number of proteins in the asymmetric unit from the preliminary X-ray diffraction data analysis alone and attempts to determine the crystal structure of PA3885 are currently under way

  16. Formation of large colonies: a defense mechanism of Microcystis aeruginosa under continuous grazing pressure by flagellate Ochromonas sp.

    Directory of Open Access Journals (Sweden)

    Fanxiang Kong

    2012-01-01

    Full Text Available Induced colony formation under grazing pressure has been reported in our previous results. However, the colonies induced in these studies comprised only tens of cells which are far smaller than the naturally occurring colonies. In this work, unicellular Microcystis aeruginosa Kützing were co-cultivated with flagellate Ochromonas sp. for 50 d to investigate colony formation in M. aeruginosa under continuous grazing pressure. Results revealed that colonial M. aeruginosa formed on the 10th d under the grazing pressure of flagellate. These algal colonies resulted from the daughter cells of freshly dividing cells that failed to separate during the reproductive process. The diameters and cell numbers of the colonies increased slowly with time. Under continuous grazing pressure by Ochromonas sp. for 50 d, the diameter of some colonies reached over 180 μm. Analysis showed that the extracellular polysaccharide (EPS content and relative gas vesicle (RGV of each cell increased significantly after colony formation. However, there was no significant difference on the monosaccharide composition between unicellular and colonial M. aeruginosa. The loose aggregation of cells in the floating colonies suggests that a correlation probably exists between cell compactness and colony buoyancy.

  17. Regulation of Initial Attachment of P. aeruginosa

    Science.gov (United States)

    2010-12-08

    shown to play a role in promoting biofilm formation in diverse P. aeruginosa strains (Friedman and Kolter , 2004; Jackson et al., 2004; Ryder et al...34House of Biofilm Cells". J. Bacteriol. 189: 7945-7947. Friedman, L., and Kolter , R. (2004) Genes involved in matrix formation in Pseudomonas

  18. Different Dose-Dependent Modes of Action of C-Type Natriuretic Peptide on Pseudomonas aeruginosa Biofilm Formation

    Directory of Open Access Journals (Sweden)

    Florie Desriac

    2018-04-01

    Full Text Available We have previously shown that the C-type Natriuretic Peptide (CNP, a peptide produced by lungs, is able to impact Pseudomonas aeruginosa physiology. In the present work, the effect of CNP at different concentrations on P. aeruginosa biofilm formation was studied and the mechanisms of action of this human hormone on P. aeruginosa were deciphered. CNP was shown to inhibit dynamic biofilm formation in a dose-dependent manner without affecting the bacterial growth at any tested concentrations. The most effective concentrations were 1 and 0.1 µM. At 0.1 µM, the biofilm formation inhibition was fully dependent on the CNP sensor protein AmiC, whereas it was only partially AmiC-dependent at 1 µM, revealing the existence of a second AmiC-independent mode of action of CNP on P. aeruginosa. At 1 µM, CNP reduced both P. aeruginosa adhesion on glass and di-rhamnolipid production and also increased the bacterial membrane fluidity. The various effects of CNP at 1 µM and 0.1 µM on P. aeruginosa shown here should have major consequences to design drugs for biofilm treatment or prevention.

  19. Phenotypic characterization and colistin susceptibilities of carbapenem-resistant of Pseudomonas aeruginosa and Acinetobacter spp.

    Science.gov (United States)

    Mohanty, Srujana; Maurya, Vijeta; Gaind, Rajni; Deb, Monorama

    2013-11-15

    Pseudomonas aeruginosa and Acinetobcter spp. are important nosocomial pathogens and carbapenem resistance is an emerging threat. Therapeutic options for infections with these isolates include colistin. This study was conducted to determine the prevalence of carbapenem resistance in P. aeruginosa and Acinetobacter spp. bloodstream isolates, phenotypically characterize the resistance mechanisms and evaluate the in vitro activity of colistin. Consecutive 145 (95 P.aeruginosa and 50 Acinetobacter spp.) non-repeat isolates were included. Antibiotic susceptibility testing was performed per CLSI guidelines. MIC for carbapenems and colistin was performed using Etest. Isolates showing reduced susceptibility or resistance to the carbapenems were tested for metallo-β-lactamase (MBL) production using imipenem-EDTA combined disk and MBL Etest. Carbapenem resistance was observed in 40% P. aeruginosa and 66.0% Acinetobacter spp. Carbapenem-resistant (CA-R) isolates were significantly (p carbapenem-susceptible isolates. Approximately half of the CA-R strains were multidrug-resistant, and 3.1-5.5% were resistant to all antibiotics tested. MBL was found in 76.3% and 69.7% of the P. aeruginosa and Acinetobacter spp., respectively. Colistin resistance was observed in three (6.0%) Acinetobacter isolates and eight (8.4%) P. aeruginosa. MIC50 for carbapenems were two to four times higher for MBL-positive compared to MBL-negative isolates, but no difference was seen in MIC for colistin. Carbapenem resistance was observed to be mediated by MBL in a considerable number of isolates. Colistin is an alternative for infections caused by CA-R isolates; however, MIC testing should be performed whenever clinical use of colistin is considered.

  20. Production of extracellular lipase by the phytopathogenic fungus Fusarium solani FS1 Produção de lipase extracelular pelo fungo fitopatogênico Fusarium solani FS1

    Directory of Open Access Journals (Sweden)

    Maria de Mascena Diniz Maia

    1999-12-01

    Full Text Available A Brazilian strain of Fusarium solani was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.l-1 of lipase after 72 hours of cultivation at 25oC in shake-flask at 120 rpm in a medium containing 3% (w/v peptone plus 0.5% (v/v olive oil. Glucose (1% w/v was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3% (w/v resulted in a reduced lipase production while increased olive oil concentration (above 0.5% did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30oC and a good enzyme stability (80% activity retention was observed at pH ranging from 7.6 to 8.6, and the activity rapidly dropped at temperatures above 50oC. Lipase activity was stimulated by the addition of n-hexane to the culture medium supernatants, in contrast to incubation with water-soluble solvents.

  1. Effect of gamma irradiation and environmental factors on the production of extracellular cellulase enzyme by trichoderma Spp. using banana waste under solid state bio processing

    International Nuclear Information System (INIS)

    El-Shafey, H.M.; Matar, Z.A.I.; Ghanem, S.M.A.

    2007-01-01

    Fungal strains were isolated from degraded banana waste including leaves, pseudo stems and skins. Many isolated strains showed cellulolytic activities using the plate screening medium. The hyper cellulolytic isolates were selected on the basis of the diameter of the hydrolysis zone surrounding the colonies and identified to the genus level. The identified strains were found to belong to one of the genera Trichoderma, Aspergillus, Pleurotus or Penicillium. The strain with the larger diameter of the hydrolysis zone was found to belong to the genus Trichoderma. It was further identified to be Trichoderma harzianum, which was selected to be studied. Banana waste including leaves and pseudo stems were inoculated by the selected fungus and the production of the carboxymethyl cellulase (CMCase) and filter paper cellulase (FPCase) was followed during changes of the growth conditions under solid state fermentation. It was found that the two enzymes shared the same incubation temperature (25 degree C) and incubation period (18 days) for the maximum enzyme production. The gamma radiation dose of 1.5 KGy increased the production of CMCase produced on leaves by 4.0% and on pseudo stems by 5.6% and the production of FPCase produced on leaves by 2.4% and on pseudo stems by 2.3%. The results also suggest that FPCase and CMCase enzymes produced on leaves were higher than those produced from pseudo stems and the level of CMCase enzyme produced was higher than that of FPCase

  2. N-Acyl-L-homoserine lactone autoinducers control production of an extracellular lipopeptide biosurfactant required for swarming motility of Serratia liquefaciens MG1

    DEFF Research Database (Denmark)

    Lindum, Peter Wurtz; Anthoni, U; Christophersen, Carsten

    1998-01-01

    A nonswarming Serratia liquefaciens mutant deficient in serrawettin W2 production was constructed by transposon mutagenesis. Sequence homology indicated that insertion had occurred in gene swrA, which encodes a putative peptide synthetase. Expression of swrA is controlled by quorum sensing....

  3. Exploring the In Vitro Thrombolytic Activity of Nattokinase From a New Strain Pseudomonas aeruginosa CMSS.

    Science.gov (United States)

    Chandrasekaran, Subathra Devi; Vaithilingam, Mohanasrinivasan; Shanker, Ravi; Kumar, Sanjeev; Thiyur, Swathi; Babu, Vaishnavi; Selvakumar, Jemimah Naine; Prakash, Suyash

    2015-10-01

    Thrombolytic therapy has become a conventional treatment for acute myocardial infarction (AMI), yet currently, clinically prescribed thrombolytic drugs have problems such as delayed action and other side effects. Fibrinolytic enzymes have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process, including plasmin activation. Nattokinase (NK) is a potent fibrinolytic agent for thrombosis therapy. The aim of this study was to enhance the production of NK from Pseudomonas aeruginosa CMSS by media optimization and strain improvement. In the present study, a potent NK-producing strain was isolated from cow milk and identified. To enhance the yield of NK, effect of various parameters such as pH, temperature, carbon source, nitrogen source and inoculum size were optimized. Strain improvement of P. aeruginosa CMSS was done by random UV-mutagenesis. Nattokinase was partially purified and the activity was determined by the casein digestion method, blood clot lysis and fibrin degradation assay. Based on morphological, biochemical and molecular characterization, the strain was confirmed as P. aeruginosa (GenBank accession number: JX112657), designated as P. aeruginosa CMSS. The optimum condition at pH 7 and temperature at 25˚C showed activity of NK as 1514 U mL(-1) and 1532 U mL(-1), respectively. Sucrose as the carbon source and shrimp shell powder (SSP) as the nitrogen source expressed NK activity of 1721 U mL(-1) and 2524 U mL(-1), respectively. At 1% inoculum size, the maximum rate of enzyme production was achieved with 2581 U mL(-1). The NK activity of the mutant strain UV60 was 4263 U mL(-1), indicating a two-fold increase in activity compared to the wild strain (2581 UmL(-1)). Nattokinase produced from mutant strain P. aeruginosa CMSS UV60 showed 94% blood clot lysis at ten minutes. The degradation of fibrin clot by the produced NK was observed after two hours of incubation. Sodium dodecyl sulfate polyacrylamide gel

  4. Effects of RU486 and indomethacin on meiotic maturation, formation of extracellular matrix, and progesterone production by porcine oocyte-cumulus complexes.

    Science.gov (United States)

    Nagyova, E; Scsukova, S; Kalous, J; Mlynarcikova, A

    2014-07-01

    This study was designed to determine whether inhibition of either cyclooxygenase-2 (COX-2) by indomethacin or progesterone receptor (PR) by PR antagonist, RU486, affects oocyte maturation, progesterone production, and covalent binding between hyaluronan (HA) and heavy chains of inter-α trypsin inhibitor, as well as expression of cumulus expansion-associated proteins (HA-binding protein, tumor necrosis factor α-induced protein 6, pentraxin 3) in oocyte-cumulus complexes (OCCs). The experiments were based on freshly isolated porcine OCC cultures in which the consequences of PR and COX-2 inhibition on the final processes of oocyte maturation were determined. Granulosa cells (GCs) and OCCs were cultured in medium supplemented with FSH/LH (both 100 ng/mL) in the presence/absence of RU486 or indomethacin. Western blot analysis, (3)H-glucosamine hydrochloride assay, immunofluorescence, and radioimmunoassay were performed. Only treatment with RU486 (25 μM) caused a decrease in the number of oocytes that reached germinal vesicle breakdown and metaphase II stage compared with indomethacin (100 μM) or FSH/LH treatment alone after 44 h. All treated OCCs synthesized an almost equal amount of HA. Heavy chains (of inter-α trypsin inhibitor)-HA covalent complexes were formed during in vitro FSH/LH-stimulated expansion in RU486- or indomethacin-treated OCCs. Follicle-stimulating hormone/LH-induced progesterone production by OCCs was increased in the presence of RU486 after 44 h. In contrast, a decrease of FSH/LH-stimulated progesterone production by GCs was detected in the presence of either RU486 or indomethacin after 72 h. We suggest that the PR-dependent pathway may be involved in the regulation of oocyte maturation. Both PR and COX-2 regulate FSH/LH-stimulated progesterone production by OCCs and GCs. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Alkaliphilic Bacillus species show potential application in concrete crack repair by virtue of rapid spore production and germination then extracellular calcite formation.

    Science.gov (United States)

    Sharma, T K; Alazhari, M; Heath, A; Paine, K; Cooper, R M

    2017-05-01

    Characterization of alkaliphilic Bacillus species for spore production and germination and calcite formation as a prelude to investigate their potential in microcrack remediation in concrete. Conditions, extent and timing of endospore production was determined by dark-field light microscopy; germination induction and kinetics were assessed by combining reduction in optical density with formation of refractile bodies by phase-contrast microscopy. Bacillus pseudofirmus was selected from several species as the most suitable isolate. Levels and timing of calcium carbonate precipitated in vitro by B. pseudofirmus were evaluated by atomic absorption spectroscopy and structural identity confirmed as calcite and aragonite by Raman spectroscopy and FTIR. The isolate produced copious spores that germinated rapidly in the presence of germinants l-alanine, inosine and NaCl. Bacterial cells produced CaCO 3 crystals in microcracks and the resulting occlusion markedly restricted water ingress. By virtue of rapid spore production and germination, calcium carbonate formation in vitro and in situ, leading to sealing of microcracks, B. pseudofirmus shows clear potential for remediation of concrete on a commercial scale. Microbial sealing of microcracks should become a practicable and sustainable means of increasing concrete durability. © 2017 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of The Society for Applied Microbiology.

  6. Enzyme-mediated quenching of the Pseudomonas quinolone signal (PQS promotes biofilm formation of Pseudomonas aeruginosa by increasing iron availability

    Directory of Open Access Journals (Sweden)

    Beatrix Tettmann

    2016-12-01

    Full Text Available The 2-alkyl-3-hydroxy-4(1H-quinolone 2,4-dioxygenase HodC was previously described to cleave the Pseudomonas quinolone signal, PQS, which is exclusively used in the complex quorum sensing (QS system of Pseudomonas aeruginosa, an opportunistic pathogen employing QS to regulate virulence and biofilm development. Degradation of PQS by exogenous addition of HodC to planktonic cells of P. aeruginosa attenuated production of virulence factors, and reduced virulence in planta. However, proteolytic cleavage reduced the efficacy of HodC. Here, we identified the secreted protease LasB of P. aeruginosa to be responsible for HodC degradation. In static biofilms of the P. aeruginosa PA14 lasB::Tn mutant, the catalytic activity of HodC led to an increase in viable biomass in newly formed but also in established biofilms, and reduced the expression of genes involved in iron metabolism and siderophore production, such as pvdS, pvdL, pvdA and pvdQ. This is likely due to an increase in the levels of bioavailable iron by degradation of PQS, which is able to sequester iron from the surrounding environment. Thus, HodC, despite its ability to quench the production of virulence factors, is contraindicated for combating P. aeruginosa biofilms.

  7. The novel virulence-related gene nlxA in the lipopolysaccharide cluster of Xanthomonas citri ssp. citri is involved in the production of lipopolysaccharide and extracellular polysaccharide, motility, biofilm formation and stress resistance.

    Science.gov (United States)

    Yan, Qing; Hu, Xiufang; Wang, Nian

    2012-10-01

    Lipopolysaccharide (LPS) is an important virulence factor of Xanthomonas citri ssp. citri, the causative agent of citrus canker disease. In this research, a novel gene, designated as nlxA (novel LPS cluster gene of X. citri ssp. citri), in the LPS cluster of X. citri ssp. citri 306, was characterized. Our results indicate that nlxA is required for O-polysaccharide biosynthesis by encoding a putative rhamnosyltransferase. This is supported by several lines of evidence: (i) NlxA shares 40.14% identity with WsaF, which acts as a rhamnosyltransferase; (ii) sodium dodecylsulphate-polyacrylamide gel electrophoresis analysis showed that four bands of the O-antigen part of LPS were missing in the LPS production of the nlxA mutant; this is also consistent with a previous report that the O-antigen moiety of LPS of X. citri ssp. citri is composed of a rhamnose homo-oligosaccharide; (iii) mutation of nlxA resulted in a significant reduction in the resistance of X. citri ssp. citri to different stresses, including sodium dodecylsulphate, polymyxin B, H(2)O(2), phenol, CuSO(4) and ZnSO(4). In addition, our results indicate that nlxA plays an important role in extracellular polysaccharide production, biofilm formation, stress resistance, motility on semi-solid plates, virulence and in planta growth in the host plant grapefruit. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  8. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

    Science.gov (United States)

    Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

    2016-09-01

    Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

  9. Extracellular matrix structure.

    Science.gov (United States)

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Extracellular electron transfer mechanisms between microorganisms and minerals

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Liang; Dong, Hailiang; Reguera, Gemma; Beyenal, Haluk; Lu, Anhuai; Liu, Juan; Yu, Han-Qing; Fredrickson, James K.

    2016-08-30

    Electrons can be transferred from microorganisms to multivalent metal ions that are associated with minerals and vice versa. As the microbial cell envelope is neither physically permeable to minerals nor electrically conductive, microorganisms have evolved strategies to exchange electrons with extracellular minerals. In this Review, we discuss the molecular mechanisms that underlie the ability of microorganisms to exchange electrons, such as c-type cytochromes and microbial nanowires, with extracellular minerals and with microorganisms of the same or different species. Microorganisms that have extracellular electron transfer capability can be used for biotechnological applications, including bioremediation, biomining and the production of biofuels and nanomaterials.

  11. Detection of bla-IMP-1 and bla-IMP-2 Genes Among Metallo-β-lactamase-Producing Pseudomonas Aeruginosa Isolated from Burn Patients in Isfahan

    Directory of Open Access Journals (Sweden)

    M. Pourbabaee

    2016-02-01

    Full Text Available Background: Pseudomonas aeruginosa is a nosocomial pathogen which especially causes infections among burn patients. Carbapenems are extensively used for the treatment of infections caused by multidrug-resistant P. aeruginosa isolates. The emergence of carbapenemases producing isolates is an outcome of increased utilization of carbapenems. The aim of this study was to determine the bla-IMP-1 and bla-IMP-2 genes in metallo-β-lactamase (MBL -producing Pseudomonas aeruginosa isolated from burn patients in Isfahan. Material and Methods: A total of 150 P. aeruginosa were isolated from burn patients hospitalized in Imam-Mousakazem hospital in Isfahan. Antimicrobial susceptibility was determined using disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI guidelines. Double Disk Synergy Test (DDST was carried out for screening of MBL production in imipenem-resistant strains. PCR assays were used for detection of bla-IMP-1 and bla-IMP-2 genes among metallo-β-lactamase-producing Pseudomonas aeruginosa isolates. The purified PCR products were sequenced. Results: Of 150 Pseudomonas aeruginosa isolates, %100 identified as multi-drug resistant strains. The most resistance rates were seen against ciprofloxacin, tobromycin, meropenem and imipenem. All of 144 imipenem-resistant Pseudomonas aeruginosa isolates were MBL producing by DDST test. Twenty-nine (19.3% and 8(5.3% MBL producing Pseudomonas aeruginosa isolates harbored bla-IMP-1 and bla-IMP-2 genes respectively. Conclusions: According to results of this study high level resistance to imipenem and MBl genes carriage was seen among Pseudomonas aeruginosa isolated from burn patient infections in our region.

  12. A Phytoanticipin Derivative, Sodium Houttuyfonate, Induces in Vitro Synergistic Effects with Levofloxacin against Biofilm Formation by Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Jing Shao

    2012-09-01

    Full Text Available Antibiotic resistance has become the main deadly factor in infections, as bacteria can protect themselves by hiding in a self-constructed biofilm. Consequently, more attention is being paid to the search for “non-antibiotic drugs” to solve this problem. Phytoanticipins, the natural antibiotics from plants, could be a suitable alternative, but few works on this aspect have been reported. In this study, a preliminary study on the synergy between sodium houttuyfonate (SH and levofloxacin (LFX against the biofilm formation of Pseudomonas aeruginosa was performed. The minimal inhibitory concentrations (MIC of LFX and SH, anti-biofilm formation and synergistic effect on Pseudomonas aeruginosa, and quantification of alginate were determined by the microdilution method, crystal violet (CV assay, checkerboard method, and hydroxybiphenyl colorimetry. The biofilm morphology of Pseudomonas aeruginosa was observed by fluorescence microscope and scanning electric microscope (SEM. The results showed that: (i LFX and SH had an obvious synergistic effect against Pseudomonas aeruginosa with MIC values of 0.25 μg/mL and 128 μg/mL, respectively; (ii ½ × MIC SH combined with 2 × MIC LFX could suppress the biofilm formation of Pseudomonas aeruginosa effectively, with up to 73% inhibition; (iii the concentration of alginate decreased dramatically by a maximum of 92% after treatment with the combination of antibiotics; and (iv more dead cells by fluorescence microscope and more removal of extracellular polymeric structure (EPS by SEM were observed after the combined treatment of LFX and SH. Our experiments demonstrate the promising future of this potent antimicrobial agent against biofilm-associated infections.

  13. Cell death in Pseudomonas aeruginosa biofilm development

    DEFF Research Database (Denmark)

    Webb, J.S.; Thompson, L.S.; James, S.

    2003-01-01

    Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids....... However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death...... occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development...

  14. Lipopolysaccharides of the cyanobacterium Microcystis aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Raziuddin, S.; Siegelman, H.W.; Tornabene, T.G.

    1983-01-01

    Lipopolysaccharides (LPS) of two isolates of Microcystis aeruginosa were extracted with phenol/water and purified. Cesium chloride gradient ultracentrifugation of these preparations yielded only one fraction. The LPS contained significant amounts of 3-deoxy-D-manno-octulosonic acid, glucose, 3-deoxy sugars, glucosamine, fatty acids, fatty acid esters, hexoses, and phosphate. Heptose, a characteristic sugar component of the polysaccharide moiety of LPS of most gram-negative bacteria was absent. Lipopolysaccharides and lipid A hydrolysate of LPS preparations were active in mouse lethality and Limulus lysate gelation. The lipid A moiety was slightly less active in toxicity and Limulus lysate gelation assay than the intact LPS. The LPS and lipid A moiety of the two isolates of M. aeruginosa were less active in toxicity in mice and Limulus test than LPS of Salmonella abortus equi. 37 references, 1 figure, 3 tables.

  15. Pseudomonas aeruginosa ventilator-associated pneumonia management

    Science.gov (United States)

    Ramírez-Estrada, Sergio; Borgatta, Bárbara; Rello, Jordi

    2016-01-01

    Ventilator-associated pneumonia is the most common infection in intensive care unit patients associated with high morbidity rates and elevated economic costs; Pseudomonas aeruginosa is one of the most frequent bacteria linked with this entity, with a high attributable mortality despite adequate treatment that is increased in the presence of multiresistant strains, a situation that is becoming more common in intensive care units. In this manuscript, we review the current management of ventilator-associated pneumonia due to P. aeruginosa, the most recent antipseudomonal agents, and new adjunctive therapies that are shifting the way we treat these infections. We support early initiation of broad-spectrum antipseudomonal antibiotics in present, followed by culture-guided monotherapy de-escalation when susceptibilities are available. Future management should be directed at blocking virulence; the role of alternative strategies such as new antibiotics, nebulized treatments, and vaccines is promising. PMID:26855594

  16. Pseudomonas aeruginosa biofilms in cystic fibrosis

    DEFF Research Database (Denmark)

    Høiby, Niels; Ciofu, Oana; Bjarnsholt, Thomas

    2010-01-01

    The persistence of chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients is due to biofilm-growing mucoid (alginate-producing) strains. A biofilm is a structured consortium of bacteria, embedded in a self-produced polymer matrix consisting of polysaccharide, protein...... and DNA. In CF lungs, the polysaccharide alginate is the major part of the P. aeruginosa biofilm matrix. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and resist phagocytosis, as well as other components of the innate and the adaptive immune system....... As a consequence, a pronounced antibody response develops, leading to immune complex-mediated chronic inflammation, dominated by polymorphonuclear leukocytes. The chronic inflammation is the major cause of the lung tissue damage in CF. Biofilm growth in CF lungs is associated with an increased frequency...

  17. Pseudomonas aeruginosa endophthalmitis masquerading as chronic uveitis

    Directory of Open Access Journals (Sweden)

    Kalpana Badami Nagaraj

    2013-01-01

    Full Text Available A 65-year-old male presented with decreased vision in the left eye of 15-day duration after having undergone an uneventful cataract surgery 10 months back. He had been previously treated with systemic steroids for recurrent uveitis postoperatively on three occasions in the same eye. B-scan ultrasonography showed multiple clumplike echoes suggestive of vitreous inflammation. Aqueous tap revealed Pseudomonas aeruginosa sensitive to ciprofloxacin. The patient was treated with intravitreal ciprofloxacin and vancomycin along with systemic ciprofloxacin with good clinical response. Even a virulent organism such as P.aeruginosa can present as a chronic uveitis, which, if missed, can lead to a delay in accurate diagnosis and appropriate management.

  18. Candida albicans ethanol stimulates Pseudomonas aeruginosa WspR-controlled biofilm formation as part of a cyclic relationship involving phenazines.

    Directory of Open Access Journals (Sweden)

    Annie I Chen

    2014-10-01

    Full Text Available In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP, and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C. albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis.

  19. Incidence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in diabetes and cancer patients

    Directory of Open Access Journals (Sweden)

    Varaiya Ami

    2008-04-01

    Full Text Available Metallo-beta-lactamase (MBL-producing Pseudomonas aeruginosa strains have been reported to be an important cause of nosocomial infections. There is not enough information from India regarding their prevalence in diabetic and cancer patients. The present study was undertaken over a period of one year from January to December 2006 to study the incidence of MBL P. aeruginosa and the clinical outcome in diabetes and cancer patients admitted to S.L. Raheja Hospital, Mumbai. Two hundred and thirty isolates of P. aeruginosa were obtained from different samples of patients. These isolates were subjected to susceptibility testing to anti-pseudomonal drugs as per CLSI guidelines. They were further screened for the production of MBL by disc potentiation testing using EDTA-impregnated imipenem and meropenem discs. Of the 230 isolates of P. aeruginosa, 60 (26% isolates were found resistant to carbapenems (both imipenem and meropenem and 33 (14.3% were found to be MBL producers. Of the 33 MBL-producing isolates, 24 (72.7% were diabetic patients, six (18.1% were cancer patients and three (9% patients had both diabetes and cancer. Five (15.1% patients responded to the combination therapy of colistin, piperacillin with tazobactam and amikacin, while 28 (84.8% patients responded to the combination therapy of amikacin, piperacillin with tazobactam and gatifloxacin. Thus, the rapid dissemination of MBL producers is worrisome and necessitates the implementation of not just surveillance studies but also proper and judicious selection of antibiotics, especially carbapenems.

  20. Maternal effects of inducible tolerance against the toxic cyanobacterium Microcystis aeruginosa in the grazer Daphnia carinata

    International Nuclear Information System (INIS)

    Jiang, Xiaodong; Yang, Wei; Zhao, Shiye; Liang, Huishuang; Zhao, Yunlong; Chen, Liqiao; Li, Rui

    2013-01-01

    Cyanobacterial blooms are becoming potent agents of natural selection in aquatic ecosystems because of their high production of some toxins and increased frequency in recent decades with eutrophication and climate change. Maternal exposure to the toxic Microcystis aeruginosa significantly increased the intrinsic rates of population increase, average life span, and net reproductive rates of a clone of the planktonic grazer Daphnia carinata in an offspring environment where cyanobacteria were present, but not for two additional clones. Offspring from mothers exposed to M. aeruginosa had lower intrinsic rates of population increase, average life span, and net reproductive rates than individuals from unexposed mothers when fed exclusively a green alga. These results suggest that benefits, costs, and clonal variations of maternal effects of inducible tolerance should be considered when trying to understand ecological consequences of cyanobacterial blooms since they can shape the trophic interactions between cyanobacteria and daphnids. -- Highlights: •Maternal exposure to Microcystis aeruginosa significantly increased the offspring tolerance in a Daphnia carinata clone. •Another two clones, however, failed to response to maternal exposure. •Offspring from exposed mothers had lower fitness when fed exclusively a green alga. -- Capsule: Maternal exposure to the toxic Microcystis aeruginosa increased offspring fitness in one of three Daphnia carinata clones and carried a cost

  1. Structure and activity of the Pseudomonas aeruginosa hotdog-fold thioesterases PA5202 and PA2801.

    Science.gov (United States)

    Gonzalez, Claudio F; Tchigvintsev, Anatoli; Brown, Greg; Flick, Robert; Evdokimova, Elena; Xu, Xiaohui; Osipiuk, Jerzy; Cuff, Marianne E; Lynch, Susan; Joachimiak, Andrzej; Savchenko, Alexei; Yakunin, Alexander F

    2012-06-15

    The hotdog fold is one of the basic protein folds widely present in bacteria, archaea and eukaryotes. Many of these proteins exhibit thioesterase activity against fatty acyl-CoAs and play important roles in lipid metabolism, cellular signalling and degradation of xenobiotics. The genome of the opportunistic pathogen Pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. We have found that two P. aeruginosa hotdog proteins display high thioesterase activity against 3-hydroxy-3-methylglutaryl-CoA and glutaryl-CoA (PA5202), and octanoyl-CoA (PA2801). Crystal structures of these proteins were solved (at 1.70 and 1.75 Å for PA5202 and PA2801 respectively) and revealed a hotdog fold with a potential catalytic carboxylate residue located on the long α-helix (Asp(57) in PA5202 and Glu(35) in PA2801). Alanine residue replacement mutagenesis of PA5202 identified four residues (Asn(42), Arg(43), Asp(57) and Thr(76)) that are critical for its activity and are located in the active site. A P. aeruginosa PA5202 deletion strain showed an increased secretion of the antimicrobial pigment pyocyanine and an increased expression of genes involved in pyocyanin biosynthesis, suggesting a functional link between PA5202 activity and pyocyanin production. Thus the P. aeruginosa hotdog thioesterases PA5202 and PA2801 have similar structures, but exhibit different substrate preferences and functions.

  2. Diversity of Antimicrobial Resistance and Virulence Determinants in Pseudomonas aeruginosa Associated with Fresh Vegetables

    Directory of Open Access Journals (Sweden)

    Kashina Allydice-Francis

    2012-01-01

    Full Text Available With the increased focus on healthy eating and consuming raw vegetables, this study assessed the extent of contamination of fresh vegetables by Pseudomonas aeruginosa in Jamaica and examined the antibiotic susceptibility profiles and the presence of various virulence associated determinants of P. aeruginosa. Analyses indicated that vegetables from retail markets and supermarkets were widely contaminated by P. aeruginosa; produce from markets were more frequently contaminated, but the difference was not significant. Lettuce and carrots were the most frequently contaminated vegetables, while tomatoes were the least. Pigment production (Pyoverdine, pyocyanin, pyomelanin and pyorubin, fluorescein and alginate were common in these isolates. Imipenem, gentamicin and ciprofloxacin were the most inhibitory antimicrobial agents. However, isolates were resistant or showed reduced susceptibility to ampicillin, chloramphenicol, sulphamethoxazole/trimethoprim and aztreonam, and up to 35% of the isolates were resistant to four antimicrobial agents. As many as 30% of the isolates were positive for the fpv1 gene, and 13% had multiple genes. Sixty-four percent of the isolates harboured an exoenzyme gene (exoS, exoT, exoU or exoY, and multiple exo genes were common. We conclude that P. aeruginosa is a major contaminant of fresh vegetables, which might be a source of infection for susceptible persons within the community.

  3. Pseudomonas aeruginosa mutations in lasl and rhll quorum sensing systems result in milder chronic lung infection

    DEFF Research Database (Denmark)

    Wu, H.; Song, Z.J.; Givskov, Michael Christian

    2001-01-01

    To understand the importance of quorum sensing in chronic Pseudomonas aeruginosa lung infection, the in vivo pathogenic effects of the wild-type P aeruginosa PAO1 and its double mutant, PAO1 lasI rhlI, in which the signal-generating parts of the quorum sensing systems are defective were compared....... The rat model of P. aeruginosa lung infection was used in the present study. The rats were killed on days 3, 7, 14 and 28 after infection with the P. aeruginosa strains. The results showed that during the early stages of infection, the PAO1 double mutant induced a stronger serum antibody response, higher...... number of lung bacteria, and minor serum IgG and IgG1 responses but increased lung interferon gamma production were detected in the group infected with the PAO1 double mutant when compared with the PAO1-infected group. Delayed immune responses were observed in the PAO1-infected group and they might...

  4. Subinhibitory concentration of kanamycin induces the Pseudomonas aeruginosa type VI secretion system.

    Directory of Open Access Journals (Sweden)

    Cerith Jones

    Full Text Available Pseudomonas aeruginosa is a Gram-negative bacterium found in natural environments including plants, soils and warm moist surfaces. This organism is also in the top ten of nosocomial pathogens, and prevalent in cystic fibrosis (CF lung infections. The ability of P. aeruginosa to colonize a wide variety of environments in a lasting manner is associated with the formation of a resistant biofilm and the capacity to efficiently outcompete other microorganisms. Here we demonstrate that sub-inhibitory concentration of kanamycin not only induces biofilm formation but also induces expression of the type VI secretion genes in the H1-T6SS cluster. The H1-T6SS is known for its role in toxin production and bacterial competition. We show that the antibiotic induction of the H1-T6SS only occurs when a functional Gac/Rsm pathway is present. These observations may contribute to understand how P. aeruginosa responds to antibiotic producing competitors. It also suggests that improper antibiotic therapy may enhance P. aeruginosa colonization, including in the airways of CF patients.

  5. Nosocomial outbreak of Pseudomonas aeruginosa endophthalmitis.

    Science.gov (United States)

    Mateos, I; Valencia, R; Torres, M J; Cantos, A; Conde, M; Aznar, J

    2006-11-01

    We describe an outbreak of nosocomial endophthalmitis due to a common source, which was determined to be trypan blue solution prepared in the hospital's pharmacy service. We assume that viable bacteria probably gained access to the trypan blue stock solution during cooling after autoclaving. The temporal cluster of Pseudomonas aeruginosa endophthalmitis was readily perceived on the basis of clinical and microbiological findings, and an exogenous source of contamination was unequivocally identified by means of DNA fingerprinting.

  6. Genetics of Persister Formation in Pseudomonas aeruginosa

    Science.gov (United States)

    2012-12-14

    situation where P. aeruginosa infects the airways. Intubated patients are at risk for developing ventilator-associated pneumonia ( VAP ), which can develop...infects the airways. Intubated patients are at risk for developing ventilator-associated pneumonia ( VAP ), which can develop into a chronic...Department of the Army position , policy or decision, unless so designated by other documentation. 12. DISTRIBUTION AVAILIBILITY STATEMENT Approved for Public

  7. Effects of ginseng on Pseudomonas aeruginosa motility and biofilm formation

    DEFF Research Database (Denmark)

    Wu, Hong; Lee, Baoleri; Yang, Liang

    2011-01-01

    protected animal models from developing chronic lung infection by P. aeruginosa. In the present study, the effects of ginseng on the formation of P. aeruginosa biofilms were further investigated in vitro and in vivo. Ginseng aqueous extract at concentrations of 0.5-2.0% did not inhibit the growth of P......Biofilm-associated chronic Pseudomonas aeruginosa lung infections in patients with cystic fibrosis are virtually impossible to eradicate with antibiotics because biofilm-growing bacteria are highly tolerant to antibiotics and host defense mechanisms. Previously, we found that ginseng treatments....... aeruginosa, but significantly prevented P. aeruginosa from forming biofilm. Exposure to 0.5% ginseng aqueous extract for 24 h destroyed most 7-day-old mature biofilms formed by both mucoid and nonmucoid P. aeruginosa strains. Ginseng treatment enhanced swimming and twitching motility, but reduced swarming...

  8. Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

    Directory of Open Access Journals (Sweden)

    Ajantha Nadesalingam

    2018-03-01

    Full Text Available Tonicity of saline (NaCl is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA, Gram-negative bacterial cell wall component lipopolysaccharide (LPS, calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus, and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus. We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12, and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol. This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic

  9. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

    Science.gov (United States)

    Cross, Megan; Biberacher, Sonja; Park, Suk-Youl; Rajan, Siji; Korhonen, Pasi; Gasser, Robin B; Kim, Jeong-Sun; Coster, Mark J; Hofmann, Andreas

    2018-04-24

    The opportunistic bacterium Pseudomonas aeruginosa has been recognized as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas, which is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of 2 TPP genes with either chromosomal or extrachromosomal location. Both TPPs were produced as recombinant proteins, and characterization of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of T6P. The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through β-sheet expansion of the individual monomers, which possess the overall fold of halo-acid dehydrogenases.-Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R. B., Kim, J.-S., Coster, M. J., Hofmann, A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

  10. Antivirulence activity of azithromycin in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Francesco eImperi

    2014-04-01

    Full Text Available Antibiotics represent our bulwark to combat bacterial infections, but the spread of antibiotic resistance compromises their clinical efficacy. Alternatives to conventional antibiotics are urgently needed in order to complement the existing antibacterial arsenal. The macrolide antibiotic azithromycin (AZM provides a paradigmatic example of an unconventional antibacterial drug. Besides its growth-inhibiting activity, AZM displays potent anti-inflammatory properties, as well as antivirulence activity on some intrinsically resistant bacteria, such as Pseudomonas aeruginosa. In this bacterium, the antivirulence activity of AZM mainly relies on its ability to interact with the ribosome, resulting in direct and/or indirect repression of specific subsets of genes involved in virulence, quorum sensing, biofilm formation and intrinsic antibiotic resistance. Both clinical experience and clinical trials have shown the efficacy of AZM in the treatment of chronic pulmonary infections caused by P. aeruginosa. The aim of this review is to combine results from laboratory studies with evidence from clinical trials in order to unify the information on the in vivo mode of action of AZM in P. aeruginosa infection.

  11. Extracellular signaling and multicellularity in Bacillus subtilis.

    Science.gov (United States)

    Shank, Elizabeth Anne; Kolter, Roberto

    2011-12-01

    Bacillus subtilis regulates its ability to differentiate into distinct, co-existing cell types in response to extracellular signaling molecules produced either by itself, or present in its environment. The production of molecules by B. subtilis cells, as well as their response to these signals, is not uniform across the population. There is specificity and heterogeneity both within genetically identical populations as well as at the strain-level and species-level. This review will discuss how extracellular signaling compounds influence B. subtilis multicellularity with regard to matrix-producing cannibal differentiation, germination, and swarming behavior, as well as the specificity of the quorum-sensing peptides ComX and CSF. It will also highlight how imaging mass spectrometry can aid in identifying signaling compounds and contribute to our understanding of the functional relationship between such compounds and multicellular behavior. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Intraclonal Genome Stability of the Metallo-β-lactamase SPM-1-producing Pseudomonas aeruginosa ST277, an Endemic Clone Disseminated in Brazilian Hospitals

    Directory of Open Access Journals (Sweden)

    Ana Paula Barbosa Nascimento

    2016-12-01

    Full Text Available Carbapenems represent the mainstay therapy for the treatment of serious P. aeruginosa infections. However, the emergence of carbapenem resistance has jeopardized the clinical use of this important class of compounds. The production of SPM-1 metallo-β-lactamase has been the most common mechanism of carbapenem resistance identified in P. aeruginosa isolated from Brazilian medical centres. Interestingly, a single SPM-1-producing P. aeruginosa clone belonging to the ST277 has been widely spread within the Brazilian territory. In the current study, we performed a next-generation sequencing of six SPM-1-producing P. aeruginosa ST277 isolates. The core genome contains 5 899 coding genes relative to the reference strain P. aeruginosa PAO1. A total of 26 genomic islands were detected in these isolates. We identified remarkable elements inside these genomic islands, such as copies of the blaSPM-1 gene conferring resistance to carbapenems and a type I-C CRISPR-Cas system, which is involved in protection of the chromosome against foreign DNA. In addition, we identified single nucleotide polymorphisms causing amino acid changes in antimicrobial resistance and virulence-related genes. Together, these factors could contribute to the marked resistance and persistence of the SPM-1-producing P. aeruginosa ST277 clone. A comparison of the SPM-1-producing P. aeruginosa ST277 genomes showed that their core genome has a high level nucleotide similarity and synteny conservation. The variability observed was mainly due to acquisition of genomic islands carrying several antibiotic resistance genes.

  13. Did Mineral Surface Chemistry and Toxicity Contribute to Evolution of Microbial Extracellular Polymeric Substances?

    Science.gov (United States)

    Campbell, Jay M.; Zhang, Nianli; Hickey, William J.

    2012-01-01

    Abstract Modern ecological niches are teeming with an astonishing diversity of microbial life in biofilms closely associated with mineral surfaces, which highlights the remarkable success of microorganisms in conquering the challenges and capitalizing on the benefits presented by the mineral–water interface. Biofilm formation capability likely evolved on early Earth because biofilms provide crucial cell survival functions. The potential toxicity of mineral surfaces toward cells and the complexities of the mineral–water–cell interface in determining the toxicity mechanisms, however, have not been fully appreciated. Here, we report a previously unrecognized role for extracellular polymeric substances (EPS), which form biofilms in shielding cells against the toxicity of mineral surfaces. Using colony plating and LIVE/DEAD staining methods in oxide suspensions versus oxide-free controls, we found greater viability of wild-type, EPS-producing strains of Pseudomonas aeruginosa PAO1 compared to their isogenic knockout mutant with defective biofilm-producing capacity. Oxide toxicity was specific to its surface charge and particle size. High resolution transmission electron microscopy (HRTEM) images and assays for highly reactive oxygen species (hROS) on mineral surfaces suggested that EPS shield via both physical and chemical mechanisms. Intriguingly, qualitative as well as quantitative measures of EPS production showed that toxic minerals induced EPS production in bacteria. By determining the specific toxicity mechanisms, we provide insight into the potential impact of mineral surfaces in promoting increased complexity of cell surfaces, including EPS and biofilm formation, on early Earth. Key Words: Mineral toxicity—Bacteria—EPS evolution—Biofilms—Cytotoxicity—Silica—Anatase—Alumina. Astrobiology 12, 785–798. PMID:22934560

  14. Coproduction of Novel 16S rRNA Methylase RmtD and Metallo-β-Lactamase SPM-1 in a Panresistant Pseudomonas aeruginosa Isolate from Brazil▿

    OpenAIRE

    Doi, Yohei; de Oliveira Garcia, Doroti; Adams, Jennifer; Paterson, David L.

    2006-01-01

    Serious infections with Pseudomonas aeruginosa are frequently treated with the combination of a β-lactam antimicrobial and an aminoglycoside. P. aeruginosa strain PA0905 was isolated in 2005 from an inpatient in Brazil. It showed a panresistant phenotype that included resistance to β-lactams, aminoglycosides, and fluoroquinolones. The β-lactam resistance was conferred by the production of the metallo-β-lactamase SPM-1. No inhibitory zone was observed when a disk diffusion test was performed w...

  15. Positive signature-tagged mutagenesis in Pseudomonas aeruginosa: tracking patho-adaptive mutations promoting airways chronic infection.

    Directory of Open Access Journals (Sweden)

    Irene Bianconi

    2011-02-01

    Full Text Available The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection.

  16. Extracellular communication in bacteria

    DEFF Research Database (Denmark)

    Chhabra, S.R.; Philipp, B.; Eberl, L.

    2005-01-01

    molecules, in different Gram-positive and Gram-negative bacteria they control pathogenicity, secondary metabolite production, biofilm differentiation, DNA transfer and bioluminescence. The development of biosensors for the detection of these signal molecules has greatly facilitated their subsequent chemical...

  17. Dermal extracellular lipid in birds.

    Science.gov (United States)

    Stromberg, M W; Hinsman, E J; Hullinger, R L

    1990-01-01

    A light and electron microscopic study of the skin of domestic chickens, seagulls, and antarctic penguins revealed abundant extracellular dermal lipid and intracellular epidermal lipid. Dermal lipid appeared ultrastructurally as extracellular droplets varying from less than 1 micron to more than 25 microns in diameter. The droplets were often irregularly contoured, sometimes round, and of relatively low electron density. Processes of fibrocytes were often seen in contact with extracellular lipid droplets. Sometimes a portion of such a droplet was missing, and this missing part appeared to have been "digested away" by the cell process. In places where cells or cell processes are in contact with fact droplets, there are sometimes extracellular membranous whorls or fragments which have been associated with the presence of fatty acids. Occasionally (in the comb) free fat particles were seen in intimate contact with extravasated erythrocytes. Fat droplets were seen in the lumen of small dermal blood and lymph vessels. We suggest that the dermal extracellular lipid originates in the adipocyte layer and following hydrolysis the free fatty acids diffuse into the epidermis. Here they become the raw material for forming the abundant neutral lipid contained in many of the epidermal cells of both birds and dolphins. The heretofore unreported presence and apparently normal utilization of abundant extracellular lipid in birds, as well as the presence of relatively large droplets of neutral lipid in dermal vessels, pose questions which require a thorough reappraisal of present concepts of the ways in which fat is distributed and utilized in the body.

  18. A Metabolic Trade-Off Modulates Policing of Social Cheaters in Populations of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Huicong Yan

    2018-02-01

    Full Text Available Pseudomonas aeruginosa uses quorum sensing (QS to regulate the production of public goods such as the secreted protease elastase. P. aeruginosa requires the LasI–LasR QS circuit to induce elastase and enable growth on casein as the sole carbon and energy source. The LasI–LasR system also induces a second QS circuit, the RhlI–RhlR system. During growth on casein, LasR-mutant social cheaters emerge, and this can lead to a population collapse. In a minimal medium containing ammonium sulfate as a nitrogen source, populations do not collapse, and cheaters and cooperators reach a stable equilibrium; however, without ammonium sulfate, cheaters overtake the cooperators and populations collapse. We show that ammonium sulfate enhances the activity of the RhlI–RhlR system in casein medium and this leads to increased production of cyanide, which serves to control levels of cheaters. This enhancement of cyanide production occurs because of a trade-off in the metabolism of glycine: exogenous ammonium ion inhibits the transformation of glycine to 5,10-methylenetetrahydrofolate through a reduction in the expression of the glycine cleavage genes gcvP1 and gcvP2, thereby increasing the availability of glycine as a substrate for RhlR-regulated hydrogen cyanide synthesis. Thus, environmental ammonia enhances cyanide production and stabilizes QS in populations of P. aeruginosa.

  19. Promysalin Elicits Species-Selective Inhibition of Pseudomonas aeruginosa by Targeting Succinate Dehydrogenase.

    Science.gov (United States)

    Keohane, Colleen E; Steele, Andrew D; Fetzer, Christian; Khowsathit, Jittasak; Van Tyne, Daria; Moynié, Lucile; Gilmore, Michael S; Karanicolas, John; Sieber, Stephan A; Wuest, William M

    2018-02-07

    Natural products have served as an inspiration to scientists both for their complex three-dimensional architecture and exquisite biological activity. Promysalin is one such Pseudomonad secondary metabolite that exhibits narrow-spectrum antibacterial activity, originally isolated from the rhizosphere. We herein utilize affinity-based protein profiling (AfBPP) to identify succinate dehydrogenase (Sdh) as the biological target of the natural product. The target was further validated in silico, in vitro, in vivo, and through the selection, and sequencing, of a resistant mutant. Succinate dehydrogenase plays an essential role in primary metabolism of Pseudomonas aeruginosa as the only enzyme that is involved both in the tricarboxylic acid cycle (TCA) and in respiration via the electron transport chain. These findings add credence to other studies that suggest that the TCA cycle is an understudied target in the development of novel therapeutics to combat P. aeruginosa, a significant pathogen in clinical settings.

  20. Pseudomonas aeruginosa can be detected in a polymicrobial competition model using impedance spectroscopy with a novel biosensor.

    Directory of Open Access Journals (Sweden)

    Andrew C Ward

    Full Text Available Electrochemical Impedance Spectroscopy (EIS is a powerful technique that can be used to elicit information about an electrode interface. In this article, we highlight six principal processes by which the presence of microorganisms can affect impedance and show how one of these--the production of electroactive metabolites--changes the impedance signature of culture media containing Pseudomonas aeruginosa. EIS, was used in conjunction with a low cost screen printed carbon sensor to detect the presence of P. aeruginosa when grown in isolation or as part of a polymicrobial infection with Staphylococcus aureus. By comparing the electrode to a starting measurement, we were able to identify an impedance signature characteristic of P. aeruginosa. Furthermore, we are able to show that one of the changes in the impedance signature is due to pyocyanin and associated phenazine compounds. The findings of this study indicate that it might be possible to develop a low cost sensor for the detection of P. aeruginosa in important point of care diagnostic applications. In particular, we suggest that a development of the device described here could be used in a polymicrobial clinical sample such as sputum from a CF patient to detect P. aeruginosa.

  1. Sequestration and Distribution Characteristics of Cd(II) by Microcystis aeruginosa and Its Role in Colony Formation.

    Science.gov (United States)

    Bi, Xiangdong; Yan, Ran; Li, Fenxiang; Dai, Wei; Jiao, Kewei; Zhou, Qixing; Liu, Qi

    2016-01-01

    To investigate the sequestration and distribution characteristics of Cd(II) by Microcystis aeruginosa and its role in Microcystis colony formation, M. aeruginosa was exposed to six different Cd(II) concentrations for 10 days. Cd(II) exposure caused hormesis in the growth of M. aeruginosa . Low concentrations of Cd(II) significantly induced formation of small Microcystis colonies ( P bEPS) contents of M. aeruginosa significantly ( P 93% of Cd(II) was sequestrated in the groups with lower added concentrations of Cd(II). More than 80% of the sequestrated Cd(II) was bioadsorbed by bEPS. The Pearson correlation coefficients of exterior and interior factors related to colony formation of M. aeruginosa revealed that Cd(II) could stimulate the production of IPS and bEPS via increasing Cd(II) bioaccumulation and bioadsorption. Increased levels of cross-linking between Cd(II) and bEPS stimulated algal cell aggregation, which eventually promoted the formation of Microcystis colonies.

  2. Evolutionary conservation of essential and highly expressed genes in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Scharfe Maren

    2010-04-01

    Full Text Available Abstract Background The constant increase in development and spread of bacterial resistance to antibiotics poses a serious threat to human health. New sequencing technologies are now on the horizon that will yield massive increases in our capacity for DNA sequencing and will revolutionize the drug discovery process. Since essential genes are promising novel antibiotic targets, the prediction of gene essentiality based on genomic information has become a major focus. Results In this study we demonstrate that pooled sequencing is applicable for the analysis of sequence variations of strain collections with more than 10 individual isolates. Pooled sequencing of 36 clinical Pseudomonas aeruginosa isolates revealed that essential and highly expressed proteins evolve at lower rates, whereas extracellular proteins evolve at higher rates. We furthermore refined the list of experimentally essential P. aeruginosa genes, and identified 980 genes that show no sequence variation at all. Among the conserved nonessential genes we found several that are involved in regulation, motility and virulence, indicating that they represent factors of evolutionary importance for the lifestyle of a successful environmental bacterium and opportunistic pathogen. Conclusion The detailed analysis of a comprehensive set of P. aeruginosa genomes in this study clearly disclosed detailed information of the genomic makeup and revealed a large set of highly conserved genes that play an important role for the lifestyle of this microorganism. Sequencing strain collections enables for a detailed and extensive identification of sequence variations as potential bacterial adaptation processes, e.g., during the development of antibiotic resistance in the clinical setting and thus may be the basis to uncover putative targets for novel treatment strategies.

  3. Molybdate transporter ModABC is important for Pseudomonas aeruginosa chronic lung infection.

    Science.gov (United States)

    Périnet, Simone; Jeukens, Julie; Kukavica-Ibrulj, Irena; Ouellet, Myriam M; Charette, Steve J; Levesque, Roger C

    2016-01-12

    Mechanisms underlying the success of Pseudomonas aeruginosa in chronic lung infection among cystic fibrosis (CF) patients are poorly defined. The modA gene was previously linked to in vivo competitiveness of P. aeruginosa by a genetic screening in the rat lung. This gene encodes a subunit of transporter ModABC, which is responsible for extracellular uptake of molybdate. This compound is essential for molybdoenzymes, including nitrate reductases. Since anaerobic growth conditions are known to occur during CF chronic lung infection, inactivation of a molybdate transporter could inhibit proliferation through the inactivation of denitrification enzymes. Hence, we performed phenotypic characterization of a modA mutant strain obtained by signature-tagged mutagenesis (STM_modA) and assessed its virulence in vivo with two host models. The STM_modA mutant was in fact defective for anaerobic growth and unable to use nitrates in the growth medium for anaerobic respiration. Bacterial growth and nitrate usage were restored when the medium was supplemented with molybdate. Most significantly, the mutant strain showed reduced virulence compared to wild-type strain PAO1 according to a competitive index in the rat model of chronic lung infection and a predation assay with Dictyostelium discoideum amoebae. As the latter took place in aerobic conditions, the in vivo impact of the mutation in modA appears to extend beyond its effect on anaerobic growth. These results support the modABC-encoded transporter as important for the pathogenesis of P. aeruginosa, and suggest that enzymatic machinery implicated in anaerobic growth during chronic lung infection in CF merits further investigation as a potential target for therapeutic intervention.

  4. Biodegradation of crude oil by Pseudomonas aeruginosa in the presence of rhamnolipids*

    OpenAIRE

    Zhang, Guo-liang; Wu, Yue-ting; Qian, Xin-ping; Meng, Qin

    2005-01-01

    The potential biodegradation of crude oil was assessed based on the development of a fermentative process with a strain of Pseudomonas aeruginosa which produced 15.4 g/L rhamnolipids when cultured in a basal mineral medium using glycerol as a sole carbon source. However, neither cell growth nor rhamnolipid production was observed in the comparative culture system using crude oil as the sole carbon source instead. As rhamnolipid, an effective biosurfactant, has been reported to stimulate the b...

  5. Increased concentration of Pseudomonas aeruginosa and Staphylococcus sp. in small animals exposed to aerospace environments

    Science.gov (United States)

    Guthrie, R. K.

    1976-01-01

    The effects of increased concentrations of PSEUDOMONAS AERUGINOSA AND STAPHYLOCOCCUS in the total bacterial flora of small animals exposed to simulated spacecraft environments were evaluated. Tests to detect changes in infectivity, effects of antibiotic treatments, immune responses to bacterial antigens, and effectiveness of immune responses in the experimental environment were conducted. The most significant results appear to be the differences in immune responses at simulated altitudes and the production of infection in the presence of a specific antibody.

  6. Optimization of extracellular polysaccharide production in ...

    African Journals Online (AJOL)

    sunny

    2014-11-26

    Nov 26, 2014 ... Llamas I (2007). Characterization of exopolysaccharides produced by three moderately halophilic bacteria belonging to the family. Alteromonadaceae. J. Appl. Microbiol. 105:521-528. Mishra, Jha (2013). Microbial Exopolysaccharides In: Rosenberg, E.,. The Prokaryotes –Applied Bacteriology and ...

  7. Extracellular Palladium Nanoparticle Production using Geobacter sulfurreducens

    KAUST Repository

    Yates, Matthew D.; Cusick, Roland D.; Logan, Bruce E.

    2013-01-01

    verified that Pd(0) nanoparticles were predominantly in the EPS surrounding cells in H2-fed cultures, with only a small number of particles visible inside the cell. Separation of the cells and EPS by centrifugation allowed reuse of the cell suspensions

  8. Optimization of extracellular polysaccharide production in ...

    African Journals Online (AJOL)

    sunny

    2014-11-26

    Nov 26, 2014 ... textiles, detergents, adhesives, cosmetics, pharmaceu- ticals, food additives ... Isolation and identification of EPS producing halophilic bacteria ..... The present study lead to the optimization of key culture conditions with CCD ...

  9. Production, optimization and characterization of extracellular ...

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-04-27

    Apr 27, 2016 ... chemical hydrolysis of starch in starch processing industry (Pandey et .... were carried out in a thermal cycler (Multigene Optimax, Labnet international .... Lactose gave the lowest amylase enzyme activity (1.34. U/mg protein) ...

  10. EXTRACELLULAR CELLULOLYTIC COMPLEXES PRODUCTION BY MICROSCOPIC FUNGI

    Directory of Open Access Journals (Sweden)

    S. O. Syrchin

    2015-10-01

    Full Text Available The aim of this work was to screen and to study the effect of inducers on the synthesis of the cellulolytic enzyme complexes by microscopic fungi. Cellulolytic and xylanolytic activities were determined by reducing sugar with DNS reagent, and β-glucosidase activity by pNPG hydrolysis. The enzyme preparations were obtained by ammonium sulphate precipitation. Among 32 studied strains of microscopic fungi 14 produced cellulo- and xylanolytic enzyme complexes. Fusarium sp. 5 and Fennellia sp. 2806 demonstrated the highest levels of all studied enzyme activities. Enzyme preparations with high endo-, exoglucanase, xylanase and β-glucosidase activities were obtained from these strains. Fusarium sp. 5 and Fennellia sp. 2806 were active producers of cellulase enzyme complexes during growth on natural substrates. It was shown that inductors of cellulolytic enzymes in Fusarium sp. 5 and Fennellia sp. 2806 differed from the ones in Trichoderma reesei.

  11. growth and extracellular enzyme production by microorganisms

    African Journals Online (AJOL)

    Okorie

    2013-06-26

    Jun 26, 2013 ... 1Federal Institute of Industrial Research Oshodi, Lagos, Nigeria. 2Department of ... of Bacillus subtilis (Bs2) were able to produce lipase enzyme. The study ... However, most commercial starter cultures originated from those ... The traditional method of preparing Ugba was employed in the laboratory to ...

  12. Utility of in vivo transcription profiling for identifying Pseudomonas aeruginosa genes needed for gastrointestinal colonization and dissemination

    DEFF Research Database (Denmark)

    Koh, Andrew Y; Mikkelsen, Per J; Smith, Roger S

    2010-01-01

    these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI......, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia....

  13. Carbapenem inactivation: a very affordable and highly specific method for phenotypic detection of carbapenemase-producing Pseudomonas aeruginosa isolates compared with other methods.

    Science.gov (United States)

    Akhi, Mohammad Taghi; Khalili, Younes; Ghotaslou, Reza; Kafil, Hossein Samadi; Yousefi, Saber; Nagili, Behroz; Goli, Hamid Reza

    2017-06-01

    This investigation was undertaken to compare phenotypic and molecular methods for detection of carbapenemase-producing Pseudomonas aeruginosa. A total of 245 non-duplicated isolates of P. aeruginosa were collected from hospitalized patients. Disc diffusion method was used to identify carbapenem-resistant bacteria. Three phenotypic methods, including Modified Hodge Test (MHT), Modified Carba NP (MCNP) test and Carbapenem Inactivation Method (CIM) were used for investigation of carbapenemase production. In addition, polymerase chain reaction (PCR) was used to detect carbapenemase encoding genes. Of 245 P. aeruginosa isolates investigated, 121 isolates were carbapenem-resistant. Among carbapenem-resistant isolates, 40, 39 and 35 isolates exhibited positive results using MHT, MCNP test and CIM, respectively. PCR indicated the presence of carbapenemase genes in 35 of carbapenem-resistant isolates. MHT showed low sensitivity and specificity for carbapenemase detection among P. aeruginosa isolates in comparison to PCR. CIM was most affordable and highly specific than MCNP test compared with the molecular method.

  14. Crude petroleum-oil biodegradation efficiency of Bacillus subtilis and Pseudomonas aeruginosa strains isolated from a petroleum-oil contaminated soil from North-East India.

    Science.gov (United States)

    Das, Kishore; Mukherjee, Ashis K

    2007-05-01

    The efficiency of Bacillus subtilis DM-04 and Pseudomonas aeruginosa M and NM strains isolated from a petroleum contaminated soil sample from North-East India was compared for the biodegradation of crude petroleum-oil hydrocarbons in soil and shake flask study. These bacterial strains could utilize crude petroleum-oil hydrocarbons as sole source of carbon and energy. Bioaugmentation of TPH contaminated microcosm with P. aeruginosa M and NM consortia and B. subtilis strain showed a significant reduction of TPH levels in treated soil as compared to control soil at the end of experiment (120 d). P. aeruginosa strains were more efficient than B. subtilis strain in reducing the TPH content from the medium. The plate count technique indicated expressive growth and biosurfactant production by exogenously seeded bacteria in crude petroleum-oil rich soil. The results showed that B. subtilis DM-04 and P. aeruginosa M and NM strains could be effective for in situ bioremediation.

  15. Detection of Chloramphenicol Resistance Genes (cat in Clinical Isolates of Pseudomonas aeruginosa with Polymerase Chain Reaction Method

    Directory of Open Access Journals (Sweden)

    Tiana Milanda

    2014-12-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic Gram negative bacteria, which may cause infection in eyes, ears, skin, bones, central nervous system, gastrointestinal tract, circulatory system, heart, respiratory system, and urinary tract. Recently, chloramphenicol is no longer used as the main option of the therapy due of its resistance case. The aim of this research was to detect the presence of gene which is responsible to chloramphenicol resistance in clinical isolates of P.aeruginosa. These bacteria isolated from pus of external otitis patients in Hasan Sadikin Hospital in Bandung City. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were performed to detect this resistance gene. Electropherogram from PCR products showed that the chloramphenicol resistance in clinical isolates of P. aeruginosa was caused by cat gene (317 bp. Based on this research, cat gene may be used to detect the chloramphenicol resistance in patients with external ostitis.

  16. Chromosomal organization and segregation in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Isabelle Vallet-Gely

    2013-05-01

    Full Text Available The study of chromosomal organization and segregation in a handful of bacteria has revealed surprising variety in the mechanisms mediating such fundamental processes. In this study, we further emphasized this diversity by revealing an original organization of the Pseudomonas aeruginosa chromosome. We analyzed the localization of 20 chromosomal markers and several components of the replication machinery in this important opportunistic γ-proteobacteria pathogen. This technique allowed us to show that the 6.3 Mb unique circular chromosome of P. aeruginosa is globally oriented from the old pole of the cell to the division plane/new pole along the oriC-dif axis. The replication machinery is positioned at mid-cell, and the chromosomal loci from oriC to dif are moved sequentially to mid-cell prior to replication. The two chromosomal copies are subsequently segregated at their final subcellular destination in the two halves of the cell. We identified two regions in which markers localize at similar positions, suggesting a bias in the distribution of chromosomal regions in the cell. The first region encompasses 1.4 Mb surrounding oriC, where loci are positioned around the 0.2/0.8 relative cell length upon segregation. The second region contains at least 800 kb surrounding dif, where loci show an extensive colocalization step following replication. We also showed that disrupting the ParABS system is very detrimental in P. aeruginosa. Possible mechanisms responsible for the coordinated chromosomal segregation process and for the presence of large distinctive regions are discussed.

  17. The Versatile Mutational Resistome of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Carla López-Causapé

    2018-04-01

    Full Text Available One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including β-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

  18. The Versatile Mutational Resistome of Pseudomonas aeruginosa.

    Science.gov (United States)

    López-Causapé, Carla; Cabot, Gabriel; Del Barrio-Tofiño, Ester; Oliver, Antonio

    2018-01-01

    One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS) data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including β-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility) between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

  19. Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4.

    Science.gov (United States)

    Cheng, Minyi; Takenaka, Shinji; Aoki, Shunsuke; Murakami, Shuichiro; Aoki, Kenji

    2009-04-01

    A bacterial strain, ME-4, isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH(4))(2)SO(4), as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the P1 position and Lys at the P2 position. It also preferred hydrophobic amino acid residues at the P1' and P2' positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan.

  20. Chronic Pseudomonas aeruginosa lung infection in normal and athymic rats

    DEFF Research Database (Denmark)

    Johansen, H K; Espersen, F; Pedersen, S S

    1993-01-01

    We have compared a chronic lung infection with Pseudomonas aeruginosa embedded in alginate beads in normal and athymic rats with an acute infection with free live P. aeruginosa bacteria. The following parameters were observed and described: mortality, macroscopic and microscopic pathologic changes...

  1. Pseudomonas aeruginosa burn wound infection in a dedicated ...

    African Journals Online (AJOL)

    Background. Pseudomonas aeruginosa infection is a major cause of morbidity in burns patients. There is a paucity of publications dealing with this infection in the paediatric population. We describe the incidence, microbiology and impact of P. aeruginosa infection in a dedicated paediatric burns unit. Methods.

  2. Novel Targets for Treatment of Pseudomonas aeruginosa Biofilms

    DEFF Research Database (Denmark)

    Alhede, Morten; Alhede, Maria; Bjarnsholt, Thomas

    2014-01-01

    Pseudomonas aeruginosa causes infection in all parts of the human body. The bacterium is naturally resistant to a wide range of antibiotics. In addition to resistance mechanisms such as efflux pumps, the ability to form aggregates, known as biofilm, further reduces Pseudomonas aeruginosa...

  3. Ap-PCR typing of carbapenem sensitive Pseudomonas aeruginosa ...

    African Journals Online (AJOL)

    In this study the antibiotic susceptibility of 51 P. aeruginosa strains isolated from clinical samples were detected by the disc diffusion test. The susceptibility of P. aeruginosa strains were found as respectively 55% amicacin, 43% aztreonam, 75% netilmycin, 68% sefepim, 73% ceftazidim, 76% ciproflaxacin, 37% gentamicin, ...

  4. A Carbenicillin R Factor from Pseudomonas aeruginosa | van ...

    African Journals Online (AJOL)

    Of 64 carbenicillin-resistant Pseudomonas aeruginosa strains 40 transferred this resistance to Escherichia coli. R factor RP-638 isolated from Ps. aeruginosa strain 638 conferred resistance to ampicillin, carbenicillin, kanamycin, neomycin and tetracycline. This R factor was transferred at frequencies 01 10-7 to 10-4 between ...

  5. Plant flavones enhance antimicrobial activity of respiratory epithelial cell secretions against Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Benjamin M Hariri

    Full Text Available Flavones are a class of natural plant secondary metabolites that have anti-inflammatory and anti-bacterial effects. Some flavones also activate the T2R14 bitter taste receptor, which is expressed in motile cilia of the sinonasal epithelium and activates innate immune nitric oxide (NO production. Flavones may thus be potential therapeutics for respiratory infections. Our objective was to examine the anti-microbial effects of flavones on the common sinonasal pathogens Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, evaluating both planktonic and biofilm growth. Flavones had only very low-level antibacterial activity alone. They did not reduce biofilm formation, but did reduce production of the important P. aeruginosa inflammatory mediator and ciliotoxin pyocyanin. However, flavones exhibited synergy against P. aeruginosa in the presence of antibiotics or recombinant human lysozyme. They also enhanced the efficacy of antimicrobials secreted by cultured and primary human airway cells grown at air-liquid interface. This suggests that flavones may have anti-gram-negative potential as topical therapeutics when combined with antibiotics or in the context of innate antimicrobials secreted by the respiratory or other epithelia. This may have an additive effect when combined with T2R14-activated NO production. Additional studies are necessary to understand which flavone compounds or mixtures are the most efficacious.

  6. Targeting quorum sensing in Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Jakobsen, Tim Holm; Bjarnsholt, Thomas; Jensen, Peter Østrup

    2013-01-01

    Bacterial resistance to conventional antibiotics combined with an increasing acknowledgement of the role of biofilms in chronic infections has led to a growing interest in new antimicrobial strategies that target the biofilm mode of growth. In the aggregated biofilm mode, cell-to-cell communication...... alternative antibacterial strategies. Here, we review state of the art research of quorum sensing inhibitors against the opportunistic human pathogen Pseudomonas aeruginosa, which is found in a number of biofilm-associated infections and identified as the predominant organism infecting the lungs of cystic...

  7. A three-phase in-vitro system for studying Pseudomonas aeruginosa adhesion and biofilm formation upon hydrogel contact lenses

    Directory of Open Access Journals (Sweden)

    Kohlmann Thomas

    2010-11-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is commonly associated with contact lens (CL -related eye infections, for which bacterial adhesion and biofilm formation upon hydrogel CLs is a specific risk factor. Whilst P. aeruginosa has been widely used as a model organism for initial biofilm formation on CLs, in-vitro models that closely reproduce in-vivo conditions have rarely been presented. Results In the current investigation, a novel in-vitro biofilm model for studying the adherence of P. aeruginosa to hydrogel CLs was established. Nutritional and interfacial conditions similar to those in the eye of a CL wearer were created through the involvement of a solid:liquid and a solid:air interface, shear forces and a complex artificial tear fluid. Bioburdens varied depending on the CL material and biofilm maturation occurred after 72 h incubation. Whilst a range of biofilm morphologies were visualised including dispersed and adherent bacterial cells, aggregates and colonies embedded in extracellular polymer substances (EPS, EPS fibres, mushroom-like formations, and crystalline structures, a compact and heterogeneous biofilm morphology predominated on all CL materials. Conclusions In order to better understand the process of biofilm formation on CLs and to test the efficacy of CL care solutions, representative in-vitro biofilm models are required. Here, we present a three-phase biofilm model that simulates the environment in the eye of a CL wearer and thus generates biofilms which resemble those commonly observed in-situ.

  8. Bacteriophage-derived enzyme that depolymerizes the alginic acid capsule associated with cystic fibrosis isolates of Pseudomonas aerugino